PubMed Health⌕ Search

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 739 records · Page 41Linked to original sources
PubMed2000

Detection of mycobacterium tuberculosis in paraffin-embedded pleural biopsy specimens by commercial ribosomal RNA and DNA amplification kits.

STUDY OBJECTIVES: To evaluate the utility of two gene amplification systems in historical paraffin-embedded pleural biopsy (PEB) tissues from patients with pleural tuberculosis, and to compare the results to those obtained with conventional histologic and microbiological methods. DESIGN: A retrospective study. PATIENTS AND METHODS: Seventy-four formalin-fixed PEB tissues collected and stored over 12 years (1984 through 1995) were retrieved. Gene amplifications were performed in 57 tissues from patients with diagnoses of pleural tuberculosis and in 17 from patients with carcinoma as controls, using the first version of the Amplified Mycobacterium tuberculosis Direct Test (AMTDT; Gen-Probe; San Diego, CA) and the LCx Mycobacterium tuberculosis Assay (LCxMTB; Abbott Laboratories; Abbott Park, IL). RESULTS: The sensitivities of the AMTDT and LCxMTB were 52.6% and 63.2%, respectively (p = not statistically significant). The specificity of both tests was 100%. Twenty tissue samples (35.1%) were positive by both systems, and 10 tissues (17.5%) were positive only by the AMTDT, while 16 tissues (28.1%) were positive only by the LCxMTB. Both tests gave negative results for 11 specimens (19.3%). When both tests were used, a positive diagnosis was achieved in 80.7% of the samples. Diagnosis of 73.7% of patient conditions had previously been made by smear examination of pleural biopsy and sputum, pleural liquid, or biopsy culture. The overall diagnostic yield with both culture and amplification techniques was 96.5% (55 of 57 patients) for pleural tuberculosis, with amplification techniques adding 22.8% of the diagnoses. CONCLUSIONS: Amplification techniques are useful in archival PEB tissues, providing additional diagnoses beyond culturing, although the sensitivity should be improved, possibly by standardizing protocols.

Biopsy↗
PubMed1990

DNA ploidy analysis of endometrial adenocarcinoma using a flow cytometry from paraffin-embedded tissues.

In this study, DNA ploidy using a flow cytometry from paraffin-embedded tissues was evaluated to establish the DNA heterogeneity and risk factor of endometrial adenocarcinoma. Seventy-six samples from 31 patients with endometrial adenocarcinoma and 6 controls with normal endometrium were measured for DNA content. DNA ploidy of endometrial adenocarcinoma was also analyzed in three different sites of a tumor, viz. (1) the primary endometrial lesion, (2) the site of myometrial invasion and, (3) metastatic lesions. In over 22% of 27 patients with endometrial adenocarcinoma, intratumor variations in DNA ploidy were demonstrated of a different sites of a tumor. It was found that all 6 controls with normal endometrium were diploid, while 28.6% of 70 samples of endometrial adenocarcinoma had aneuploid tumors. Poorly differentiated tumors had a significantly higher incidence of aneuploidy than well or moderately differentiated tumors. DNA ploidy was related to the surgical stage and histological grade, although it was not indicated that DNA ploidy has reliable prognostic value for endometrial adenocarcinoma. It was suggested that the myometrial invasion of endometrial adenocarcinoma is not an important factor in DNA content heterogeneity, because the incidence of aneuploidy in three different sites of a tumor was as follows: myometrial invasion less than primary endometrial lesion less than metastatic lesions.

Adenocarcinoma↗
PubMed1997

Numerical aberrations of chromosome 7 detected in 15 microns paraffin-embedded tissue sections of primary cutaneous melanomas by fluorescence in situ hybridization and confocal laser scanning microscopy.

Fluorescence in situ hybridization (FISH) to sections of formalin-fixed paraffin-embedded archival tissues allows the detection of gene or chromosome copy number changes in interphase cell nuclei within the histological context and thus may be of particular interest in tumor pathology. In this report, we describe the application of FISH to thick (15 microns) paraffin sections of 7 primary cutaneous malignant melanomas. A chromosome 7-specific centromeric DNA probe was used to detect numerical aberrations of chromosome 7. By optical sectioning using confocal laser scanning microscopy (CLSM) only complete, uncut interphase cell nuclei were scored. The mean percentage (+/- SEM) of melanoma cell nuclei with three hybridization spots was (20.7 +/- 2.8)%; (6.8 +/- 1.0)% of nuclei showed one spot and (5.0 +/- 1.2)% four or more spots. The frequency distribution of spot numbers among melanoma cell nuclei and normal keratinocyte nuclei was significantly different (chi(2) = 176.8, df = 5, p < 0.001). Trisomy 7 was detected in all 7 cases analyzed, mostly associated with monosomy 7 or polysomy 7. The approach used in our study and the data obtained could be useful for further studies designed to investigate a possible involvement of chromosome 7 in melanocytic tumor progression.

Adult↗
PubMed1994

Detection of human herpesvirus-6 in paraffin-embedded tissue of cervical cancer by polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect human herpesvirus-6 (HHV-6) DNA sequences from paraffin-embedded tissue from cervical cancer patients. Two of eight cases were positive for HHV-6 using two sets of HHV-6 primers. Hybridization of PCR products with specific radioisotope-labeled oligonucleotide probes confirmed the results. Furthermore, HHV-6 typing was possible by adapting restriction endonuclease digestion of PCR product. This method is useful for retrospective studies in investigating the etiologic role of HHV-6 in the development of human diseases.

Base Sequence↗
PubMed1991

A simple method for isolation of DNA from formalin-fixed paraffin-embedded samples for PCR.

A simple method of processing formalin-fixed, paraffin-embedded tissue sections for DNA amplification by polymerase chain reaction (PCR) is described. In this procedure, deparaffinized sections are readily subjected to DNA isolation simply by boiling and the released DNA can be directly employed for PCR. The method allows analysis of single-copy genes or viral sequences at least up to 300 base pairs long in one working day. This method is particularly useful in analysing retrospective materials when the simplicity and low cost of the assay are preferable. Furthermore, the simplicity of the procedure reduces the risk of contamination.

Base Sequence↗
PubMed1978

A method for specific diagnosis of Rocky Mountain spotted fever on fixed, paraffin-embedded tissue by immunofluorescence.

For specific demonstration of Rickettsia rickettsii in fixed, paraffin-embedded tissues, the technique of trypsin digestion of deparaffinized, rehydrated sections was investigated. It was determined that 3.5 hr of digestion was optimal for achieving discrete, bright, green immunofluorescence of rickettsiae. At autopsy kidneys from seven of 10 cases of probably Rocky Mountain spotted fever contained structures that were specifically stained and that had the size and shape of rickettsiae. These structures were strictly limited to the endothelium and vascular walls of renal capillaries, veins, and arteries. Results from controls indicated that specificity of the immunofluorescence. R. rickettsii may be demonstrated by this method with greater sensitivity and specificity than by current histological methods. The technique allows retrospective analysis of certain organs for pathogenesis of involvement in Rocky Moutain spotted fever and offers a specific diagnostic test.

Animals↗
PubMed1997

DNA extraction from formalin-fixed, paraffin-embedded tissues: protein digestion as a limiting step for retrieval of high-quality DNA.

Several DNA extraction methods have been used for formalin-fixed, paraffin-embedded tissues, with variable results being reported regarding the suitability of DNA obtained from such sources to serve as template in polymerase chain reaction (PCR)-based genetic analyses. We present a method routinely used for archival material in our laboratory that reliably yields DNA of sufficient quality for PCR studies. This method is based on extended proteinase K digestion (250 micrograms/ml in an EDTA-free calcium-containing buffer supplemented with mussel glycogen) followed by phenol-chloroform extraction. Agarose gel electrophoresis of both digestion buffer aliquots and PCR amplification of the beta-globin gene tested the suitability of the retrieved DNA for PCR amplification.

Adrenal Glands↗
PubMed1999

Non-isotopic in situ hybridization of CD44 transcript in formalin-fixed paraffin-embedded sections.

We established a protocol for the non-isotopic in situ detection of adhesion molecule CD44 messenger RNA (mRNA) in archival formalin-fixed paraffin-embedded sections of human surgical materials. Four brain tumor samples with different histopathologies (a metastatic adenocarcinoma, a metastatic squamous carcinoma, a glioblastoma and a craniopharyngioma) were thus studied using a 157 nt digoxigenin-labeled RNA probe complementary to the common mRNA region to all the CD44 isoforms. The CD44 transcript was detected in the cytoplasm of glioma and such epithelial tumor cells as metastatic carcinoma and craniopharyngioma. A competitive hybridization study confirmed the specificity of the CD44 probe. The optimization of critical conditions are also discussed. This protocol should therefore be useful in making an accurate evaluation of mRNA localization and may also facilitate the successful completion of extensive retrospective studies on a large number of archival samples.

Brain Neoplasms↗
PubMed1999

Detection and genetic relationship of dengue virus sequences in seventeen-year-old paraffin-embedded samples from Cuba.

This study describes the use of the reverse transcriptase-polymerase chain reaction (RT-PCR) to generate dengue 2 amplicons from paraffin-embedded autopsy tissues collected in Cuba 17 years ago. The presumptive diagnoses had been made only by clinical evolution without serologic confirmation. This study confirms once again that dengue 2 virus was directly associated with the fatal cases in children and illustrates the potential of the RT-PCR for retrospective diagnosis of dengue cases 17 years after death. A close similarity in the genomic sequences of the dengue 2 RNA detected in tissue samples from fatal cases and those dengue 2 Cuban strains that had been previously investigated confirms the appropriate genomic classification of the etiologic agent associated with the 1981 dengue hemorrhagic fever Cuban epidemic.

Amino Acid Sequence↗
PubMed1996

Automated assessment of numerical chromosomal aberrations in paraffin embedded prostate tumor cells stained by in situ hybridization.

We investigated the feasibility of automated counting of in situ hybridization signals (ISH) in interphase cells isolated from paraffin embedded prostate tissue. In total, 34 specimens from 7 patients with prostate cancer were stained with probes specific for the centromeric regions of chromosomes Y, 1, 7, 8, 10, and 15, using an immunoperoxidase based technique suitable for bright-field microscopy. Enumeration of the number of ISH spots of 500 nuclei per specimen was performed (1) using an automatic system developed without any human intervention and (2) using the same system, but including verification of the counts based on visual inspection of the stored images. As reference from each specimen, 200 cell nuclei were evaluated manually, using conventional microscopy. A typical analysis procedure (including user verification) took 35 min. The difference (root mean error) between the automated counting and the counting after visual interaction was relatively small (15%). The percentage of cells with incorrect counts by automated analysis was 20.2%, a number that could easily be improved by user interaction. Detection of cells with aneusomy proved to be more sensitive compared to the routine manual counting, in cases where aberrant frequencies were low. Automated counting of samples with low frequencies (< 10%) resulted in a higher frequency of aberrant cells in 9 of 11 cases, probably due to the fact that an unbiased cell selection is guaranteed. Automated assessment of ISH signals is considered useful for the evaluation of chromosomal aberrations in prostate tumor cells, provided that the counts are visually confirmed.

Aged↗
PubMed2004

DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution.

Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5-12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.

Antigens↗
PubMed2003

Detection of bovine viral diarrhea virus in formalin fixed paraffin embedded tissue sections by real time RT-PCR (Taqman).

Real time quantitative RT-PCR (Taqman) identified specifically BVD virus in freshly processed formalin-fixed paraffin-embedded tissue sections and archival samples up to 7 years old. Samples included tissue from both acutely and persistently infected animals. To assess RNA degradation due to tissue handling and processing, freshly collected tissues from a calf persistently infected with BVD virus were stored at 4 degrees C or room temperature for up to 1 week prior to 24 h formalin fixation and routine histologic processing. Samples stored at 4 degrees C for up to a week prior to fixation were positive while samples stored at room temperature were positive at 74 h but became negative after 96 h. Fixation of fresh tissue in formalin for 1 week prior to processing resulted in a mild decrease in signal strength compared with tissue fixed for 24-48 h. Real time RT-PCR improves diagnosis of BVD infection by allowing prospective and retrospective identification of BVD virus in tissues processed routinely and stored for histologic evaluation.

Animals↗
PubMed2005

Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS.

There exists a need for robust approaches for tandem mass spectrometry (MS/MS)-based identification of proteins in formalin-fixed paraffin-embedded (FFPE) material. We demonstrate herein the identification of proteins in FFPE material using enzymatic cleavage for extraction of peptides from the FFPE specimen and liquid chromatography (LC) followed by MS/MS. We identified 324 proteins from a 3-year-old FFPE cell-block of a human lymphoma cell line. The identified proteins were assigned to the membrane, cytosol and nucleus, with diverse cellular functions. The results were comparable to those obtained with lysates from a fresh specimen of the lymphoma cell line. Western blotting analysis and immunofluorescence microscopy confirmed the expression of selected proteins. The functional significance of one protein (PKC eta) was validated using a PKC inhibitory peptide which resulted in lymphoma cell death in vitro. The ability to identify proteins from FFPE specimens has significant implications for MS/MS-based proteomics of vast repositories of archival primary tissue samples for disease-related discovery research.

Cell Line, Tumor↗
PubMed1995

The use of the polymerase chain reaction in the diagnosis of B-cell lymphomas from formalin-fixed paraffin-embedded tissue.

In this study the use of the polymerase chain reaction (PCR) to detect monoclonality in B-cell lymphoid proliferations in archival formalin-fixed paraffin-embedded tissue was assessed. Using consensus primers against the framework 3 (FR 3) region of the immunoglobulin heavy chain gene (IgH), PCR analysis was performed on 29 low grade B-cell non-Hodgkin's lymphomas. Cases of benign lymphoid hyperplasia served as polyclonal controls. Sequenced cases of acute lymphoblastic leukaemia served as positive controls. In the lymphomas, monoclonality could be demonstrated in 18 of 29 (62%) cases. Only five of 11 (45%) follicle centre cell lymphomas were positive by this method whilst the success rate for the remainder was 13 of 18 (72%). None of the polyclonal controls gave false positive results although occasional non-specific dominant bands were present which disappeared on repeating the experiments. These results show that this method will identify monoclonality in 62% of low grade B-cell non-Hodgkin's lymphomas in archival material. The success rate is increased to 72% if follicle centre cell lymphomas are excluded. Thus, this method is a useful adjunctive test to aid diagnosis in lymphoid infiltrates when standard morphology and immunohistochemistry are equivocal.

Base Sequence↗
PubMed1994

Methods in pathology. An improved method for DNA diagnosis of leprosy using formaldehyde-fixed, paraffin-embedded skin biopsies.

To improve the sensitivity of the previously reported polymerase chain reaction (PCR) for the detection of Mycobacterium (M.) leprae in the formaldehyde-fixed, paraffin-embedded tissues, we adapted the PCR designed to amplify an internal 372 bp fragment of a M. leprae-specific repetitive sequence to 39 skin biopsies taken from patients with leprosy of the lepromatous type and tuberculoid type. Crude DNA samples were prepared from tissue sections that were deparaffinized and subjected to proteinase-K digestion without any further treatment for DNA purification. Overcoming a false-negative reaction by an elongation of the period for enzymatic digestion and an appropriate dilution of the samples, an amplification of the target sequence was obtained as a single band with all 39 skin biopsies tested. The fragments specifically amplified by the PCR were subjected to direct sequencing and were confirmed to be identical with an internal 372 bp of M. leprae-specific repetitive sequence. Although in nine of 24 nonleprosy control samples, a false-positive amplification was observed as from one to several bands, they were distinguishable from the specific one by the electrophoretic pattern. This PCR makes up for the classic histological methods used in the diagnosis of leprosy.

Base Sequence↗
PubMed2003

Identification of porcine macrophages with monoclonal antibodies in formalin-fixed, paraffin-embedded tissues.

Here we report the successful use of monoclonal antibodies (mAbs) BL1H7, BA1C11 (anti-SWC3) and 4E9 for immunohistochemical identification of macrophages in formalin-fixed, paraffin-embedded porcine tissues. Retrieval of antigen reactivity was achieved by heating the slides in a domestic pressure cooker, which makes the technique suitable for the routine laboratory. This method allows to perform retrospective studies in routinely processed tissues and may be useful to investigate the role of macrophages in different pathological processes.

Animals↗
PubMed1982

An improved method for HBcAg demonstration in paraffin-embedded liver tissue.

Factors influencing the preservation of HBcAg in formalin-fixed, paraffin-embedded liver tissue have been analysed. Fixation time, choice of the embedding medium, deparaffinization and selection of anti-HBc proved to be critical variables. For optimal results, fixation in 4% buffered formalin for no longer than 24-48 h, the use of wax with a low melting point, a heating step prior to deparaffinization, and a search for anti-HBc specifically reactive for paraffinated HBcAg are recommended.

Fixatives↗