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Results for “Paraffin Embedding”

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PubMed1994

Methods in pathology. p53 expression in formalin-fixed, paraffin-embedded archival specimens of intrahepatic cholangiocarcinoma: retrieval of p53 antigenicity by microwave oven heating of tissue sections.

Immunohistochemical demonstration of p53 is thought to reflect mutations of the p53 gene. Although p53 expression or mutation has been investigated in a variety of carcinomas, it has not been examined in intrahepatic cholangiocarcinoma (CC). We investigated expression of p53 in formalin-fixed, paraffin-embedded archival specimens of 40 CCs (22 autopsy cases and 18 surgical cases) by immunohistochemistry using four antibodies (PAb1801, DO-7, BP53-12, CM1). We also attempted to enhance p53 expression by pretreatments of tissue sections by pepsin digestion as well as by microwave oven heating. Formalin-fixed, paraffin-embedded archival surgical specimens of 15 colon carcinomas were used as controls. In surgical cases, p53 expression was abolished by pepsin predigestion, although it was greatly enhanced by pretreatment of microwave oven heating in all immunostainings (PAb1801, DO-7, BP53-12, CM1). In surgical cases immunostained with microwave oven heating, DO-7, BP53-12, and CM1 showed frequent p53 expression (22% in CC; 60-67% in colon carcinoma), whereas PAb1801 showed low p53 expression (0% in CC; 13% in colon carcinoma). In contrast to the surgical cases, all 22 CCs of autopsy cases showed no p53 expression by any antibodies as well as by any pretreatments. These results shows that a pretreatment of tissue sections by microwave oven heating is a very good method for demonstrating p53 protein in formalin-fixed, paraffin-embedded archival materials and that DO-7, BP53-12, and CM1 are useful antibodies for detection of p53 in formalin-fixed, paraffin-embedded archival materials. No expression of p53 in autopsy cases of CC suggests that p53 antigenicity is lost during autopsy procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMedDate not supplied

The use of MAB 1977 monoclonal antibody for the immunohistochemical localization of beta 1 integrins in paraffin-embedded human kidney.

AIMS AND BACKGROUND: Integrins are widely known cell membrane receptors for extracellular matrix molecules. The beta 1 integrin subgroup is mainly expressed by kidney cells; immunolocalization of these molecules is usually carried out on cryostatic sections. A commercial monoclonal antibody directed against the human beta 1 integrin was tested in order to design a method for the detection of this antigen in formalin-fixed, paraffin-embedded human kidney tissue. METHODS: Specimens obtained from nephrectomies were fixed with 10% formalin and embedded in paraffin. Three different detection protocols were applied after incubation with the anti-human beta 1 integrin monoclonal antibody (MAB 1977): 1) immunoperoxidase with labeled streptavidin biotin (LSAB), using biotinylated secondary antibodies, peroxidase-labeled biotin-streptavidin, and 3,3'-diaminobenzidine tetra-hydrochloride (DAB) as the revealing system; 2) immunoperoxidase with tyramide signal amplification (TSA), using biotinylated secondary antibodies, streptavidin-peroxidase, tyramide, streptavidin-peroxidase again and DAB; 3) indirect immunofluorescence with fluorescein-labeled anti-mouse immunoglobulins. RESULTS: The beat results were obtained with the LSAB detection protocol preceded by a predetection step with proteinase k. Proteinase k pretreatment did not significantly damage the tissue morphology and successfully unmasked beta 1 integrin antigens. Nonspecific background staining was reduced by a blocking step with swine serum. Similar results were obtained with the TSA detection method; however, although lower concentrations of anti-beta 1 integrin immunoglobulins and of secondary biotinylated antibody were employed, there was more undesired background staining than with the LSAB protocol. CONCLUSIONS: The method reported and discussed here may represent a valid tool for research and diagnostic applications based upon detection of beta 1 integrin in paraffin-embedded human tissues.

Adenocarcinoma, Clear Cell↗

PubMed1994

Molecular biological analysis of paraffin-embedded tissues.

Molecular biology techniques have been adapted to the analysis of paraffin-embedded tissues (PETs), expanding their clinical utility. In vitro amplification with the polymerase chain reaction (PCR) promises to be the most useful means of retrospective analysis because it can be performed successfully on nucleic acids that have been partially degraded during fixation, paraffin embedding, and the extraction process. Five clinical situations in which DNA analysis of PETs can be helpful are: (1) confirmatory molecular diagnosis of lymphoma in which fresh tissue has not been obtained at the time of surgery, (2) identification of infectious agents, (3) genetic characterization of a putative inherited disease in which the affected individual has died, (4) confirmation of donor cell malignancy in transplant recipients, and (5) specimen identification. The role of the pathologist in molecular diagnosis will grow because of the feasibility of using PETs, a venue unique to our profession.

DNA↗

PubMed1995

In situ hybridization for vasopressin mRNA in the human supraoptic and paraventricular nucleus; quantitative aspects of formalin-fixed paraffin-embedded tissue sections as compared to cryostat sections.

In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.

Aged↗

PubMed1993

Expression of the human progenitor cell antigen CD34 (HPCA-1) distinguishes dermatofibrosarcoma protuberans from fibrous histiocytoma in formalin-fixed, paraffin-embedded tissue.

BACKGROUND: Histologic distinction of dermatofibrosarcoma protuberans (DFSP) from fibrous histiocytoma (FH) may be difficult. In addition, differential diagnosis is hampered by the lack of appropriate immunohistochemical markers that reliably distinguish between these two entities. OBJECTIVE: This study is aimed at the introduction of a monoclonal antibody (anti-human progenitor cell antigen-1; anti-CD34) that distinguishes between DFSP and FH in formalin-fixed, paraffin-embedded tissue. METHODS: Paraffin-embedded specimens of DFSP, FH, and other soft-tissue tumors were investigated for CD34 expression by anti-human progenitor cell antigen-1/alkaline phosphatase-antialkaline phosphatase immunostaining. RESULTS: Strong CD34 reactivity was present in each DFSP (n = 19) but was consistently absent from FH (n = 45) and other soft-tissue tumors (n = 47). CONCLUSION: CD34 immunostaining of paraffin-embedded specimens may be useful in differentiating between DFSP and FH.

Antigens, CD↗

PubMed2003

Differential gene expression analysis using paraffin-embedded tissues after laser microdissection.

Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen, a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.

Acetic Acid↗

PubMed2001

Polymerase chain reaction analysis for viruses in paraffin-embedded myocardium from dogs with dilated cardiomyopathy or myocarditis.

OBJECTIVE: To perform polymerase chain reaction (PCR) analysis on paraffin-embedded myocardium from dogs with dilated cardiomyopathy (DCM) and dogs with myocarditis to screen for canine parvovirus, adenovirus types 1 and 2, and herpesvirus. SAMPLE POPULATION: Myocardial specimens from 18 dogs with an antemortem diagnosis of DCM and 9 dogs with a histopathologic diagnosis of myocarditis were evaluated. PROCEDURE: Paraffin-embedded myocardial specimens were screened for viral genome by PCR analysis. Positive-control specimens were developed from cell cultures as well as paraffin-embedded tissue specimens from dogs with clinical and histopathologic diagnoses of viral infection with canine parvovirus, adenovirus types 1 and 2, and herpesvirus. The histologic characteristics of all myocardial specimens were classified regarding extent, location, and type of inflammation and fibrosis. RESULTS: Canine adenovirus type 1 was amplified from 1 specimen from a dog with DCM. Canine parvovirus, adenovirus type 2, and herpesvirus were not amplified from any myocardial specimens. Histologic analysis of specimens from dogs with DCM revealed variable amounts of fibrosis; myocardial inflammation was observed in 1 affected dog. Histopathologic analysis of specimens from dogs with myocarditis disclosed variable degrees of inflammation and fibrosis. CONCLUSIONS AND CLINICAL RELEVANCE: Viral agents canine parvovirus, adenovirus types 1 and 2, and herpesvirus are not commonly associated with DCM or active myocarditis in dogs. Additional studies evaluating for nucleic acid from viruses that less commonly affect dogs or different types of infectious agents may be warranted to gain insight into the cause of DCM and myocarditis in dogs.

Animals↗

PubMed2005

[Detection of FUS-CHOP fusion gene in paraffin-embedded tissues and its clinicopathologic significance for myxoid/round cell liposarcomas].

OBJECTIVE: To explore the feasibility of detecting FUS-CHOP fusion gene in formalin-fixed, paraffin-embedded tissue and its application in the diagnosis and differential diagnosis of myxoid/round cell liposarcomas (MRCLs). METHODS: Forty-four formalin-fixed, paraffin-embedded MRCL samples and 60 control cases (atypical/well-differentiated liposarcoma, pleomorphic liposarcoma, low-grade myofibrosarcoma, etc.) retrieved from the archival files were studied. Nested reverse transcription-polymerase chain reaction (RT-PCR) technique was employed to detect the FUS-CHOP mRNA expression, followed by DNA sequencing confirmation of the PCR product. Housekeeping gene PGK was used to assess the quality of the mRNA templates. RESULTS: PGK mRNA was detected in 93 of 104 tumor cases (89.4%), including 39 MRCLs cases (39/44, 88.6%) and 90% of the negative control cases. Type II FUS-CHOP fusion transcript was successfully detected in 20 out of 39 (51.3%) MRCL cases. Type I FUS-CHOP fusion transcript was not detected in any MRCLs in this study. All 60 negative control cases were negative for the FUS-CHOP fusion gene transcripts. CONCLUSIONS: (1) Nested RT-PCR can be used to detect FUS-CHOP mRNA in formalin-fixed, paraffin-embedded tissues. (2) FUS-CHOP is considered a specific molecular and genetic hallmark for MRCLs. Nested RT-PCR is a sensitive and specific technique in detecting FUS-CHOP gene, and can be used in the diagnosis and differential diagnosis of MRCLs.

Adolescent↗

PubMed2002

Detection of SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction.

OBJECTIVE: To assess the feasibility of detecting SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: RT-PCR was used to amplify the SYT-SSX fusion transcripts using archival formalin-fixed paraffin-embedded tumor specimens from a series of 37 synovial sarcoma cases. To investigate the specificity of the SYT-SSX fusion transcripts, a variety of non-synovial sarcoma tumors were included in the study as negative controls. The detected messages derived from fusion genes were confirmed by subsequent sequence analysis. RESULTS: SYT-SSX fusion transcripts were detected in 33 of 37 (89.2%) synovial sarcomas. None of the 34 cases of non-synovial sarcoma tumors showed amplified products of SYT-SSX fusion transcripts, although PBGD mRNA was detected in all specimens. Among 33 SYT-SSX-positive synovial sarcomas, 22 tumors had an SYT-SSX 1 fusion transcript, whereas 6 tumors had an SYT-SSX2 fusion transcript. Fusion types can not be distinguished in the remaining 5 cases. There was a significant relationship between SYT-SSX fusion type and histologic subtype. All 10 biphasic synovial sarcomas had the SYT-SSX1 fusion, whereas all tumors with SYT-SSX2 were of monophasic morphology (P < 0.05). CONCLUSIONS: RT-PCR can be applied to archival formalin-fixed paraffin-embedded tumor tissues as a sensitive and reliable technique for the diagnosis and differential diagnosis of synovial sarcoma. There is an association between SYT-SSX fusion type and histological subtype. SYT-SSX2 fusion transcripts can only be found in monophasic synovial sarcoma.

Adolescent↗

PubMed1979

Electron spin resonance (ESR) spectrum of paraffin embedded human liver tissue. A possible method for the estimation of copper content.

Human liver tissues embedded in paraffin wax for histological examination have been studied by Electron Spin Resonance (ESR) spectroscopy. A signal was detected at g approximately 2.05 section of the spectrum. The amplitude of this signal was correlated with the copper content of the embedded specimens measured by flame atomic absorption technique. The positive correlation which has been found can make ESR spectroscopy suitable for estimating the copper content of tissues without damaging the sample. The limits and errors of this method have also been analysed.

Copper↗

PubMed1999

Detection of mammosomatotrophs in paraffin-embedded specimens of various pituitary adenomas.

BACKGROUND: There are various classification systems for pituitary adenomas based on whether mammosomatotroph cells, which simultaneously express both prolactin (PRL) and growth hormone (GH), can be found. Until the present, the identification of such cells required special techniques and could not be performed in paraffin-embedded specimens. This hindered large-scale studies for detection of mammosomatotrophs in various pituitary adenomas and, as a result, such classification has remained controversial. To establish a methodology for the detection of mammosomatotrophs in paraffin-embedded specimens and to propose a more logical classification for pituitary adenomas, the authors conducted this retrospective study. METHODS: We performed double immunofluorescence staining of PRL and GH in paraffin-embedded specimens of various pituitary adenomas with subsequent observation with a confocal laser-scanning microscope. RESULTS: Mammosomatotrophs were found in four of the 10 GH-secreting adenomas and one of the 10 clinically nonfunctioning adenomas. However, mammosomatotrophs were not identified in all 10 cases of prolactinoma. CONCLUSIONS: This is the first report in the literature that successfully demonstrates the presence of mammosomatotrophs in routine paraffin-embedded pituitary adenomas. The new methodology is important for future study of the function and role of these cells. A large-scale study for mammosomatotrophs in various pituitary adenomas with this method and a more logical classification of pituitary adenomas are proposed.

Adenoma↗

PubMed1993

Retrieval of p53 protein in paraffin-embedded head and neck tumor tissues.

OBJECTIVES: To improve the detection of p53 protein in formalin-fixed, paraffin-embedded head and neck tumor tissues. DESIGN: Cohort. SETTING: University and Veterans Administration medical centers. PATIENTS OR OTHER PARTICIPANTS: Retrospective samples. INTERVENTION: Surgery for head and neck carcinoma. MAIN OUTCOME MEASURES: Retrieval of p53 antigen. Hypothesis formulated after data collection. RESULTS: An antigen retrieval method facilitated the unmasking of previously inaccessible p53 antigenic determinants in formalin-fixed, paraffin-embedded tissues. This approach has made possible a much more reliable and sensitive immunohistochemical detection of p53 antigen. The procedure is simple, requiring only microwave heating of tissue sections to 100 degrees C in the presence of a zinc sulfate solution. CONCLUSIONS: Antigen retrieval method in formalin-fixed, paraffin-embedded tissue demonstrated a significant increase in p53 immunostaining.

Genes, p53↗

PubMed1995

Comparison of immunoreactivity of neu oncoprotein in fine-needle aspirates and paraffin-embedded materials.

Expression of the neu oncogene has been extensively examined in frozen and paraffin section breast cancers; however, very few studies examine neu oncoprotein oncoprotein expression in fine-needle aspirates. To this effect, we compared the expression of neu oncoprotein in formalin-fixed paraffin-embedded breast cancers and the corresponding fine-needle aspirates of these cancers. There was 100% correlation between the expression of neu oncoprotein in the paraffin-embedded breast cancers and the fine-needle aspirates, indicating the suitability of fine-needle aspirates for the expression of neu oncoprotein in breast cancers.

Biopsy, Needle↗

PubMed1995

Effects of fixation and paraffin embedding on the immunohistological detection of cell-associated HIV-1 by different monoclonal antibodies.

This study evaluates a panel of monoclonal antibodies (MAbs) to HIV-1 antigens (DuPont anti-gp120, gp41, p24; Olympus anti-gp120/160, gp41, p24A, p24B, p55, p18A, p18B, reverse transcriptase) for their ability to detect the virus in tissues after exposure to various fixatives (100% acetone, 10% formaldehyde, 2.5% glutaraldehyde, 4% paraformaldehyde/1% glutaraldehyde, Bouin's fluid) and after paraffin embedding. Acetone, 10% formaldehyde, and Bouin's fluid all preserved a wide range of viral epitopes compared with other fixatives. The most robust MAbs were DuPont p24 and Olympus p55, which produced excellent staining regardless of the fixative used. Embedding in paraffin variability influenced the capacity of MAbs to detect HIV-1 epitopes on fixed cells. Certain antibodies (e.g., DuPont gp24, Olympus p24B) produced good staining, whereas other epitopes (e.g., DuPont gp120, formaldehyde) were destroyed. In some cases, paraffin embedding revealed antigenic sites that had been formerly masked (e.g., Olympus gp120 and p24A; formaldehyde and glutaraldehyde fixation). These results indicate that HIV-1 antigens can be detected by immunohistology on cells exposed to most common fixatives. Therefore, retrospective analysis of pathological material is possible, provided that the antibodies are matched to the fixative used to preserve the tissue.

Acetates↗

PubMed1996

Detection of paraneoplastic anti-neuronal autoantibodies on paraffin-embedded tissues.

Anti-neuron-specific autoantibodies are widely recognised as useful, though non-specific, diagnostic markers of paraneoplastic neurological disorders. However, controversies on the best way to detect these autoantibodies have recently arisen, and the use of different procedures for their detection by different laboratories has made results difficult to compare. The aim of this study was to adapt the existing immunohistochemical techniques used for the detection of anit-neuron autoantibodies to improve their visualisation and to facilitate a wide application of these procedures. Sera and cerebrospinal fluid (CSF) were obtained from 15 patients known to carry paraneoplastic anti-neuronal autoantibodies; in addition, one serum with "atypical" anti-neuron autoantibody and 18 control sera were studied. Paraformaldehyde-fixed, paraffin-embedded rat nervous tissue and formalin-fixed, paraffin-embedded human nervous tissue treated in a microwave oven were used as substrate; the reactions were developed by immunoperoxidase methods. At the dilutions used for diagnostic purposes, all the sera and CSFs showed staining whose intensity and specificity was comparable to that obtained using frozen tissue; the end-point dilutions were, however, reduced. The atypical pattern of staining of one serum was confirmed and better emphasised using these procedures; all control sera and CSFs were negative. The morphology was improved by the use of paraffin-embedded tissues; moreover, the results obtained are permanent because of peroxidase staining, which makes it possible to use them as standards for further investigations and for comparison between different laboratories. The convenience of using paraffin-embedded material could facilitate a wide application of these procedures in clinical neurology.

Animals↗

PubMed1993

Immunoblot analysis of the placental form of glutathione S-transferase in protein extracted from paraffin-embedded human glioma tissue.

Protein extracted from conventional formalin-fixed and paraffin-embedded tissue sections of human gliomas was examined for immunoblot analysis using antibody against the placental form of glutathione S-transferase (GST-pi). Four benign astrocytomas, five anaplastic astrocytomas and four glioblastomas were used in this study. The preliminary study demonstrated that immunoreactivity of GST-pi was well preserved in normal brain tissue and normal term placenta fixed in acetone, formalin or buffered formalin (pH 7.4). GST-pi in gliomas fixed in formalin also had a good immunoreactivity and showed clear bands on nitrocellulose membranes processed by the method of Western blotting using anti-GST-pi antibody. The results of immunoblot analysis for GST-pi indicate that the intensity of immunoreactivity of benign astrocytoma, anaplastic astrocytoma and glioblastoma increases with the advance of malignancy of these neoplasms. Western blot analysis for GST-pi can be performed using protein extracted from formalin-fixed and paraffin-embedded tissue sections, and the immunoreactive bands can be analyzed quantitatively by densitometric scanning.

Blotting, Western↗

PubMed2005

Protein-embedding technique: a potential approach to standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections.

A serial study was performed to develop a protein-embedding technique for standardization of immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) tissue sections. A protein carrier matrix must have two phases, a liquid phase to allow uniform mixing of a protein and a solid phase forming a 'block' that can be fixed and processed in the same manner as human tissue. This standardized protein block would serve as a source of thin sections for control of IHC and therefore must also withstand the boiling conditions of antigen retrieval (AR). After multiple experiments, a method was developed utilizing polymer microsphere (beads) as a support medium for protein. The method showed particular promise for quantitative IHC.

Animals↗

PubMed1984

[Detectability of poisons in paraffin-embedded organ parts].

The detectability of metals and drugs in histological organ parts embedded in paraffin was tested in 24 cases, which had been analyzed before. The liver, kidney and brain parts were separated from the paraffin by heating the paraffin up to 110 degrees C and were homogenized by means of Ultra-Turrax blendor and ultrasound. Analysis was realized using gas chromatography, atomic absorption and mass spectrometry. Only 1.5%-4% of the original quantities of metals (As, Tl, Hg) was found in the embedded organs. Drugs could not be detected except for a low percentage of phenobarbital (2 cases) and bromine (3 cases). Reliable estimations cannot be made about the concentrations in the organs before the embedding procedure.

Arsenic Poisoning↗