Direct PCR from paraffin-embedded tissue.
Explore the source record for details and available documents.
Appendix↗
SEARCH · Search PubMed
Results for “Tissue Embedding”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Immunohistochemical detection of peste des petits ruminants viral antigen in formalin-fixed, paraffin-embedded tissues from cases of naturally occurring infection.
Explore the source record for details and available documents.
Animals↗
A simple method using alizarin red S for the detection of calcium in epoxy resin embedded tissue.
This report presents a simple procedure for staining 1-2 microns epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue O solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralization. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated. Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.
Animals↗
Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis.
From a practical point of view, one of the most difficult issues in the standardization of IHC for FFPE tissue is the adverse influence of formalin upon antigenicity, as well as the great variation in fixation/processing procedures. Based on previous study, an additional study using four markers demonstrated the potential for obtaining equivalent IHC staining among FFPE tissue sections with periods of formalin fixation ranging from 6 hr to 30 days. On this basis, the following hypothesis is proposed. "The use of optimized AR protocols permits retrieval of specific proteins (antigens) from FFPE tissues to a defined and reproducible degree (expressed as R%), with reference to the amount of protein present in the original fresh/unfixed tissue". This hypothesis may also be presented mathematically: the protein amount in a fresh cell/tissue, expressed as Pf, produces an IHC signal in fresh tissue of integral(Pf). When the identical IHC staining plus AR treatment is applied to a FFPE tissue section, the IHC signal may be represented as integral (Pffpe). The degree of retrieval after AR (R%) is calculated as follows: R% = integral (Pffpe)/ integral (Pf) x 100%. The amount of protein in the FFPE tissue may then be derived as follows: Pffpe = Pf x R%. In a situation where optimized AR is 100% effective, the IHC signal would then be of equal strength in fresh tissue and FFPE tissue, and Pffpe= Pf. Further studies are designed to test the limitations of the proposed hypothesis.
Antigens↗
Enhanced detection of RNA from paraffin-embedded tissue using a panel of truncated gene-specific primers for reverse transcription.
Explore the source record for details and available documents.
DNA Primers↗
Flow cytometric DNA content analysis of paraffin-embedded tissue derived from cervical carcinoma.
PURPOSE: Flow cytometric deoxyribonucleic acid (DNA) content analysis has been shown to be of prognostic importance in some cancers. There have been recent reports of a prognostic importance for DNA content analysis in cervical carcinoma. METHODS AND MATERIALS: We retrospectively reviewed the hospital and radiation oncology records of cervical carcinoma patients who presented between 1984-1990. RESULTS: A total of 101 archival paraffin-embedded blocks were processed, of which 77 were of technical quality for analysis. Thirty-five percent were found to be DNA content aneuploid (DNA-A) and 65% DNA content diploid (DNA-D). No statistical difference was found between the two groups in age at diagnosis, % S-phase, coefficient of variation (CV), or proliferative index (PI). A statistical difference was noted in the G2M phase between the two groups (p = 0.004). The median % S-phase was 8.4% in the DNA-D group. A statistical difference (p = 0.017) in survival was noted between the low and high % S-phase DNA-D groups. In patients who received radiation alone, high-PI patients had improved survival compared to low-PI patients. No statistical difference in survival was noted in the high % S-phase DNA-D group and DNA-A group (p = 0.28). Proportional Hazard (Cox) Regression found clinical stage the only independent prognostic indicator for survival. CONCLUSION: Flow cytometric DNA content analysis is being used more frequently in the management of different malignant tumors. Our study shows that DNA content analysis is useful in determining the prognosis and survival outcomes in cervical carcinomas and may aid in predicting outcome to certain types of treatment regimens.
Aneuploidy↗
Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5' nuclease quantitative reverse transcription-polymerase chain reaction.
Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.
Base Sequence↗
Genetic analysis of multiple loci in microsamples of fixed paraffin-embedded tissue.
Molecular analysis of alterations in genomic DNA is essential for understanding mechanisms by which chemical agents induce or modify tumor development. The assessment of microsatellite polymorphisms, loss of heterozygosity, mutations, and gene rearrangement allows specific comparisons of tumors to premalignant lesions or normal tissue or between similar tumors seen in laboratory species and humans. Utilization of these techniques is frequently limited by minute quantities of available tissue, often restricted to small formalin-fixed tumors or biopsies in paraffin blocks. To address these limitations, we have combined recently developed methodologies for selective recovery, amplification, and analysis of DNA. These techniques provide sufficient materials of high quality for analysis of DNA alterations in microscale amounts of starting material. By combining whole genome amplification through primer extension preamplification with locus-specific heminested PCR, we are able to analyze multiple genetic loci from as little as 1 mm2 of a 3-micron-thick formalin-fixed paraffin section. From 10 to greater than 100 loci can be analyzed per tissue section, and locus-specific PCR products may be further evaluated by a variety of techniques (e.g., SSCP, sequencing). Integrating these methodologies into situations where evaluation of very small tissue samples is necessary provides a powerful approach for elucidating molecular events that may be causally related to chemically induced cellular transformation and tumorigenesis.
Animals↗
Malignant histiocytosis. Immunohistochemical characterization on paraffin embedded tissue.
Clinically, malignant histiocytosis is a malignant neoplasia with poor prognosis. Diseased are lymphnodes (especially cervical nodes), liver, spleen and bones. Few cases become leukemic. The cells show characteristic pale roundish, often indented nuclei, without large nucleoli and with abundant ill-defined cytoplasm. Phagocytosis of erythrocytes and leukocytes, as well as, hemosiderin deposits may serve as indicators for histiocytic, respectively macrophagic qualities. On touch preparation, tumor cells previously had been marked by acid phosphatase and non-spevific esterase, as being histiocytic. - A comparable marking could be carried out on paraffin embedded material with lysozyme (muramidase) and alpha1-antichymotrypsin, by the indirect immuno-peroxidase technique. No correlation could be proven between any special shape of tumor cells or between different grades of cellular atypism and presence or absence of the immunohistochemical reaction. The reaction with lysozyme and alpha1-antichymotrypsin was also tested in other tumors and was found to be positive in a variety of different tumor cells showing degenerative changes, respectively necrobiosis. - But lysozyme and alpha1-antichymotrypsin are markers characteristically found in histiocytes, respectively histiocytic tumor cells. They are apparently less distinct in MH with a larger number of immature histiocytic tumor cells.
Adolescent↗
X-ray microanalysis of calcium containing organelles in resin embedded tissue.
The localization of calcium in cell organelles at the electron microscope level is often achieved through cytochemical techniques, and verified by X-ray microanalysis. Various methods have been used to cytochemically detect calcium or calcium-binding sites: calcium loading, calcium substitution by strontium, barium, or even lead, and calcium precipitation by oxalate, phosphate, fluoride, or pyroantimonate. Their results may have heuristic value, particularly in preliminary studies of poorly known cell types. A complementary and more physiological approach is offered by quantitative measurement of the total calcium content of organelles after cryofixation. Resin embedding is less demanding than cryomicrotomy and gives better images: it can be used after cryosubstitution in the presence of oxalic acid. This technique was tested, and applied to several cell types.
Acrylic Resins↗
Detection of mycobacterial DNA in formalin-fixed, paraffin-embedded tissue specimens by duplex polymerase chain reaction: application to histopathologic diagnosis.
Granuloma is a chronic inflammatory process associated with noninfectious agents or infectious diseases such as tuberculosis. Determination of the causative agent might be occasionally difficult in histopathologic sections. In this study, we examined 60 specimens of granuloma or inflammatory lesions that were originally diagnosed as 51 cases of granulomatous inflammation, 6 of leprosy, and 3 of atypical mycobacteriosis. The diagnoses in the last two categories were made both histologically and clinically. All of the sections and DNA were prepared from formalin-fixed, paraffin-embedded blocks. Histopathologic and immunohistochemical findings were compared with the results of duplex polymerase chain reaction (PCR) using two primers to amplify mycobacterial-common 383-base pair (bp) DNA and Mycobacterium tuberculosis-complex-specific 240-bp DNA. Six samples of leprosy and three of atypical mycobacteriosis showed the 383-bp but not the 240-bp band. Among the 51 specimens of granulomatous inflammations, nine showed no band of even the beta-globin, the cases being excluded from this analysis. The 42 specimens of granulomatous inflammation were subdivided into three categories by PCR: (1) 383- and 240-bp positive; (2) 383-bp positive and 240-bp negative; and (3) both negative. Category 1 included 32 specimens (76.2%), being considered as tuberculosis. One specimen was classified into Category 2, indicating possible atypical mycobacterium. Category 3 included nine specimens, composed of five of sarcoidosis and four other agent-induced granulomas, when compared with histologic and clinical findings. These findings indicate that the PCR assay using DNA extracted from paraffin-embedded materials provides useful information to differentiate tuberculosis from other type of granulomas.
DNA Primers↗
Southwestern histochemistry as a molecular histochemical tool for analysis of expression of transcription factors: application to paraffin-embedded tissue sections.
Southwestern histochemistry is used to localize transcription regulatory factors that bind to specific sequences of DNA and regulate the transcriptional activity of the genes, using a haptenized double-stranded DNA. This method has been developed in fresh frozen sections, but it was difficult to detect a specific signal in paraffin sections. In this review, we focus on the use of Southwestern histochemistry in paraffin sections. To detect a reliable signal in paraffin sections, we autoclaved or microwaved the sample before incubation with probe DNA. As a model system, we assessed estrogen receptor (ER) in paraffin sections of mouse ovary. ER was detected by Southwestern histochemistry in autoclaved or microwaved paraffin sections, and no signal was detected without such pretreatment. The ER staining pattern detected by Southwestern histochemistry was well consistent with that of ER alpha and ER beta detected by immunohistochemistry. Next, we analyzed the expression of cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB) in regenerating livers of rats and mice. CREB was detected in hepatocyte nuclei in autoclaved paraffin sections by Southwestern histochemistry. Immunohistochemistry of CREB displayed homogeneous staining in hepatocyte nuclei, while the signal detected by Southwestern histochemistry showed a heterogeneous speckled pattern in the nuclei. CREB expression increased prior to the induction of proliferating cell nuclear antigen (PCNA). Because the PCNA gene has a cAMP-responsive element in its promoter, it can be suggested that CREB may activate the PCNA gene and lead to the induction of hepatocyte DNA synthesis. Our results indicate that autoclave or microwave treatment is very useful for Southwestern histochemistry in paraffin-embedded sections.
Animals↗
Microwave irradiation of paraffin-embedded tissue sensitizes the TUNEL method for in situ detection of apoptotic cells.
Apoptosis is a morphologically distinct form of programmed cell death that plays an important role in the growth regulation of a variety of tissues and also in the elimination of self-reacting immunocompetent cells. Several techniques for the qualitative and quantitative detection of this process have been established; recently, an in situ nick end-labelling technique based on the detection of DNA fragmentation, which is a molecular characteristic of apoptotic cell death, was described. Applying this method to paraffin sections of human tissues, sensitivity was observed to be inconsistently low with regard to the expected number of apoptotic cells. In the present study we show that irradiation of the tissue sections in 10 mM citrate buffer, pH 6.0, by microwaves at 750 W considerably enhances the sensitivity of this nick end-labelling technique.
Apoptosis↗
Immunocytochemical detection of cyclin A and cyclin D in formalin-fixed, paraffin-embedded tissues: novel, pertinent markers of cell proliferation.
Cyclin proteins in association with cyclin-dependent protein kinase subunits represent a new class of potentially oncogenic serine/threonine protein kinases that function to execute critical cell cycle transitions in all eukaryotic cells. Characterized by dramatic fluctuations in abundance, which occur in accordance with the periodicity of the cell cycle, the expression patterns of specific cyclins provide a unique and relevant indicator of cellular activation and cell cycle progression. In this study, we introduce a series of monospecific antibodies that are selective for human cyclin A and cyclin D, respectively, and we assess the feasibility of utilizing these reagents for immunocytochemical analyses. Conditions were optimized for detecting cyclin A and cyclin D in formalin-fixed, paraffin-embedded sections of the postnatal human palatine tonsil, in which normal cell proliferation is well characterized. Subsequent studies demonstrated the performance of these antibodies in the examination of pediatric bone tumors, in which decalcification methods are additionally performed. In both cases, the proliferative status of individual cells was monitored with an exceedingly high degree of resolution. Taken together with the available biochemical data, the results of these studies reveal a novel means of characterizing the proliferative status of normal as well as neoplastic tissues. The demonstrated utility of these immunochemical reagents will potentially facilitate retrospective studies aimed at examining cell proliferation in a wide variety of archival histopathologic specimens.
Antibodies↗
Markers for dysplasia of the upper aerodigestive tract. Suprabasal expression of PCNA, p53, and CK19 in alcohol-fixed, embedded tissue.
Recognition of premalignant lesions in the oral epithelium has the potential to increase survival rates for squamous cell carcinoma of the oral cavity. It has previously been reported that cytokeratin 19 (CK19), a 40-kd epithelial cytoskeletal protein within the suprabasal squamous epithelium, is a specific marker of moderate-to-severe dysplasia and carcinoma in situ in oral cavity squamous epithelium. In contrast, normal epithelium and hyperplastic lesions reportedly express CK19 only in the basal layer if at all. The authors chose to test and extend this hypothesis by studying suprabasal CK19 expression and dysplasia of the oral cavity and upper aerodigestive tract in paraffin-embedded specimens that had been fixed in alcohol, a superior fixative for the preservation of cytokeratins. The authors examined 56 alcohol-fixed, paraffin-embedded specimens including 37 from the oral cavity, using two antibodies specific for CK19 (Ks19.1 and 4.62), an antibody to the nuclear proliferation marker, proliferating cell nuclear antigen (PCNA) (19A2), and an antibody to the putative tumor suppressor gene, p53 (pAb1801). The lesions were classified as normal, hyperplasia, mild dysplasia, moderate dysplasia, severe dysplasia/carcinoma in situ, or invasive squamous cell carcinoma, following standard histologic criteria. Immunocytochemically stained sections were scored for the presence or absence of suprabasal CK19, suprabasal PCNA, and p53 positivity, regardless of location. The immunostaining patterns of the two anti-CK19 antibodies were essentially equivalent. Except for one laryngeal specimen, normal epithelium, when positive, showed CK19 expression only in scattered cells throughout the basal layer. Proliferating cell nuclear antigen-positive nuclei were found exclusively in the basal layer. In areas of hyperplasia, CK19 immunostaining was absent or confined to the basal layer in 20 of 38 specimens and was expressed in suprabasal cells in 18 of 38 hyperplastic specimens. Proliferating cell nuclear antigen immunostaining in all cases of hyperplasia was limited to the basal layer. Severe dysplasia and carcinoma in situ showed suprabasal CK19 staining in six of nine specimens and no CK19 staining in three of nine specimens. In contrast, suprabasal PCNA immunostaining was found in all dysplasia and carcinoma in situ cases. p53 expression was detected in three of nine severe dysplasia/CIS specimens and was immunocytochemically undetectable in all normal, hyperplasia, and mild to moderate dysplasia specimens. The authors conclude that suprabasal CK19 expression is neither a sensitive nor a specific marker of premalignancy in oral epithelium and cannot be used to distinguish hyperplasia from dysplasia. In contrast, a strong correlation between suprabasal expression of PCNA, a marker for proliferating cells, and dysplasia/carcinoma in situ was evident.(ABSTRACT TRUNCATED AT 400 WORDS)
Antigens, Neoplasm↗
Molecular diagnosis of mantle cell lymphoma in paraffin-embedded tissue.
Mantle cell lymphoma (formerly known as intermediate lymphocytic lymphoma, diffuse centrocytic lymphoma, or diffuse small cleaved lymphoma) is one of the small cell non-Hodgkin lymphoma entities that is clinically more aggressive than small lymphocytic lymphoma, and needs to be separated from it. Mantle cell lymphoma is strongly associated with the t(11;14) chromosomal translocation that rearranges the bcl-1 oncogene (PRAD-1 gene) and immunoglobulin heavy chain gene. In this study, we developed a nested polymerase chain reaction system to evaluate the t(11;14) translocation. The study material consisted of 10 mantle cell lymphomas fulfilling the criteria suggested by other authors (P. M. Banks et al. Surg Pathol 16:637, 1992). A novel nested polymerase chain reaction system was used to evaluate the bcl-1 breaks in the major translocation cluster using two successive polymerase chain reaction amplifications. This reaction yielded a background-free single product of the size of 200 to 300 base pairs in four of 10 mantle cell lymphomas. The identity of the product from the nested polymerase chain reaction was confirmed by Southern blotting followed by hybridization with a specific probe. The amplification products were also evaluated by Sst-I, Alu-I, Dde-I, and Ita-I restriction enzymes and showed different patterns of digestion reflecting individual differences between the MTC/ IgH junctions. A selection of other low-grade lymphomas, including lymphocytic, follicular, and mucosa-associated lymphoid tissue lymphoma, and hairy cell leukemia and 29 hyperplastic lymph nodes were negative. This nested polymerase chain reaction system for the t(11;14) translocation involving major translocation cluster offers a convenient specific identification for mantle cell lymphoma. However, this test has a limited diagnostic power because only about half of the mantle cell lymphomas show the bcl-1 breaks in the major translocation cluster. The test performs well in formaldehyde-fixed and paraffin-embedded material, allowing the study of large numbers of retrospective cases of mantle cell lymphomas.
Aged↗
Detection of fusion genes in sarcomas using paraffin-embedded tissues.
Many sarcomas are characterized by specific recurrent chromosomal translocations resulting in gene fusions. The genes involved in almost all of these translocations have been cloned, greatly changing sarcoma diagnosis. At the biological level, these chromosomal translocations produce highly specific fusion genes that encode key molecules for tumor development. The clinical correlation between these translocation-derived genetic markers and discrete histopathological entities has been remarkable. Today, detection of fusion genes plays a crucial role in the diagnosis of sarcomas that harbor atypical clinical or pathological presentations. The focus of this brief review is the recent impact that cytogenetic and molecular detection of these translocations has had on sarcoma diagnosis using paraffin-embedded sections.
Animals↗
Multiplex RT-PCR for the detection of common BCR-ABL fusion transcripts in paraffin-embedded tissues from patients with chronic myeloid leukemia and acute lymphoblastic leukemia.
Diagnosis of chronic myeloid leukemia and acute lymphoblastic leukemia requires the investigation of the Philadelphia chromosome translocation t(9;22) or the molecular detection of BCR-ABL fusion transcripts. Determination of the type of fusion transcript is crucial for quantitative molecular monitoring the course of the disease during treatment. Histopathologists, who usually use formalin-fixed tissues, may be confronted with the need to investigate the BCR-ABL rearrangement when evaluating tumor forming infiltrates and bone marrow trephines from patients presenting with chronic myeloproliferative disorders. Therefore, we have established a one-tube multiplex RT-PCR for the detection of common BCR-ABL fusion transcripts (b2a2, b3a2, e1a2) in routinely processed tissues and bone marrow trephines with respect to the inevitable fragmentation of ribonucleic acids in these specimens. RT-PCR products allow distinct and unequivocal differentiation of the underlying fusion in either the Major- or minor-breakpoint cluster region. Detection of BCR-ABL fusion transcripts by multiplex RT-PCR in routinely processed and fixed tissues is a time- and cost-sparing tool for definite diagnosis of typical chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia.
Bone Marrow Cells↗