SEARCH · PubMed Health

Results for “Paraffin Embedding”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources

PubMed1994

Determination of B-cell clonality in paraffin-embedded endoscopic biopsy specimens of abnormal lymphocytic infiltrates and gastrointestinal lymphoma by polymerase chain reaction.

External and seminested polymerase chain reaction techniques were used to determine B-cell clonality in paraffin-embedded biopsy specimens of abnormal lymphocytic infiltrates and malignant lymphomas of the gastrointestinal tract. Using consensus primers for the variable and joining regions, the authors detected clonal immunoglobulin heavy-chain gene rearrangements in five of eight endoscopic biopsy specimens (62.5%) and six of eight resection specimens (75%) of well-characterized B-cell gastrointestinal lymphomas. No clonal rearrangements were detected in 21 negative controls including 7 cases of chronic gastritis and 7 cases of Crohn's disease. In endoscopic biopsy specimens of eight patients with abnormal lymphocytic infiltrates, clonal immunoglobulin heavy-chain gene rearrangement was found in three of six cases (50%) in whom gastrointestinal involvement by lymphoma was subsequently established. Therefore, polymerase chain reaction may be used to demonstrate B-cell clonality in paraffin-embedded endoscopic biopsy specimens of abnormal lymphocytic infiltrates and may circumvent the need for more invasive procedures.

B-Lymphocytes↗

PubMed2002

[Study on detection of the Mycobacteria DNA in formalin-fixed, paraffin-embedded tissue samples by triplex polymerase chain reaction].

To supply an additional differential diagnostic method for pathological diagnosis of Mycobacterium tuberculosis complex and nontuberculous mycobacteria infections in formalin-fixed, paraffin-embedded tissue samples by triplex-PCR. Three pairs of oligonucleotide primer were used in triplex-PCR. A 383 bp DNA fragment encoding part of the 65 kD mycobacterial surface antigen, a 123 bp fragment corresponding to a specific Mycobacterium tuberculosis complex sequence which was the insertion sequence 6110 (IS6110) and a 268 bp fragment for human beta-globin were amplified by triplex-PCR respectively. The sensitivity of the triplex-PCR-electrophoresis for the mycobacteria DNA was 0.6 picogram. The specific bands of 383 bp and 123 bp among the amplified DNA from Mycobacterium tuberculosis, M. bovis, M. bovis BCG and M. simiae were present in the agarose gel. By contrast, only a band of 383 bp was found among the nontuberculosis mycobacteria which contained M. avium, M. chelonae, M. scrofulaceum, M. xenopi, M. kansasii, M. intracellulare and M. smegmatis. Compared with the standard strains, there was an additional 268 bp band in simulated clinic samples infected by mycobacteria, 209 formalin-fixed, paraffin-embedded tissue samples of the patients diagnosed as scrofula by clinic doctor at first visit were examined by triplex polymerase chain reaction. Among them, 193 tissue samples of the patients pathologically diagnosed as scrofula, tuberculous granulomatous tissue or tuberculous granulomatous inflammation were positive: the specific hands of 383 bp, 123 bp and 268 bp were present in the agarose gel and this tallied with Mycobacterium tuberculosis complex infection. Of 16 tissue samples of the patients pathologically diagnosed as suspicious scrofula, 15 samples were same positive results and this tallied with Mycobacterium tuberculosis complex infection, too; 1 sample could find the specific bands of 383 bp and 268 bp which were present in the agarose gel and this tallied with nontuberculous mycobacteria infection. The results showed that the triplex-PCR could detect and identify the DNA of Mycobacterium tuberculosis complex and nontuberculous mycobacteria except M. simiae. It is a valuable detecting method which has high sensitivity and specificity.

Adolescent↗

PubMed2002

Detection of Chlamydia trachomatis infection with DNA extracted from formalin-fixed paraffin-embedded tissues.

It is a well-known fact that tubal stenosis and/or peritubal adhesion are often associated with Chlamydia trachomatis infection. Although tubal pregnancy may be attributed to this infection, there are only extremely rare cases in which the presence of C. trachomatis has been confirmed by immumo-histochemical examination on tissues isolated from patients with tubal pregnancy. We thus tried to confirm the presence of C. trachomatis infection by detecting DNA of the organism in tissues surgically isolated from patients with tubal pregnancy. Two detection methods, a ligase chain reaction (LCR) method and an immuno-histochemical staining which detects an antigen of C. trachomatis, were compared using paraffin-embedded tissue samples which were surgically isolated from patients with tubal pregnancy or hydrosalpinx. The LCR method was capable of detecting DNA of C. trachomatis in tissue samples in which the C. trachomatis-specific antigen could not be detected using immuno-histochemical staining. This was due to the fact that immuno-histochemical staining methods are applicable to the analysis of sequences the length of which range from 200 to 400 bp (base pairs), while the LCR method used here allows the analysis of sequences as small as 48 bp. This fact makes the LCR method a very convenient tool, as compared with immuno-histochemical methods, for analysis of the paraffin embedded tissue samples where by effects of formalin--used for fixation for pathologic diagnosis--the risk of fragmenting the DNA samples is relatively high. Presence of C. trachomatis DNA as detected by LCR method in surgically isolated samples from patients with tubal pregnancy supports the involvement of Chlamydia trachomatis infection in the occurrence of tubal pregnancy. Accordingly the LCR method is capable of detecting DNA of C. trachomatis in paraffin-embedded samples of tubal tissue in which presence of C. trachomatis could not be confirmed by an immuno-histochemical staining method.

Adult↗

PubMed2003

Differentiation between porcine epidemic diarrhea virus and transmissible gastroenteritis virus in formalin-fixed paraffin-embedded tissues by multiplex RT-nested PCR and comparison with in situ hybridization.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) were detected and differentiated in formalin-fixed, paraffin-embedded tissues from experimentally and naturally infected pigs by multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR). The results of this new method were compared with in situ hybridization. A method based on xylene deparaffinization followed by proteinase K digestion yielded RNA of a suitable quality for reliable and consistent multiplex RT-nPCR analyses. PEDV and TGEV cDNAs were detected in jejunal tissues from experimentally and naturally infected pigs by multiplex RT-nPCR. Distinct positive signals for PEDV and TGEV were also detected in the same jejunal tissues by in situ hybridization. The rate of conformity between multiplex RT-nPCR and in situ hybridization was 100% for the detection of PEDV and TGEV in formalin-fixed paraffin-embedded jejunal tissues.

Animals↗

PubMed2004

Electrochemical antigen-retrieval of formaldehyde fixed and paraffin-embedded archived leprosy skin biopsies.

While formaldehyde fixation preserves tissue morphology, it often hinders immunodetection of antigens in paraffin-embedded tissue because the antigens are masked. Antigen unmasking can be achieved with treatments such as microwave irradiation but they often lead to excessive tissue damage. Therefore, an electrochemical antigen-retrieval method (EAR) was devised in which an alternating electric current is passed through the tissue in a chamber containing an electrolyte buffer. The results obtained with this method were compared to those after microwave irradiation using archived samples of formaldehyde-fixed and paraffin-embedded lepromatous leprosy skin. The efficacy of the two unmasking procedures was assessed by the immunodetectability of several marker antigens using 24 antibodies. Fifteen antibodies that were directed against transmembrane proteins (CD), and the remaining 9 against cytokeratins 18.6 and 19, laminin, vimentin, S100a, BCG, Ulex europaeus lectin, PCNA, and P21ras. Simple and double immunohistochemistry was performed using the universal ENVISION and LSAB + AP detection systems. After unmasking with the EAR method, immunoreactivity was clearly detected with 22 of the 24 antibodies in single labeling reactions. They include the critical antigens CD3 and CD4 for identifying the T lymphocyte lineages. In contrast, only 20 of the antibodies reacted after microwave irradiation. After double immunolabeling, immunoreactivity was quantitatively similar with both methods. However, the EAR unmasking produced a stronger labeling reaction. Thus, with double labeling immunohistochemistry, EAR made it possible to use higher antibody dilutions and shorter incubation times. Heat damage was also prevented. In conclusion, EAR treatment produces better staining results than microwave irradiation treatment.

Electrochemistry↗

PubMed1981

Enzyme histochemistry on normal and pathologic paraffin-embedded lymphoid tissues.

So far, enzyme histochemical examination has been applied, with few exceptions, either to tissue imprints or to cryostat sections of freshly collected samples. This procedure is not easily applicable in routine histopathologic examination. In this study, a simplified tissue embedding procedure is presented which can be performed using an automatic tissue changer. The paraffin embedded samples can be used for both conventional histopathologic examination and for demonstrating enzymes in sections. The enzymes studied (alkaline phosphatase, alpha-naphthyl acetate esterase, acid phosphatase, tartrate resistant acid phosphatase, ATPase, peroxidase, and chloroacetate esterase) gave comparable results in formalin-fixed cryostat sections and paraffin sections in both normal and pathologic lymphoid samples. The only exception was ATPase, which could not be demonstrated on paraffin-embedded material. The technic described has broad application in the analysis of lymphoid diseases.

Acid Phosphatase↗

PubMed2000

Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed paraffin-embedded tissues: experience with 63 markers.

Formalin fixation produces cross-links between the proteins and the fixative that alter the ability of some antibodies to recognize antigens. We used formalin-fixed, paraffin-embedded tissues to compare two different antigen retrieval methods for 63 antibodies used in the diagnosis of infectious and neoplastic diseases of animal species. Eighty-four percent of the antibodies needed some type of antigen retrieval for optimal results. Of those antibodies, 67.7% were monoclonal and 32.3% were polyclonal. Steam heat was the method of choice for 31 antibodies. Ten antibodies reacted only with steam heat, but 9 antibodies did not react when steam heat was used. Optimal results were obtained with enzyme digestion for 22 antibodies. Only 10 antibodies yielded optimal results without antigen retrieval; 64% of these antibodies were polyclonal. All antibodies against cytokeratins were optimally retrieved with proteinese K. Antigen retrieval appears to be necessary for the majority of antibodies when used with formalin-fixed, paraffin-embedded tissues.

Animal Diseases↗

PubMed1997

Detection of three transthyretin gene mutations in familial amyloidotic polyneuropathy by analysis of DNA extracted from formalin-fixed and paraffin-embedded tissues.

We identified three different missense mutations of the transthyretin (TTR) gene in three Japanese patients with familial amyloidotic polyneuropathy by analysis of their DNAs extracted from formalin-fixed and paraffin-embedded tissues. Patient 1 carried the TTR methionine-30 (Met) mutation (G to A transition at position 1679). DNA sequencing analysis of the TTR gene from patient 2 showed a G to T transversion at position 3830 in exon 3, resulting in an amino acid replacement of serine-50 (Ser) with isoleucine (Ile). Patient 3 had the novel mutation (G to T transversion at position 7314) in exon 4, resulting in an amino acid replacement of alanine-109 (Ala) with Ser. We established DNA diagnostic methods for detecting TTR Ile50 by polymerase chain reaction (PCR)-induced mutation restriction analysis and for TTR Ser109 by PCR-restriction fragment length polymorphism. Gene analysis of archival paraffin-embedded tissues is useful for the precise diagnosis of FAP and for clarifying its molecular pathogenesis in patients for whom fresh genomic DNA is not available.

Aged↗

PubMed1993

Proliferating cell nuclear antigen immunohistochemistry using monoclonal antibody 19A2 and a new antigen retrieval technique has prognostic impact in archival paraffin-embedded node-negative breast cancer.

We evaluated whether proliferating cell nuclear antigen (PCNA) immunohistochemistry with antigen retrieval could be used as a measure of cell proliferation in archival, formalin-fixed, paraffin-embedded tissues and whether the staining results have long-term prognostic significance in axillary node-negative breast cancer. Primary tumor samples obtained from 109 axillary-node-negative breast cancer cases were used for the study. The best staining results were obtained with the 19A2 antibody after microwave heating in a solution of saturated lead thiocyanate. Using this method, there was a significant correlation (linear regression, r = 0.580, P < 0.001) between the proportion of PCNA19A2-positive carcinoma cells (PCNA19A2 score) and DNA flow cytometric S phase fraction. A high PCNA19A2 score was associated with high mitotic count, DNA aneuploidy, and absence of estrogen receptors. Axillary-node-negative patients with a high PCNA19A2 score (cut-point 8%) had significantly worse prognosis than those with a low PCNA19A2 score (P = 0.008). According to a Cox multivariate analysis, PCNA19A2 score had independent prognostic value but only if S phase fraction was excluded from the analysis. In our study, the PCNAPC10 score correlated weakly only with primary tumor size (analysis of variance) and prognosis (5-year univariate survival analysis), but the significance of these findings needs further evaluation. In conclusion, PCNA immunohistochemistry with the 19A2 antibody after an appropriate antigen retrieval treatment may offer a useful alternative to DNA flow cytometry for the analysis of cell proliferation activity from formalin-fixed, paraffin-embedded breast carcinomas.

Adult↗

PubMed1993

Investigation of bone marrow lymphocyte subsets in normal, reactive, and neoplastic states using paraffin-embedded biopsy specimens.

Bone marrow lymphocyte subsets in normal and reactive states and in neoplastic diseases involving the marrow were investigated with a select panel of monoclonal antibodies reactive on routinely processed, paraffin-embedded trephine biopsy material. In all cases, the antibodies beta F1 and UCHL1 (CD45RO) stained virtually equal numbers of T cells (reactive and neoplastic), whereas antibody OPD4 stained only about one half of this number of T cells. Antibody L26 (CD20) stained B cells (reactive and neoplastic) in all specimens. The T-cell to B-cell ratio in the normal marrow was between 4:1 and 5:1, and a significant increase in T-cell numbers was observed in reactive and myelodysplastic states. A significant increase in B-cell numbers, however, was seen only in marrow infiltrated by B-cell lymphoma. Bone marrow exhibiting infiltrates of B-cell lymphoma, acute leukemia, or myeloproliferative disorders showed normal or decreased numbers of T cells. These findings show that antibodies UCHL1, beta F1, and L26 can be used to determine the numbers of B and T lymphocytes in paraffin-embedded, formalin-fixed bone marrow specimens and thus may help to distinguish reactive T lymphocytosis from B-cell lymphoma.

B-Lymphocytes↗

PubMed1987

Preparation of cells from paraffin-embedded tissue for cytometry and cytomorphologic evaluation.

A method is described for the preparation of monolayer smears from paraffin-embedded tissue. The smears are suitable for automated image analysis and DNA measurements while still allowing interpretation of nuclear morphology. The proposed technique uses enzyme treatment and syringing for cell dispersal. The preparation of cell monolayers is performed by cytocentrifugation. After staining the specimens with gallocyanin, nuclear DNA can be measured. Automated DNA measurements using the Leyden Television Analysis System (LEYTAS) showed coefficients of variation of 4.5% for the diploid cell population of suspended benign tissue. After DNA measurements, the specimens are counterstained using orange G and eosin. Since gallocyanin has spectral properties similar to those of hematoxylin, the obtained end product is comparable to specimens stained according to the routinely used Papanicolaou procedure. Using this technique, image cytometry can be applied to paraffin-embedded tissue in combination with conventional cytomorphologic study of the cells.

Cytological Techniques↗

PubMed1993

Nuclear DNA content of human breast carcinoma: a comparison of results obtained by microspectrophotometry and flow cytometry of paraffin embedded tissue.

This study compares 2 techniques for estimating the nuclear DNA content of tumor cell lines: (i) static cytometry of smears taken from fresh tissue and (ii) flow cytometry of cells extracted from paraffin embedded tissue. Parallel determinations of DNA content, using both techniques, were made on samples of tissue taken from 130 female patients with breast carcinoma. Using a simple classification into diploid and non-diploid groups, the 2 techniques yielded discrepant results in 11% of cases. The most frequent causes of disagreement were (a) the inability of static cytometry to distinguish between a diploid and a near-diploid peak and (b) for flow cytometry, the difficulty of determining whether a minor peak in the tetraploid region represented the G2 peak of a diploid cell line or the G0/G1 peak of a tetraploid cell line. If it is deemed necessary to accurately assess ploidy status, flow cytometry on paraffin embedded tissue, using modern statistical programmes, would seem to be most practical for routine use, but some neoplasms, particularly those with an equivocal ploidy peak in the tetraploid range by this method, will require static cytometry to accurately assess nuclear DNA content. Using this approach, it appears that the disagreement between the 2 techniques would be less than 5%.

Adult↗

PubMed2006

[Value of combinational detection by IgH and IgL primers in improving detection rate of lymphoma gene in paraffin-embedded tissue].

BACKGROUND & OBJECTIVE: Clonality detection through amplifying immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) is a useful tool in diagnosis of lymphoma, but the false negative rate is high, especially in paraffin-embedded tissues. This study was to explore the value of tumor tissue microdissection and combinational detection of IgH and immunoglobulin light chain (Ig kappa or Ig lambda) in diagnosis of non-Hodgkin's lymphoma (NHL). METHODS: Two pairs of conventional primers for IgH and T-cell receptor gamma (TCRgamma), 2 novel designed pairs of primers for Ig kappa and Ig lambda were used to detect 58 paraffin-embedded blocks, which had been diagnosed by pathology and histochemistry. Of the 58 cases of lymph node tissues, 39 were B-cell lymphoma, 16 were T-cell lymphoma, and 3 were reactive proliferative lymph node tissue. Lymphoma cell lines DG75 and Jurkat were used as control. RESULTS: The positive rates of IgH primers (P1) and IgL primers (P kappa/P lambda) were 79.5% and 71.8% in the 39 cases of B-cell lymphoma (P>0.05), 6.3% and 12.5% in the 16 cases of T-cell lymphoma, respectively. The positive rate was greatly increased to 92.3% in combinationally detecting the primers for IgH and P kappa/P lambda. There was no positive detection among the reactive proliferative lymph node tissues. CONCLUSION: B-cell lymphoma detection rate can be significantly improved by the combination of IgH and IgL gene rearrangement primers, which provides efficient assistant method for the diagnosis and differential diagnosis of lymphoma.

DNA Primers↗

PubMed1994

Sensitive detection of human growth hormone mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues by non-isotopic in situ hybridization.

A sensitive technique of non-isotopic in situ hybridization (NISH) is presented, which permits the detection of human growth hormone (hGH) mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues and human post mortem pituitaries; the latter were used as positive tissue controls in this study. In addition, a double staining procedure combining NISH and immunohistochemistry for the visualization of both hGH and hGH mRNA in the same paraffin section is described. Digoxigenin-labelled antisense hGH RNA was used for NISH of hGH mRNA. The NISH protocol was based upon an established radioactive method. Alkaline phosphatase and horseradish peroxidase-based immunoenzymatic procedures for the detection of digoxigenin-labelled RNA probes using different chromogens [4-nitro blue tetrazolium chloride (NBT), Fast Blue BB, New Fuchsin, and 3,3'-diaminobenzidine tetrahydrochloride (DAB) with or without intensification of the DAB staining] were compared. The proteolytic tissue pretreatment and the detection procedure were found to be the most critical steps for successful visualization of hGH mRNA. After optimization of the permeabilization conditions, hGH mRNA could be visualized in each case studied when alkaline phosphatase/NBT-based detection was employed. The NISH technique presented here, performed either separately or in combination with immunohistochemistry, permits retrospective analyses, of hGH (trans)gene expression in archival, paraffin-embedded specimens.

Animals↗

PubMed2004

PolyA PCR amplification of cDNA from RNA extracted from formalin-fixed paraffin-embedded tissue.

RNA extraction still relies almost exclusively on the use of fresh or frozen tissue, limiting the number of samples that can be analyzed, and there is a growing need for means of global mRNA analysis of archived formalin-fixed paraffin-embedded tissue (FFPET). Previous reports of RNA extraction and amplification from FFPET are limited and do not enable global cDNA amplification. This study used polyA PCR to generate globally amplified cDNA from RNA extracted from formalin-fixed paraffin-embedded samples. RNA was extracted from nine routinely processed archival FFPET samples (lymph node, nasopharynx, prostate, lung and bone marrow) using an Ambion Paraffin Block RNA Isolation Kit. Global cDNA was generated by polyA RT-PCR and used in GAPDH specific PCR and PCR for CD33, c-myb, and SNF2. PolyA cDNA was reamplified by polyA PCR and the reamplified cDNA also used in GAPDH PCR. RNA was extracted from all nine samples, but was degraded. PolyA RT-PCR generated cDNA from all samples and was positive for GAPDH PCR in seven. PCR for CD33, c-myb, and SNF2 was positive in all samples tested. Following reamplification, the polyA cDNA remained positive for GAPDH by PCR. The results demonstrate the feasibility of globally amplifying RNA isolated from archival FFPET samples using polyA RT-PCR, which generates a renewable cDNA pool that can be probed for any cDNA species and reamplified as necessary.

Adenosine↗

PubMed2004

Development of an optimized protocol for the detection of classical swine fever virus in formalin-fixed, paraffin-embedded tissues by seminested reverse transcription-polymerase chain reaction and comparison with in situ hybridization.

An optimized protocol was developed for the detection of classical swine fever virus (CSFV) in formalin-fixed, paraffin-embedded tissues obtained from experimentally and naturally infected pigs by seminested reverse transcription-polymerase chain reaction (RT-PCR). The results for seminested RT-PCR were compared with those determined by in situ hybridization. The results obtained show that the use of deparaffinization with xylene, digestion with proteinase K, extraction with Trizol LS, followed by seminested RT-PCR is a reliable detection method. An increase in sensitivity was observed as amplicon size decreased. The highest sensitivity for RT-PCR on formalin-fixed, paraffin-embedded tissues RNA was obtained with amplicon sizes less than approximately 200 base pairs. An hybridization signal for CSFV was detected in lymph nodes from 12 experimentally and 12 naturally infected pigs. The sensitivity of seminested RT-PCR compared with in situ hybridization was 100% for CSFV. When only formalin-fixed tissues are available, seminested RT-PCR and in situ hybridization would be useful diagnostic methods for the detection of CSFV nucleic acid.

Animals↗

PubMed1992

Demonstration of Epstein-Barr virus genome in neoplastic cells of Hodgkin's disease by in situ hybridization, in paraffin-embedded tissue using biotinylated probes. A study of 46 cases.

Paraffin-embedded tissue specimens from 46 cases of Hodgkin's disease (HD) were studied by in situ hybridization with biotinylated probes for the presence of EBV genomes. EBV specific DNA sequences were detected in the nuclei of Reed-Sternberg (RS) and Hodgkin cells (H), in 14 of these 46 cases. There was no correlation between positive hybridization and morphological subtype or site of tumor. By demonstrating an exclusive localization of the viral DNA in the tumor cells of HD, our study adds to the growing body of evidence to suggest an involvement of EBV in the pathogenesis of at least some cases of HD.

Adolescent↗

PubMed2005

Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA.

The ability to utilize formalin-fixed, paraffin-embedded (FFPE) archival specimens reliably for high-resolution molecular genetic analysis would be of immense practical application in the study of human disease. We have evaluated the ability of the GenomePlex whole genome amplification (WGA) kit to amplify frozen and FFPE tissue for use in array CGH (aCGH). GenomePlex gave highly representative data compared with unamplified controls both from frozen material (Pearson's R(2) = 0.898) and from FFPE (R(2) = 0.883). Artifactual amplification observed using DOP-PCR at chromosomes 1p, 3, 13q, and 16p was not seen with GenomePlex. Highly reproducible aCGH profiles were obtained using as little as 5 ng starting material from FFPE (R(2) = 0.918). This WGA method should readily lend itself to the determination of DNA copy number alterations from small fresh-frozen and FFPE clinical tumor specimens, although some care must be taken to optimize the DNA extraction procedure.

DNA, Neoplasm↗