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Results for “Paraffin Embedding”

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PubMed2001

[Detection of the expression of Smad4, transforming growth factor beta(1) and beta receptor II proteins in paraffin-embedded human pancreatic cancer tissues].

OBJECTIVE: To explore the relationships between Smad4, TGF beta1 and TbetaR II in the TGF-beta pathway and the possible mechanisms by which they effect pancreatic carcinoma. METHODS: The expression of Smad4, TGF beta1 and TbetaR II in paraffin embedded pancreatic carcinoma tissues was detected by using antibodies against Smad4, TGF beta1 and TbetaR II with EnVision immunohistochemistry. RESULTS: The positive rates of Smad4, TGF beta1 and TbetaR II were 58.93% (33), 66.07% (37) and 60.71% (34), respectively. The positive rates of the above three proteins in matched normal pancreatic tissues were 89.29% (50), 25.00% (14) and 25.00% (14) respectively. There was a significant relationship between the expression of TGF beta1, clinical stage and the metastasis of the tumor (P < 0.05). There was also a significant relationship between the expressions of TGF beta1 and TbetaR II (P < 0.05). CONCLUSIONS: The expression of Smad4 in pancreatic carcinoma tissue is significantly decreased but the expression of TGF beta1 and TbetaR II are increased. Smad4 may play an important role in the regulation of TGFbeta inducible gene expression and subsequent growth inhibition, but TGFbeta may also act in a Smad4-independent manner.

DNA-Binding Proteins↗

PubMed1998

Analysis of clonality in human endometriotic cysts based on evaluation of X chromosome inactivation in archival formalin-fixed, paraffin-embedded tissue.

The clonality of epithelial cells in ovarian endometriotic cysts was evaluated on the basis of a polymorphism of the X-linked phosphoglycerate kinase gene (PGK). The problems associated with clonality analysis of DNA samples extracted from archival formalin-fixed, paraffin-embedded tissue were addressed with the use of newly designed primers and a modified stepdown program for PCR amplification. The method relies on digestion of DNA with the methylation-sensitive restriction enzyme SnaBI, PCR amplification of PGK, and detection of a BstXI polymorphism. With this approach, only the inactive (methylated) PGK allele is selectively amplified by PCR. A total of 25 epithelial cells in endometriotic cysts and 25 matched normal ovarian stromal tissue specimens were analyzed. All of the control tissue samples yielded PCR products, with 11 of the 25 samples found to be heterozygous for the BstXI polymorphism. Ten of the 25 epithelial cells in endometriosis specimens were informative at this locus, and all of these 10 were shown to be monoclonal on the basis of PGK methylation. The results indicate that this new method of clonal analysis with archival formalin-fixed tissue is reliable and confirm that endometriotic cysts are monoclonal in origin.

Base Sequence↗

PubMed1992

Diagnostic usefulness of dipeptidyl aminopeptidase IV monoclonal antibody in paraffin-embedded thyroid follicular tumours.

Monoclonal antibodies to dipeptidyl aminopeptidase IV (DAP IV, EC 3.4.14.5) were raised and selectively applied to paraffin-embedded sections of thyroid carcinoma. Five monoclonal antibodies were found to stain paraffin sections of thyroid carcinomas. Using one of these antibodies (44-4), we studied retrospectively aberrant expression of DAP IV in thyroid carcinoma to determine whether immunohistochemical staining with DAP IV antibody is useful in pathological diagnosis. In almost all cases of thyroid follicular and papillary carcinoma, tumour cells were positive (99.0 per cent) with DAP IV, whereas the cases of follicular adenoma showed a low incidence (27.1 per cent) of positive staining. Follicular adenoma with incomplete capsular invasion had a higher positive incidence (50 per cent) than follicular adenoma without incomplete capsular invasion (9.6 per cent). In positive staining cases previously diagnosed as benign tumours, 11 benign cases reacting positively with DAP IV were rediagnosed as carcinoma after re-examination of more thyroid paraffin block sections or serial sections. These findings suggest that DAP IV monoclonal antibody is very useful in distinguishing thyroid follicular carcinoma from follicular adenoma.

Adenoma↗

PubMed2026

Spatial Total RNA Sequencing of Formalin-Fixed Paraffin-Embedded Tissue by spRandom-seq.

The molecular pathogenesis of infectious diseases and cancer is orchestrated by nanoscale of host and microbial RNA transcripts within the tissue microenvironment. Nevertheless, spatially resolving the comprehensive transcriptional landscape within complex clinical tissues, like formalin-fixed paraffin-embedded (FFPE) specimens, still poses a formidable challenge. Here, we present spRandom-seq, a random primer-based spatial total RNA sequencing technology designed to spatially resolve complete transcriptomes from host, bacteria, and even nanoscale viruses in FFPE tissues. Capitalizing on the random primer design, our technology not only facilitated the discovery of specific lncRNAs and alternative splicing events in mouse brain and olfactory bulb, but also delineated pronounced spatial heterogeneity in clinical FFPE sections-across distinct tumor regions in breast cancer and microbial infection sites in Klebsiella pneumoniae-infected tissues. Importantly, integrated analysis of host and viral RNAs in FFPE samples from hepatitis B virus (HBV)‑positive hepatocellular carcinoma (HCC) demonstrated that complement and coagulation pathways were specifically activated across expansive HBV‑infected tumor areas, which also exhibited an increased burden of copy number variations (CNVs). Owing to its compatibility with existing spatial transcriptomics platforms and minimal operational complexity, spRandom-seq represents a practical and scalable approach for clinical pathology applications and infection diagnostics.

Paraffin Embedding↗

PubMed1997

Interphase cytogenetic (in situ hybridization) analysis of astrocytomas using archival, formalin-fixed, paraffin-embedded tissue and nonfluorescent light microscopy.

Astrocytomas contain nonrandom chromosomal abnormalities that recently have been correlated with shortened patient survival. Two frequently reported aberrations are trisomy 7 and monosomy 10. We assessed the numerical complement of chromosomes 7 and 10 in formalin-fixed, paraffin-embedded brain biopsy tissue from 28 diffuse astrocytomas by in situ hybridization using a nonfluorescent enzymatic detection system. Clinical follow-up of at least 5 years was available in 26 cases (93%). Monosomy 10 was identified in 7 cases (25%): astrocytoma, 1 case; anaplastic astrocytoma, 1 case; and glioblastoma, 5 cases. Trisomy 7 was identified in 11 cases (39%): astrocytoma, 5 cases; glioblastoma, 6 cases. Multivariate analysis revealed that monosomy 10 was the most statistically significant negative predictor of patient survival. Numerical chromosomal abnormalities are detectable in astrocytomas in archival tissue using interphase cytogenetics and nonfluorescent light microscopy. Although larger studies are required, our data suggest that potentially useful prognostic information may be obtained with this approach.

Adult↗

PubMedDate not supplied

Heat-induced antigen retrieval of proliferating cell nuclear antigen and p53 protein in formalin-fixed, paraffin-embedded sections.

Immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA) and p53 protein is important, particularly for the surgical diagnosis of neoplastic disorders. An effective, simple and reproducible method was established for observing the expression of these intranuclear antigens in routinely processed, formalin-fixed paraffin-embedded sections. Dramatic improvement of the antigenicity was obtained when the deparaffinized sections were heated in a hot water bath at 90 degrees C for 120 min in 0.01 mol/L citrate buffer, pH 6.0, for PCNA and in 0.01 mol/L phosphate-buffered saline, pH 7.2, for p53 protein. These reliable pretreatments are useful for the detailed comparative analysis of the expression of PCNA and p53 protein and fine histologic architecture and for retrospective study using a large number of archival specimens.

Adenocarcinoma↗

PubMed2003

Detection of Candida albicans mRNA from formalin-fixed, paraffin-embedded mouse tissues by nested reverse transcription-PCR.

Histopathology archives represent a vast source of infectious disease specimens that can be used to elucidate important disease processes. In this report, we describe a method to detect Candida albicans gene expression from infected, formalin-fixed, paraffin-embedded mouse tissue. By use of glass beads to break open the fungal cells and proteinase K treatment, RNA was extracted routinely from tissue sections that had been fixed for up to 72 h. Upon reverse transcription of the RNA and nested PCR, the procedure detected C. albicans "housekeeping" and putative virulence genes.

Animals↗

PubMed1994

Monoclonal antibodies to Coxiella burnetii for antigenic detection in cell cultures and in paraffin-embedded tissues.

The authors developed monoclonal antibodies to Coxiella burnetti, the agent of Q fever. The selected monoclonal antibody, Cox1D8, did not cross-react with other bacteria and was used for early detection of C burnetti in shell vial cell cultures and for staining C burnetii in paraffin embedded tissues. Formalin or Bouin fixation did not alter the reactivity of the antigen with the antibody. This monoclonal antibody could be useful in the pathologic diagnosis of Q fever hepatitis and endocarditis.

Animals↗

PubMed1993

DNA cytometric and interphase cytogenetic analyses of paraffin-embedded hydatidiform moles and hydropic abortions.

The combined application of DNA cytometric and interphase cytogenetic analyses was used to find objective criteria for the differential diagnosis of complete hydatidiform mole, partial hydatidiform mole and hydropic abortion. DNA ploidy and G0/G1 exceeding rates were determined using image and flow cytometric analyses on paraffin-embedded tissues of 166 cases: 71 cases of complete mole, 20 cases of partial mole, and 75 cases of abortions. To determine the existence and histological distribution of cell subpopulations with numerical chromosome aberrations, interphase cytogenetic analysis using probes specific for chromosomes 1, X, and Y was applied to paraffin tissue sections of 23 cases: 12 cases of complete mole, 3 cases of partial mole, and 8 cases of abortions. In contrast to previously reported findings that complete moles are diploid, the results of this study showed that complete moles are DNA-polyploid (96 per cent), with high G0/G1 exceeding rates and a high frequency of numerical chromosomal aberrations in the trophoblast hyperplasia. The majority of the partial moles were DNA-triploid (55 per cent). This study, however, also showed the presence of DNA-polyploid partial moles (30 per cent). Abortions were DNA-diploid (60 per cent) or DNA-triploid (39 per cent). DNA cytometric analysis, especially image DNA cytometric analysis with determination of the G0/G1 exceeding rate, and interphase cytogenetic analysis provide objective measurements which are contributory in the differential diagnosis between complete mole, partial mole, and hydropic abortion.

DNA↗

PubMed1999

Microwave-stimulated recovery of myosin-V immunoreactivity from formalin-fixed, paraffin-embedded human CNS.

The lability of brain myosin-V (BM-V) to aldehyde-fixation has hindered immunohistochemical (IH) studies of this actin-based motor. We show here that BM-V immunoreactivity (IR) can be retrieved from formalin-fixed, paraffin-embedded human tissue. BM-V IR was optimally retrieved by boiling 5 microm cerebellar tissue sections in 10 mM sodium citrate buffer, pH 6, for 15 min, using a microwave oven set at 900 W and 2.45 GHz. A polyclonal, affinity purified anti-BM-V antibody, raised in rabbits against the tail domain of chicken BM-V, was shown here to recognize a single band in Western blots of human cortical homogenates. The combined use of this monospecific antibody and of the antigen retrieval (AR) method above allowed us to verify that BM-V IR is strongly expressed in human Purkinje cell bodies and dendrites, and in granule cells. The same pattern of BM-V IR expression was consistently and maximally detected in tissues stored in 10% formalin from 1 week to 2.5 months. The AR protocol for BM-V described here permits its IH study in formaldehyde-fixed tissues. It is a valuable tool to study BM-V in well fixed tissues, as occurs with the large collection of human archival tissue available.

Adult↗

PubMed2006

Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells.

Array-based comparative genomic hybridisation (aCGH) has diverse applications in cancer gene discovery and translational research. Currently, aCGH is performed primarily using high molecular weight DNA samples and its application to formalin-fixed and paraffin-embedded (FFPE) tissues remains to be established. To explore how aCGH can be reliably applied to archival FFPE tissues and whether it is possible to apply aCGH to small numbers of cells microdissected from FFPE tissue sections, we have systematically performed aCGH on 15 pairs of matched frozen and FFPE astrocytic tumour tissues using a well-established in-house human 1 Mb BAC/PAC genomic array. By spiking tumour DNA with normal DNA, we demonstrated that at least 70% of tumour DNA was required for reliable aCGH analysis. Using aCGH data from frozen tissue as a reference, it was found that only FFPE astrocytic tumour tissues that supported PCR amplification of >300 bp DNA fragment provided high quality, reproducible aCGH data. The presence of necrosis in a tissue specimen had an adverse effect on the quality of aCGH, while fixation in formalin for up to 96 h of fresh tissue did not appear to affect the quality of the result. As little as 10-20 ng DNA from frozen or FFPE tissues could be readily used for aCGH analysis following whole genome amplification (WGA). Furthermore, as few as 2000 microdissected cells from haematoxylin-stained slides of archival FFPE tissues could be successfully used for aCGH investigations when WGA was used. By careful assessment of DNA integrity and review of histology, to exclude necrosis and select specimens with a high proportion of tumour cells, it is feasible to preselect archival FFPE tissues adequate for aCGH analysis. With the help of microdissection and WGA, it is also possible to apply aCGH to histologically defined lesions, such as carcinoma in situ.

Astrocytoma↗

PubMed1998

Expression of MDR1 mRNA and encoding P-glycoprotein in archival formalin-fixed paraffin-embedded gall bladder cancer tissues.

The aim of this study was to examine the expression of P-glycoprotein (Pgp) and MDR1 mRNA, in gall bladder carcinoma, a chemo-resistant tumour. 26 cases of gall bladder cancer and nine samples of normal gall bladder archival paraffin blocks were investigated for the presence of Pgp protein with immunohistochemistry (IHC) and MDR1 RNA by reverse transcription-polymerase chain reaction (RT-PCR). Monoclonal antibodies JSB-1 and UIC-2, recognising separate epitopes of Pgp, were used for IHC. For RT-PCR, total RNA was extracted from paraffin-embedded tissue. After RT, the samples were subjected to nested PCR (NPCR) using primers specific for the MDR1 gene, and evaluated by electrophoresis. In gall bladder carcinoma, the percentage of positive cases expressing Pgp (77% for JSB-1, 69% for UIC-2) and MDR1 mRNA (52%) was significantly higher than those in normal gall bladder. In earlier TNM stages Pgp and MDR1 mRNA were more frequently expressed (non-significant) than in advanced stages. The results of this study suggested that overexpression of MDR1 mRNA and Pgp in gall bladder carcinoma tissue probably is a very important reason why gall bladder cancer is generally not responsive to chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

A new and improved "quick-hot Gram-chromotrope" technique that differentially stains microsporidian spores in clinical samples, including paraffin-embedded tissue sections.

OBJECTIVE: This report describes a new and improved "quick-hot Gram-chromotrope" staining technique that detects microsporidian spores in clinical specimens, such as stool, urine, saliva, nasopharyngeal fluid, and bronchoalveolar lavage samples, as well as in formalin-fixed and paraffin-embedded tissue sections. DESIGN: In this procedure, the samples are stained in heated (50 degrees C to 55 degrees C) solutions of crystal violet and iodine used in Gram's stain, followed by a modified chromotrope solution (heated to 50 degrees C to 55 degrees C). The modified stain is composed of chromotrope 2R (1%), fast green (0.15%), and phosphotungstic acid (0.25%). RESULTS: With this stain and the new protocol, microsporidian spores are stained dark violet against a pale green background, and the total staining time is shortened to 5 minutes. CONCLUSIONS: This new technique is fast, reliable, and simple. It can be easily adapted for use in clinical laboratories.

Animals↗

PubMed2006

Exploring allelic imbalance within paraffin-embedded tumor biopsies using pyrosequencing technology.

BACKGROUND: The comparison of molecular genetic changes in healthy and pathological tissues has historically led to the identification of oncogenes and tumor suppressor genes. It is very common that studies investigating loss of heterozygosity are carried out retrospectively on paraffin-embedded samples. METHODS: In this study, we evaluated the power of pyrosequencing for determining the loss of heterozygotic regions. The present method uses the fact that pyrosequencing is an accurate, sensitive and reproducible technique. The method is also simple to perform, with results available in 96-well format, making the assays amenable to automation. Thus, we analyzed nine single nucleotide polymorphisms along 1 Mb between the EMSY and PAK1 genes on 11q13, a region frequently rearranged in different tumors and cell lines. We assessed the study using samples from breast cancer and thyroid cancer biopsies. RESULTS AND CONCLUSIONS: We conclude that this technique is capable of detecting variations of >10% in allele loss. However, strong allele imbalances were detected, depending on the origin of the sample. Seven out of the nine markers used exhibited differential allele amplification, depending on the DNA quality (p<0.01).

Allelic Imbalance↗

PubMed2004

Limited numbers of apoptotic cells in fresh paraffin embedded bone marrow samples of patients with myelodysplastic syndrome.

In myelodysplasia (MDS) the precise mechanism of ineffective erythropoiesis is not fully elucidated, but it is suggested that apoptosis may contribute to this process. We performed TdT-mediated dUTP-nick end labelling (TUNEL) staining of paraffin embedded bone marrow specimens to assess the amount of apoptotic cells in 21 MDS patients (7 RA, 3 RARS, 5 RAEB, 3 RAEB-T, 3 CMML) and five normal controls. In 10 MDS patients the TUNEL assay was performed in combination with immunostaining for Glycophorin-A (GpA) to determine apoptosis in the maturing erythroid compartment. To assess the proliferation of the bone marrow cells the expression of Ki-67 antigen was used as a marker. The mean apoptotic index (AI) in MDS patients was not increased (2.3 +/- 3.0% in MDS versus 4.8 +/- 1.2% in normal controls (P < 0.05)). Moreover, no significant difference in mean AI was observed in the GpA+ compartment between MDS and normal controls (0.8 +/- 0.2% versus 0.6 +/- 0.1%). In addition the different FAB-classifications and the different International Prognostic Scoring System (IPSS)-risk groups showed no significant differences between the subgroups. The expression of Ki-67, as marker for proliferative activity, in the GpA+ compartment from MDS did not differ significantly from normal controls (84.0 +/- 12.2% versus 79.9 +/- 20.2%). Our findings suggest that the observed increased apoptosis in in vitro culture assays is related to the detachment of the cells from the microenvironment leading to an increased susceptibility to apoptosis.

Adult↗

PubMed1997

Detection of porcine reproductive and respiratory syndrome virus in paraffin-embedded tissues: comparison of immunohistochemistry and in situ hybridization.

A non-radioactive in situ hybridization (ISH) method developed recently and an immunohistochemical method, the immunogold silver staining (IGSS), were compared for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in formalin-fixed paraffin-embedded tissues. Serial sections from 98 tissues, representing different organs from PRRSV experimentally infected pigs, and serial sections from 46 lung tissues from field cases were tested by both methods. Results obtained on tissues from experimentally infected pigs and tissues from field cases demonstrated that ISH was more sensitive than immunohistochemistry. More tissues were positive by ISH compared to IGSS and also a greater number of labelled cells and a stronger signal in stained cells were observed in ISH-treated sections. The ISH method described, using a 254 bp digoxigenin-labelled cDNA probe, is a rapid, highly specific and sensitive detection method which can be used for the diagnosis of PRRSV in routinely fixed and processed tissues.

Animals↗

PubMed2003

Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients.

Improved extraction techniques combined with sensitive real-time reverse transcriptase-polymerase chain reaction may allow detection of mRNA in formalin-fixed, paraffin-embedded (FFPE) materials, but the factors affecting mRNA quantification in clinical material using these methods have not been systematically analyzed. We designed analyses using real-time reverse transcriptase-polymerase chain reaction for quantification of MART-1, beta-actin, and beta(2)-microglobulin mRNAs. The analytical intra- and interassay imprecision (coefficient of variation) was in the range 10 to 20% for all three genes studied. Using these protocols, we studied the influence of tissue autolysis and length of formalin-fixation on mRNA detection in metastatic melanoma. Delay in freezing reduced detectable mRNA, although this was less than predicted and mostly occurred early in autolysis. MART-1, beta-actin, and beta(2)-microglobulin mRNAs were consistently detected in FFPE metastatic melanoma even after fixation for up to 3 weeks, although the total mRNA detected was markedly reduced in fixed compared with fresh tissues (up to 99%). Quantification of MART-1 was, however, possible if this was expressed relative to a housekeeping gene. The polymerase chain reaction product from FFPE tissues could be increased up to 100-fold amplifying short (<136 bp) compared with long amplicons. Variations in time before tissue processing and in fixation length seem to be less important sources of imprecision than previously assumed. Our findings suggest that quantitative analysis of mRNA in archive and routine diagnostic tissues may be possible.

Actins↗

PubMed1996

Investigation of specimen mislabelling in paraffin-embedded tissue using a rapid, allele-specific, PCR-based HLA class II typing method.

Mislabelling of surgical specimens can seriously affect the accuracy of histopathology reports. We describe two cases in which clinically suspected mislabelling was investigated by polymerase chain reaction (PCR)-based HLA DRB and DQB tissue typing of paraffin biopsy-derived DNA, using sequence specific primers (PCR-SSP HLA typing). In the first case, two patients underwent surgery for breast carcinoma. A subcutaneous lymph node containing metastatic carcinoma was received with the breast specimen from one patient, but was clinically considered more likely to originate from the other patient who underwent breast surgery on the same day. In the second case, histological examination of retained products of conception from a young woman revealed adenocarcinoma, but a repeat curettage specimen consisted of secretory phase endometrium. In case 1, PCR-SSP HLA typing confirmed the clinical suspicion that the subcutaneous lymph node received with tissue from one patient originated from the other patient. This result converted the first patient from lymph node positive breast carcinoma to lymph node negative disease. In case 2, there was no evidence from PCR-SSP HLA typing that the endometrial samples had originated from different patients. PCR-SSP HLA typing requires fewer steps than methods based on PCR amplification followed by oligonucleotide probing (PCR-SSOP HLA typing), and relies on the amplification of shorter sequences of DNA. Therefore, this technique can produce more rapid results than PCR-SSOP HLA typing, and is ideally suited to typing partially degraded DNA derived from formalin-fixed and paraffin-embedded tissue.

Adenocarcinoma↗