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PubMed1998

Further study of hepatitis C virus RNA detection in formalin-fixed, paraffin-embedded liver tissues by ligation-dependent polymerase chain reaction.

Ligation-dependent polymerase chain reaction (LD-PCR) is a novel, simple and sensitive PCR assay that could be useful for the molecular diagnosis of various viral infections. This assay uses two capture probes for RNA isolation and two hemiprobes for the subsequent PCR. To investigate its usefulness in the field of pathological diagnosis, further study of this new PCR technology to detect hepatitis C virus (HCV) RNA was carried out in formalin-fixed, paraffin-embedded (FFPE) liver tissues and the results compared with those obtained by the reverse transcription (RT)-PCR method. We analyzed routinely processed FFPE liver specimens of surgical origin from 13 anti-HCV-seropositive Japanese patients with hepatocellular carcinoma (HCC). As a result, HCV-RNA was detected in FFPE liver specimens in 12 of the tested patients (92.3%) by the LD-PCR method without the use of reverse transcription. In contrast, HCV-RNA was detectable in eight out of 13 cases (61.5%) by the RT-PCR method. These results indicated that LD-PCR was useful for HCV-RNA detection in routinely processed FFPE liver specimens.

Adult↗
PubMed1986

Detection of Pi Z phenotype individuals by alpha-1-antitrypsin (AAT) immunohistochemistry in paraffin-embedded liver tissue specimens.

Immunostaining for alpha-1-antitrypsin (AAT) was applied on paraffin-embedded liver specimens from 38 Pi Z phenotype (27 Pi MZ; 9 Pi ZZ; 2 Pi SZ) individuals (33 adults, 5 newborns). Histological diagnoses included normal liver, various forms of chronic hepatitis, cirrhosis. A positive hepatocytic staining for AAT was revealed in all cases, whereas on PAS-D staining 5 cases were negative. Immunostaining revealed different patterns: type I: fine solid granules or coarse laminated globules filling the whole liver cell cytoplasm; type II: positive granules marginated towards the cell's periphery; type III: positive granules restricted to focal areas in the cytoplasm. Type I and III patterns were observed in all specimens. Association of type I and III with type II was seen in all Pi MZ and Pi SZ cases, but only in 3 of the 9 Pi ZZ specimens. To check the specificity of these staining patterns for the Z allele of AAT in adults and newborns, 180 further cases were investigated blindly by immunohistochemistry. Recognition of hepatocytic staining patterns type I, II and III allowed to reliably diagnose the Pi Z phenotype in 6 cases, whose heterozygous state was confirmed by independent serum phenotyping (5 Pi MZ, 1 Pi SZ). These results allow to conclude that: (1) immunohistochemistry is a reliable technique (superior to PAS-D staining) to identify Pi Z individuals (homo- and heterozygotes) on paraffin-embedded liver sections; (2) heterozygotes Pi MZ and Pi SZ cannot be differentiated without serum phenotyping; (3) immunohistochemistry is helpful in distinguishing between Z homozygotes from heterozygotes, but determination of AAT serum concentration is required to allow confident distinction without phenotyping. AAT immunohistochemistry thus allows to diagnose the Z allele of AAT when phenotyping procedures are not available or in retrospective studies on biopsy and autopsy material.

Adult↗
PubMed2003

Flow cytometric sorting of paraffin-embedded tumor tissues considerably improves molecular genetic analysis.

The characterization of genetic aberrations in paraffin-embedded tumor material is impaired by contaminating normal cells. In the present study on the genetic causes of loss of HLA expression in diffuse large B-cell lymphoma (DLBCL), we compared the efficacy of microdissection with flow cytometric sorting of tumor cells. Single-cell suspensions from paraffin-embedded material of 5 DLBCL cases were stained for CD79a and DNA content (propidium iodide). Fluorescent in situ hybridization (FISH) using HLA class II and chromosome 6 centromeric probes and loss of heterozygosity (LOH) analysis with 5 HLA-specific microsatellite markers were performed on microdissected and flow cytometry-sorted fractions. FISH confirmed considerable enrichment of the samples after flow cytometric sorting and disclosed tumor heterogeneity in 4 cases. Moreover, lymphomas with a so-called zebra LOH pattern in the microdissected material showed unambiguous LOH after flow cytometric sorting, revealing in 1 case a biologically relevant hemizygous deletion in the HLA region.

Chromosome Aberrations↗
PubMed1979

An improved method for embedding brain tissue in albumin-gelatin.

A new modification of the Snodgrass-Dorsey (1963) albumin embedding method is described. Formalin fixed brains of various ages of rhesus monkey (Macaca mulatta) were sunk in 10% phosphate buffered formalin which contained 30% sucrose, and then embedded in a 3% gelatin, 30% egg albumin solution which had been centrifuged to ensure uniformity. The albumin-gelatin was hardened in formaldehyde fumes and blocks cut frozen at 10-40 micron. Sections thus prepared can be handled easily and mounted without damage to the tissue. Modifications of conventional cell and fiber stains produce high quality finished slides in which the stained brain tissue is surrounded by a colorless albumin-gelatin matrix.

Animals↗
PubMed1987

Ultrastructural immunocytochemistry of glia cells. Double labeling studies using LR White embedding and colloidal gold.

The introduction of acrylate resins (Lowicryl K4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class II antigens by pre-embedding method then followed by post-embedding IGS for the other intracytoplasmic antigens.

Acrylates↗
PubMed2003

A simple method for RNA isolation from formalin-fixed and paraffin-embedded lymphatic tissues.

Gene activation that lies beneath lymphoid cell differentiation has been one of the most explored issues in immunology in the recent years. However, the analysis of this molecular event in lymphoproliferative diseases is often hampered by the lack of fresh material. Most tissues available for routine histological investigation are formalin fixed and paraffin embedded. Gene expression in such specimens could be analyzed using reverse transcription of mRNA and the polymerase chain reaction (RT-PCR). Therefore we adjusted and established a method for mRNA isolation from such specimens by a combination of previously reported protocols and a modification of the phenol/chloroform extraction method. Given the significance of transcription factors in the human hemopoietic system, we investigated whether mRNA could be successfully isolated from archival tissue for a study on expression of Ikaros family transcription factors in lymphatic tissue. Although quantitative analysis of RNA isolated from archival tissue is probably not feasible due to the unpredictable degree of RNA isolation varying from sample to sample, we show here that screening analysis is possible and simple.

DNA Primers↗
PubMed1997

Fixed archival tissue. Purify DNA and primers for good PCR yield!

The analysis of archival formalin-fixed, paraffin-embedded tissue samples becomes increasingly important for molecular biology studies. As fixation and paraffin-embedding cause alterations of proteins and nucleic acids, archival sources of DNA must be handled with care. To address the need for specificity and reproducibility, we developed an improved protocol for semi-automated DNA extraction adapted to fixed, embedded tissue samples, and a PCR approach using HPLC-purified primers.

DNA↗
PubMed1991

Detection of hepatitis B virus pre-S1 and pre-S2 determinants in paraffin wax embedded liver tissue: importance of reagents used.

The presence of pre-S polypeptides in paraffin wax embedded liver sections from the biopsy specimens of 15 hepatitis B surface antigen (HBsAg) seropositive patients (five with chronic persistent hepatitis (CPH), four with chronic active hepatitis (CAH), four with cirrhosis and two "healthy" HBsAg carriers) was investigated using monoclonal antibodies directed to distinct epitopes on pre-S1 (18/7 and TO 606) and pre-S2 (5535 and Q 19/10). Pre-S1 was found in 13 cases when MA 18/7 was used but in only one specimen when TO 606 was used. Pre-S2 was detected in all the biopsy specimens with 5535 and in eight samples with Q 19/10. Mild enzymatic digestion annulled the staining of all monoclonal antibodies but Q 19/10. No association was observed between pre-S polypeptide expression and hepatitis B virus (HBV) replication or disease severity. Pre-S polypeptides can be detected readily in paraffin wax embedded material but the results obtained largely depend on the monoclonal antibodies used.

Adolescent↗
PubMed2006

Accuracy of BRCA1 and BRCA2 founder mutation analysis in formalin-fixed and paraffin-embedded (FFPE) tissue.

BACKGROUND: A major limitation in counseling unaffected women from families with inherited breast and ovarian cancer is that a "true-negative" interpretation of wild type BRCA analysis of the proband cannot be inferred in the absence of demonstration of a BRCA mutation segregating in the kindred. Documentation of familial BRCA mutations from paraffin-derived DNA of deceased patients has been limited due to reports of technical complications leading to lack of reproducibility of BRCA testing of archival material. METHODS: DNA was extracted from formalin-fixed paraffin-embedded (FFPE) morphologically normal tissue of 161 blinded, coded samples from women previously genotyped for the three Ashkenazi Jewish BRCA founder mutations from lymphocyte-derived DNA. Multiplex PCR followed by denaturing polyacrylamide gel electrophoresis was performed for the three founder mutations to determine if analysis on FFPE tissue could produce results concordant with those of the lymphocyte-derived DNA. RESULTS: After disclosure of the sample codes, the results were compared with the original lymphocyte-derived DNA genotypes. Excluding one sample unevaluable due to PCR failure, there was 100% concordance of 160 genotypes (120 mutation samples) derived from DNA from archival FFPE tissue compared to peripheral lymphocytes. CONCLUSIONS: The method described reliably detected BRCA founder mutations in archival DNA derived from FFPE tissue. These results suggests that this technique may be useful in clinical settings to inform wild type BRCA results of unaffected probands, leading to avoidance of unnecessary intensified surveillance or risk-reducing surgery. With further validation this approach can also be applied to other populations where founder mutations are observed.

DNA↗
PubMed2001

Direct in situ PCR allows rapid and sensitive detection of high risk human papillomavirus in cytologic specimens and formalin-fixed paraffin tissues by fluorescent labelling.

We developed a rapid, sensitive and robust high risk human papillomavirus (HR HPV) detection protocol based on direct in situ PCR technology and fluorochrome-modified nucleotides on cytologic specimens (cell smears) and on HPV infected tissues (CIN III). Reproducible results on both cytologic specimens and paraffin-embedded tissues were obtained, providing a powerful tool for clinical investigation on HR HPV infection. Quantitative PCR performed on the same tissue sections adjacent to those used for in situ techniques allowed us to establish the sensitivity of our methods, able to detect rare copies (about 15 in our paraffin-embedded tissues) of HPV.

Female↗
PubMed1987

Distinction between undifferentiated (small noncleaved) and lymphoblastic lymphoma. An immunohistologic study on paraffin-embedded, fixed tissue sections.

The morphologic differentiation between malignant lymphoma of the small noncleaved cell (SNC) type and lymphoblastic lymphoma (LBL) is at times difficult, particularly when fresh tissue is not available for immunologic studies. We have examined the reactivities of a panel of monoclonal and polyclonal antibodies, including LN-1, LN-2, and antibodies to immunoglobulin light chains, leukocyte common antigen (LCA), Leu-M1, vimentin, S-100 protein, lysozyme, and alpha-1-antitrypsin, in paraffin-embedded, B5- and formalin-fixed tissue involved by SNC or LBL. The immunophenotypes in all of the cases included in this study had been characterized previously in fresh-frozen sections or cell suspensions. Among 21 samples of B5-fixed SNC, LN-1 was reactive in 17 and LN-2 in 18 cases. Among 13 B5-fixed LBL, LN-1 was reactive in two cases and LN-2 was reactive in two cases. Each of 20 B5-fixed samples of SNC was reactive with at least one of the antibodies, whereas 10 of the 13 B5-fixed samples of LBL were negative for both antibodies. Lesser reactivity was evident in formalin-fixed tissues, with only nine of 27 SNC specimens positive for LN-1 and 16 of 27 positive for LN-2. Most or all of the SNC and LBL samples were negative for immunoglobulin light chains, Leu-M1, vimentin, S-100 protein, lysozyme, and alpha-1-antitrypsin. The majority of both SNC and LBL were positive for LCA. We conclude that LN-1, preferably in combination with LN-2, can be used for distinguishing between SNC and LBL in paraffin-embedded, B5-fixed tissue when fresh tissue is not available.

Adolescent↗
PubMed1990

Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify viral or oncogene sequences from frozen or formalin-fixed, paraffin-embedded tissue sections. Methods for preparing fixed, embedded colonic tissue for PCR amplification of c-K-ras sequences from genomic DNA and for amplification of viral DNA from other tissues, including brain, lung, and liver, were evaluated. The effect of formalin fixation on the efficiency of amplification was also determined. While there seemed to be only a modest variation in the efficiency of the PCR for amplification of single-copy human genes, regardless of the methods used for tissue preparation, amplification of viral DNA sequences against a human genomic DNA background was more efficient when the DNA was purified to some degree before amplification of the tissue. We used the PCR to examine frozen and fixed embedded tissue sections for the presence of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) DNA. One patient with a heart-lung transplant succumbed to a lymphoproliferative disorder, and EBV genome was present in tissues with abnormal lymphoid infiltrates. CMV was also present in bronchial lavages from the same patient, where cytologic diagnosis was not apparent. Another patient with a liver transplant showed CMV genome in multiple liver biopsies, with negative histologic results for CMV. In vitro DNA amplification with the PCR demonstrated sensitivity superior to that of histology in detecting CMV and EBV in the cases examined.

Adult↗
PubMed1985

Purification of DNA from formaldehyde fixed and paraffin embedded human tissue.

The ability to isolate DNA from preserved human tissues would provide numerous experimental opportunities. In this report it is shown that DNA can be extracted from tissues prepared for routine histopathological examination (i.e., fixed with formaldehyde and embedded in paraffin). Although the extracted DNA is not intact, it is double stranded, cleavable with restriction endonucleases, and suitable for a variety of standard techniques used in molecular biology.

Carcinoma↗
PubMed1982

[Comparative immunohistological studies of the localization of the carcinoembryonic antigen in benign and malignant breast tissue].

By means of immunoperoxidase techniques carcinoembryonic antigen (CEA) has been localized first in carcinomas of the gastrointestinal tract and then also in other malignant tissues, such as carcinomas of the female genital tract and the breast. There is still an obvious need for intensive studies of the possible effects which the initial tissue processing, i. e. different fixation procedures may have on the localization results of CEA. By using the two step HRP-labelled antibody technique, we studied comparatively the effect of the most employed fixations on tissues from patients with mammary carcinomas and benign breast lesions: 1. Formalin-fixed and paraplast-embedded tissues 2. Cryostat sections of tissues fixed in ethanol and acetone 3. Parablast embedded tissues fixed by the freeze substitution method. The latter sections were tested whether there was any difference in the CEA localization when using the immunofluorescence or immunoperoxidase staining techniques. We concluded from our studies that routinely fixed paraplast sections, stained by the two step HRP-labelled antibody sandwich technique gave sufficient results for the demonstration and localization of CEA.

Breast Neoplasms↗
PubMedDate not supplied

Immunohistochemical detection of infectious bursal disease virus in formalin-fixed, paraffin-embedded chicken tissues using monoclonal antibody.

Immunoperoxidase and immunofluorescence techniques detected and localized infectious bursal disease virus (IBDV) in formalin-fixed paraffin-embedded sections of the bursa of Fabricius of experimentally and naturally infected chickens. The primary antibody was a monoclonal antibody that bound all IBDV serologic subtypes, including the GLS isolate. Both techniques were valuable in detecting IBDV. The presence and severity of microscopic lesions in the bursa correlated with the location and number of positive IBDV-infected cells as measured by either test. Mild vaccine strains induced minimal microscopic lesions and resulted in a few cells positive by either test. In contrast, virulent IBDV produced widespread lymphoid necrosis and numerous cells positive by both assays. The immunoperoxidase test was more useful than the immunofluorescence test, since the same section could be stained and examined by immunoperoxidase and then restained and examined for microscopic pathology. The presence of immunoperoxidase-positive stained cells associated with areas of microscopic pathology suggested that microscopic lesions were induced by IBDV.

Animals↗
PubMed1993

The role of Ras mutation in pancreatic cancer, precancerous lesions, and chronic pancreatitis.

Point mutations in K-ras codon 12 were detected in 7 of 10 specimens (70%) of frozen tissues. To determine when point mutations appear in the oncogenic stage, paraffin-embedded tissues of pancreatic cancer, hyperplastic ductal lesions in pancreatic cancer and in chronic pancreatitis were used. DNA extracted from the paraffin-embedded tissues classified as cancer, atypical and nonatypical hyperplasia of the pancreatic duct epithelium, and normal tissue from pancreatic cancer patients and those classified as atypical hyperplasia of the pancreatic duct epithelium and normal tissue from chronic pancreatitis patients were amplified by the polymerase chain reaction (PCR) method. Point mutations were detected in 8 of 17 cancer cases (47.0%). In 5 of 8 point mutation positive cases, atypical hyperplasias were found in the surrounding tissues. In all 5 of these atypical hyperplasia, point mutations, that is, GGT-GTT (Gly-Val) and GGT-GAT (Gly-Asp), were detected. The same transitions were observed in the cancer of these 5 cases. These results suggest a strong relationship between cancer and atypical hyperplasia surrounding cancer. Although atypical hyperplasia around cancer was histologically very similar to that found in chronic pancreatitis, all 5 atypical and 5 nonatypical hyperplasias in chronic pancreatitis were negative for ras point mutation. Thus, the result argues against an association between chronic pancreatitis and pancreatic cancer.

Adult↗
PubMed1985

Epithelial markers for paraffin-embedded human tissues. Immunohistochemistry with monoclonal antibodies against milk fat globule antigens.

About 200 human tumours and corresponding normal tissue samples were investigated by immunoperoxidase tests for the expression of MAM-3, MAM-5 and MAM-6 antigens, which had previously been defined by monoclonal antibodies to human milk fat globule membranes. All tissue specimens had been treated for routine histopathology, i.e. fixed in formalin and embedded in paraffin. One of the antigens, MAM-6, appeared to be an important epithelial marker, present in all normal and neoplastic breast tissue samples, in about 80% of non-mammary normal tissues and in more than 90% of non-mammary epithelial tumours. It could never be detected in normal and neoplastic mesenchymal and neuroectodermal structures. Direct comparison with the distribution of Carcinoembryonic Antigen (CEA), Tissue Polypeptide Antigen (TPA) and keratin provided clear evidence that MAM-6 is different from these well known epithelial markers. MAM-3 proved to be an additional important marker exhibiting a characteristic distribution pattern in those epithelial tissues investigated. In contrast to MAM-6, it could never be detected in renal cell cancers and carcinomas of the prostate gland, thus allowing differential diagnosis on the basis of immunohistochemistry. MAM-5, known to be associated with lactoferrin, was mainly detectable in secretory organs and their tumours. In the group of breast tumours, its expression was mainly seen in lobular cancers. These findings suggest a use for these new markers for routine histopathology.

Adenocarcinoma↗