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PubMed1983

Immunohistology on formol-sublimate fixed and paraplast embedded kidney tissue with comparison to formalin fixation and to pre-embedding immunofluorescent staining.

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 micron Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.

Animals↗
PubMed1992

Preservation of the diffusible cations for secondary ion mass spectrometry. II. Artefacts in material embedded in araldite or melamine.

Flotation on hot water (about 60 degrees C) which is frequently employed to stretch semithin sections on substrates for SIMS (secondary ion mass spectrometry) microscopy, is the cause of numerous artefacts. In the case of epoxy resin-embedded tissue, one observes loss of potassium and sodium and accumulation of calcium. The relative contrast of cell nuclei in the ionic images, is rapidly affected by these ion migrations. After prolonged contact with hot water, tissue becomes uniformly emissive. In the case of hydrosoluble resin-embedded tissue, potassium and sodium do not appear to be affected by the action of water, which suggests that they are covalently bound with chelating sites buried beneath the layer of water bound to the surface of the macromolecules. Calcium accumulates, probably on widely exposed anionic sites. Moreover, the domains observed in hydrosoluble resin-embedded tissue shrink differently according to the proportion of water removed by melamine; this can provide interesting information on the initial equilibrium between water, ion sand macromolecules. Our results seem to support the assumption that bound water should play an important role in the preservation of both macromolecular architecture and ion distributions.

Animals↗
PubMed1995

Identification of abnormal chromosomal complement in formalin-fixed, paraffin-embedded placental tissue.

The objective of this project was to assess the efficacy of fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes to identify chromosome number in formalin-fixed, paraffin-embedded placental specimens. Using this approach, 75 per cent of the karyotypes in 20 formalin-fixed placental samples (comprising aneuploids, triploids, and normals) were correctly identified. As this technology improves, the ability to obtain information regarding chromosomal abnormalities in formalin-fixed, paraffin-embedded placental tissue should improve as well. This technology can potentially provide important cytogenetic information even when fresh tissue is not available for standard karyotypic analysis.

Chromosome Aberrations↗
PubMed1983

The influence of embedding media and fixation on the post-embedment ultrastructural demonstration of complex carbohydrates. II. Dialyzed iron staining.

The influence of fixation and the embedding medium on post-embedment dialyzed iron (DI) staining of acidic complex carbohydrates in mouse colon was studied at the ultrastructural level. DI staining of ultrathin sections from non-osmicated tissues embedded in epoxy resins was very weak, whereas DI staining of non-osmicated tissues embedded in non-epoxy resins such as polystyrene, polyester resins, methacrylates and newly developed embedding mixtures was strong. Brief exposure to OsO4 (5 min) abolished the DI staining in stored secretions of goblet and mucous cells, in apical cytoplasmic vesicles and at the microvillous surface of columnar absorptive cells and in the Golgi cisternae of all colonic cell types in epoxy embedded tissues, but only reduced slightly or had no effect on the DI reactivity observed in these sites in tissues embedded in non-epoxy resins. Prolonged exposure to OsO4 (60 min) prior to embedment in non-epoxy resins further reduced DI staining in all reactive sites and abolished the staining in Golgi cisternae of all colonic epithelial cells. Embedment of non-osmicated tissues in a styrene-Vestopal W mixture and of tissues briefly exposed to OsO4 after primary glutaraldehyde fixation in styrene-methacrylate is recommended for optimal post-embedment DI staining of the acidic groups of complex carbohydrates.

Animals↗
PubMed1993

Immunohistological detection of human herpes virus 6 in formalin-fixed, paraffin-embedded lung tissues.

Human herpes virus type 6 (HHV-6) infection is widespread in healthy individuals. The only definite disease association is with exanthem subitum in infants though the virus has been linked with a variety of other diseases including interstitial pneumonitis in bone marrow allograft recipients. In order to investigate the role of HHV-6 in the latter disease we have developed an optimised staining method for the demonstration of specific antigen in routinely processed post-mortem tissues. Formalin-fixed, paraffin-embedded lung tissue from 8 immunocompromised patients who died from interstitial pneumonitis was subjected to immunoperoxidase staining with monoclonal antibodies against HHV-6, cytomegalovirus (CMV), and adenovirus, using a modified avidin-biotin complex (ABC) method. Staining for HHV-6 was obtained in 6 of the 8 patients studied and was present in pneumocytes and macrophages. CMV and adenovirus antigens were identified in 4 and 6 patients, respectively. Whilst the lung tissue of 6 patients contained more than one virus, there was no evidence of cross-reactivity between the monoclonal antibodies. We demonstrated that accurate localisation of HHV-6 using monoclonal antibodies is possible in post-mortem lung tissue and conclude that either HHV-6 alone or in combination with other viruses may play a role in the development of interstitial pneumonitis following bone marrow transplantation or chemotherapy.

Adenoviridae Infections↗
PubMed2004

[Detection of ASPL-TFE3 fusion gene by reverse transcriptase polymerase chain reaction in paraffin-embedded tumor tissues of alveolar soft part sarcoma].

OBJECTIVE: To investigate the significance of detecting chimeric mRNA resulting from t(X;17)(p11.2;q25) in paraffin-embedded tumor tissues of alveolar soft part sarcoma (ASPS). METHODS: Formalin-fixed, paraffin-embedded tumor tissues from 8 cases of alveolar soft part sarcoma and 15 cases of controls (including 6 alveolar rhabdomyosarcomas, 6 renal cell carcinomas, 2 paragangliomas and 1 granular cell myoblastoma) were retrieved from the archival materials. ASPL-TFE3 fusion transcripts were analyzed in all samples by reverse transcriptase-polymerase chain reaction (RT-PCR). The quality of the mRNA was assessed using the house-keeping gene beta-actin. RESULTS: ASPL-TFE3 fusion transcripts were detected in 6 of the 8 ASPS cases (4 being type 2 and 2 being type 1). The remaining 2 cases were negative for both beta-actin and ASPL-TFE3. No ASPL-TFE3 mRNA expression was detected in all the controls. PAX3/7-FKHR fusion transcripts were also detected in 4 of the 6 alveolar rhabdomyosarcoma samples. CONCLUSIONS: The expression of ASPL-TFE3 fusion transcripts in paraffin-embedded tumor tissues can serve as an useful molecular marker in the diagnosis of ASPS. It may also be helpful in elucidating the underlying pathogenesis of ASPS in subsequent retrospective studies.

Adolescent↗
PubMed1998

Detection of EB Virus Gene Fragments in Paraffin-embedded NPC Tissue.

To detect more sensibly Epstein-Barr virus (EBV) DNase gene fragments in nasopharyngeal carcinoma (NPC) tissue, a pair of optimized primers flanking 29% of EBV-DNase gene were designed. PCR results showed that EBV-DNase gene was detected in 145 of 149(97.32%) paraffin-embedded tissue samples with NPC and 2 of 155 (1.29%) cases in high risk population. DNA sequencing proved that DNA sequence of the PCR product agreed with BGLF5 reading frame from Genbank VO1555. Paraffin-embedded NPC samples stored for less than 1.5 years under room temperature and atmosphere conditions in South China were suitable for PCR detection.

Journal Article↗
PubMed2005

[Analysis of bcl-6 protein expression and gene rearrangement in diffuse large B-cell lymphoma].

OBJECTIVE: To investigate bcl-6 protein expression and gene rearrangement patterns in diffuse large B-cell lymphoma (DLBCL) and their clinicopathologic significance. METHODS: Immunohistochemical studies for bcl-6 and CD10 proteins were performed on 51 cases of DLBCL paraffin-embedded tissues (including 22 nodal samples and 29 extranodal samples) and 10 cases of reactive lymphoid hyperplasia (RLH) paraffin-embedded tissues. Interphase fluorescence in-situ hybridization (FISH) with dual color breakapart probe was also used to identify rearrangement of bcl-6 gene in 32 cases of nodal DLBCL tissues (including 22 paraffin-embedded samples and 10 fresh samples) and 5 cases of RLH paraffin-embedded tissues. RESULTS: (1) The rates of bcl-6 protein expression in nodal DLBCL, extranodal DLBCL and RLH were 72.7% (16/22), 75.9% (22/29) and 100.0% (10/10) respectively. The rates of CD10 expression were 40.9% (9/22), 41.4% (12/29) and 100.0% (10/10) respectively. All lymphoma samples which expressed CD10 also showed co-expression of bcl-6 protein. (2) The co-expression of bcl-6 and CD10 was observed in 40.9% (9/22) nodal DLBCL and 41.4% (12/29) extranodal DLBCL. Low clinical stage (stage I and II) was more frequently observed in cases with co-expression of bcl-6 and CD10 (P < 0.05). (3) The rates of bcl-6 gene rearrangement in nodal DLBCL was 28.1% (9/32), with 27.3% (6/22) in paraffin-embedded tissues and 30.0% (3/10) in fresh tissues. There was no statistically significant difference found between the two groups (P > 0.05). Bcl-6 gene rearrangement was not found in all the 5 cases of RLH, and there was a significant difference between RLH and DLBCL (P < 0.05). CONCLUSIONS: The rate of bcl-6 protein expression is high in DLBCL cases, and the detection of bcl-6 and CD10 protein co-expression may help in the diagnosis and differential diagnosis of DLBCL. Those DLBCL cases with co-expression of bcl-6 and CD10 may also have a better prognostic implication. On the other hand, bcl-6 gene rearrangement can be identified by interphase FISH with dual color breakapart probe in both paraffin-embedded and fresh lymphoma tissues.

Diagnosis, Differential↗
PubMed2001

Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers : a new approach for the molecular analysis of paraffin-embedded cancer tissue.

We have developed a protocol for degenerate oligonucleotide-primed-polymerase chain reaction-based array comparative genomic hybridization (array CGH) that, when combined with a laser microdissection technique, allows the analysis of cancer cell populations isolated from routine, formalin-fixed, paraffin-embedded tissue samples. Comparison of copy number changes detected by degenerate oligonucleotide-primed-polymerase chain reaction-based array CGH to those detected by conventional array CGH or fluorescence in situ hybridization, demonstrated that amplifications can be reliably detected. Using a genomic microarray containing 57 oncogenes, we screened a total of 28 breast cancer samples and obtained a detailed amplicon profile that is the most comprehensive to date in human breast cancer. The array CGH method described here will allow the genetic analysis of paraffin-embedded human cancer materials for example in the context of clinical trials.

Breast Neoplasms↗
PubMed2003

Nested PCR assays for detection of Blastomyces dermatitidis DNA in paraffin-embedded canine tissue.

A Blastomyces dermatitidis nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family ONYGENACEAE: We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively. The B. dermatitidis PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy. Sequencing revealed that all PCR products were homologous to the B. dermatitidis WI-1 adhesin gene. No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma. The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively. Only six products were identified as being identical to B. dermatitidis 18S rDNA; they were exclusively obtained from specimens positive by the B. dermatitidis nested PCR. For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific H. capsulatum product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the B. dermatitidis PCR assay. In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of B. dermatitidis in canine tissue.

Animals↗
PubMed1991

Detection of hepatitis B virus DNA in paraffin-embedded liver tissues in chronic hepatitis B or non-A, non-B, hepatitis using the polymerase chain reaction.

We developed a polymerase chain reaction assay for the direct detection of hepatitis B virus in paraffin-embedded liver tissue and applied this assay to determine whether hepatitis B virus DNA exists in livers with chronic hepatitis non-A, non-B. Fifty five liver biopsy samples were studied: 11 from patients with HBeAg-positive chronic hepatitis (paraffin-embedded) and 44 from patients with chronic hepatitis non-A, non-B (21 paraffin-embedded; 25 fresh frozen). Thirty three (75%) of the non-A, non-B cases were positive for hepatitis C virus antibodies. Approximately 1 to 10 ng of DNA was extracted from the paraffin-embedded tissue and amplified using oligonucleotide (23-mer) primers specific for the S gene (positions 261 to 692). The beta-globin gene was used as an internal control for sensitivity because this is a single copy gene and allows for relative quantification. In each of the chronic hepatitis B livers, the expected 432-base-pair amplification product for hepatitis B virus DNA and beta-globin gene product were both detected. On the other hand, in the 21 paraffin-embedded chronic hepatitis non-A, non-B livers, no hepatitis B virus DNA was detected, although beta-globin gene was observed in all. Furthermore, in all 25 frozen non-A, non-B livers, beta-globin gene was observed, but no hepatitis B virus band was seen. The limit of detection of hepatitis B virus DNA by this method was estimated to be one genomic copy of hepatitis B virus DNA per cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2001

Evaluation of T cell receptor testing in lymphoid neoplasms: results of a multicenter study of 29 extracted DNA and paraffin-embedded samples.

To evaluate current diagnostic methods used for the evaluation of T cell receptor (TCR) gene rearrangements, 24 different laboratories analyzed 29 lymphoid neoplasm samples of extracted DNA and paraffin-embedded tissue and were asked to complete a technical questionnaire related to the testing. Participating laboratories performed Southern blot and polymerase chain reaction (PCR) testing for rearrangements of the TCRbeta chain gene and PCR for the TCRgamma chain gene rearrangements. Of 14 laboratories performing TCRbeta Southern blot analysis, there was complete agreement in 10 of 14 cases, with some false negative results obtained in 4 cases. No false positive results were obtained by Southern blot analysis. TCRbeta PCR analysis was only performed by two laboratories, and only 47.1% of positive samples were detected. Twenty-one laboratory results were obtained for TCRgamma PCR. This method showed an overall detection rate of 77.9% for T cell gene rearrangements with a 4.1% false positive rate, as compared to both TCRgamma Southern blot analysis results and immunophenotyping. The detection rate for TCRgamma PCR, however, significantly differed when extracted DNA samples from frozen tissue were compared to paraffin-embedded tissue (85.4% versus 65.9%; P = 0.0005). Significant differences in true positive results were obtained when laboratories using primers directed against multiple TCRgamma variable regions (V1-8 plus one to three other primer sets) were compared to laboratories that used only a single set of TCR primers directed against the V1-8 (P < 0.0001). Other technical factors significantly affecting results were also identified. These findings provide useful data on the current state of diagnostic TCR testing, highlight the risk of false negative results for TCR testing directed against only portions of the TCRgamma gene, and identify limitations of testing of paraffin-embedded tissues in some laboratories.

Blotting, Southern↗
PubMed2004

[Using PCR method to study the EB virus DNA in NPC].

OBJECTIVE: To study the EB virus DNA distribution in paraffin-embedded tissue, peripheral blood leukocyte and serum in nasopharyngeal carcinoma patients (NPC). METHOD: PCR method, in paraffin-embedded tissue of 39 patients with NPC (NPC group-1) and 20 patients with chronic nasopharyngitis (control group-1), in peripheral blood leukocyte and serum of 24 patients with NPC (NPC group-2) and 10 patients with other head and neck carcinoma (control group-2) and 10 patients with chronic nasopharyngitis (control group-3) were used to test the presence of Epstein-Barr virus DNA. At the same time, we also measured serum EB virus VCA-IgA antibodies with enzyme-immunoassay in NPC group-2, control group-2 and control group-3. RESULT: The results showed that 28 of 39 nasopharyngeal carcinoma patients (71.8%) (NPC group-1) and 3 of 20 chronic nasopharyngitis (15%) (control group-1) were EB virus DNA positive in paraffin-embedded tissues. The difference between NPC group-1 and control group-1 was statistically significant. Ten of 24 NPC group-2 (41.67%) and 1 of 10 control group-2 (10%) were EB virus DNA positive in peripheral blood leukocyte. Control group-3 EB virus DNA was negative in peripheral blood leukocyte. The difference between NPC group-2 and control group-3 was statistically significant (P < 0.05). All of the patients' EB virus DNA were negative in serum. The difference between NPC group-2 and control group-2; 2 was statistically significant in VCA-IgA titer respectively (Ridit Test P < 0.01). CONCLUSION: EB virus DNA positive in paraffin-embedded tissues is higher than that in peripheral blood leukocyte in nasopharyngeal carcinoma patients. In serum EB virus DNA is negative. The results suggest that EB virus DNA positive from paraffin-embedded tissues to peripheral blood leukocyte to serum is decrease gradually. There must be some relationship between VCA-IgA in serum and EB virus DNA in peripheral blood leukocyte.

Adolescent↗
PubMed1996

An anti-CD3 epsilon serum detects T lymphocytes in paraffin-embedded pathological tissues in many animal species.

The in situ identification of T cells has been difficult and restricted to frozen tissue samples of a limited range of species. In this study, we demonstrate that an antiserum recognizing a phylogenetically conserved part of the CD3 epsilon cytoplasmic tail detects T cells of many avian and mammalian species in formalin-fixed paraffin-embedded tissue sections. This antiserum enables the morphological characterization of normal and pathological lymphoid tissues and lymphoid infiltrations in experimental work and in animal disease.

Animals↗
PubMed1992

Staining of semithin tissue sections embedded in HPMA, quetol 523 and MMA.

Various tissues fixed in a mixture of formaldehyde and glutaraldehyde, and embedded in an improved 2-hydroxypropyl methacrylate mixture were employed for studying the fine structures of cells and tissues by light microscopy. The embedding mixture contained Quetol 523 and methyl methacrylate as a plasticizer without a cross-linker. The catalyst was QCU-1. The mixture had a low viscosity, was easy to handle and penetrated readily and completely into the specimen, producing a homogeneous block from which it was easy to cut sections of 1-2 microns in thickness. A wide variety of stains have been employed with such sections and those reported here are hematoxylin-eosin, Azan and PAS. There was excellent preservation of alkaline phosphatase activity. A method of poststaining immunoperoxidase labeling was also applied to the mouse pancreas and examples of staining with insulin are included.

Aminosalicylic Acid↗
PubMed2001

Hepatitis C virus RNA detection by in situ RT-PCR in formalin-fixed paraffin-embedded liver tissue. Comparison with serum and tissue results.

The purpose of this study was to localize HCV RNA in formalin-fixed paraffin-embedded liver biopsies of 15 patients with chronic hepatitis C using in situ RT-PCR method. The results were compared to serum and tissue extract analysis of HCV RNA. HCV RNA was detected in 80% of the sera tested, in 40% of the corresponding hepatic tissue extract and in 60% of the tissue sections tested by in situ RT-PCR. Compared to the serum positive cases, 67% of the cases were positive with in situ RT-PCR and 41% were positive with tissue extract detection. 50% of the cases in situ RT-PCR positive were also positive with tissue extract detection. These results underlined the complementarity of the different methods of viral detection for the precise diagnosis of hepatitis C.

Formaldehyde↗
PubMed1994

Thick-section fluorescence in situ hybridization on formalin-fixed, paraffin-embedded archival tissue provides a histogenetic profile.

Fluorescence in situ hybridization has become a major tool for analysis of gene and chromosome copy number in normal and malignant tissue. The technique has been applied widely to fresh tissue and dispersed formalin-fixed, paraffin-embedded archival tissue, but its use on sections of archival tissue has largely been limited to sections < 6 mu thick. This does not provide intact, uncut nuclei for accurate analysis of gene or chromosome copy number. We report here a method of hybridization to sections > 20 microns thick that overcomes these difficulties. Key developments were the use of DNA probes directly labeled with fluorochromes and optical sectioning using laser-scanning confocal microscopy.

Chromosomes, Human, Pair 1↗
PubMed2004

LM-PCR permits highly representative whole genome amplification of DNA isolated from small number of cells and paraffin-embedded tumor tissue sections.

Analysis of genetic changes is often hampered by insufficient starting DNA from limited clinical tissue specimens. We employed ligation-mediated PCR (LM-PCR) for global amplification of the genome to overcome this limitation, generating up to 5 microg of representative amplicons of genomic DNA from as little as one cell. We demonstrate successful global genome amplification in high-quality starting DNA source like laser-captured cultured cells, as well as partially degraded starting DNA from old formalin-fixed paraffin-embedded tissue sections. This process generates adaptor-tailed templates that can be repeatedly amplified almost ad infinitum. We have further modified this technique such that, instead of a single endonuclease digest, we can achieve higher amplicon coverage by combining 3 endonuclease digests prior to LM-PCR. As tested by examining amplification of STS sequences scattered genome-wide, the coverage was improved from the published 70% to 96%. The faithful representation of global losses and gains in the amplified genomic DNA was confirmed by array-comparative genomic hybridization. Further, we exemplify the utility of this technique for finer p53 point mutation analysis by PCR-SSCP. This technique is thus a clinically useful tool for globally amplifying and archiving DNA from finite sources like paraffin tissue sections, providing a potentially unlimited resource for genetic analyses.

Cell Line, Tumor↗