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Sample records for formalin-fixed paraffin embedded

  1. Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue

    DEFF Research Database (Denmark)

    Laub Petersen, Bodil; Zeuthen, Mette Christa; Pedersen, Sanni

    2004-01-01

    , such as quantitation of signals as in triploidy, it is possible to isolate nuclei from paraffin-embedded tissue. However, using formalin-fixed paraffin-embedded tissue, either in thin sections or as isolated nuclei, one encounters a range of technical problems, paralleling those met in immunohistochemistry. Variations...... nuclei and tissue sections from formalin-fixed, paraffin-embedded tissue....

  2. MicroRNA Expression in Formalin-fixed Paraffin-embedded Cancer Tissue

    DEFF Research Database (Denmark)

    Boisen, Mogens Karsbøl; Dehlendorff, Christian; Linnemann, Dorte

    2015-01-01

    BACKGROUND: Archival formalin-fixed paraffin-embedded (FFPE) cancer tissue samples are a readily available resource for microRNA (miRNA) biomarker identification. No established standard for reference miRNAs in FFPE tissue exists. We sought to identify stable reference miRNAs for normalization...

  3. Evaluating Quality of Aged Archival Formalin-Fixed Paraffin-Embedded Samples for RNA-Sequencing

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    Archival formalin-fixed paraffin-embedded (FFPE) samples offer a vast, untapped source of genomic data for biomarker discovery. However, the quality of FFPE samples is often highly variable, and conventional methods to assess RNA quality for RNA-sequencing (RNA-seq) are not infor...

  4. PrPSc detection in formalin-fixed paraffin-embedded tissue by ELISA

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    Formalin-fixed paraffin-embedded tissue is regularly employed in the diagnosis of transmissible spongiform encephalopathies (TSE) by immunohistochemistry (IHC), the standard by which all other TSE diagnostic protocols are judged. While IHC affords advantages over diagnostic approaches that typically...

  5. Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue

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    Davis, A. Sally; Richter, Anke; Becker, Steven; Moyer, Jenna E.; Sandouk, Aline; Skinner, Jeff

    2014-01-01

    Tissue autofluorescence frequently hampers visualization of immunofluorescent markers in formalin-fixed paraffin-embedded respiratory tissues. We assessed nine treatments reported to have efficacy in reducing autofluorescence in other tissue types. The three most efficacious were Eriochrome black T, Sudan black B and sodium borohydride, as measured using white light laser confocal Λ2 (multi-lambda) analysis. We also assessed the impact of steam antigen retrieval and serum application on human tracheal tissue autofluorescence. Functionally fitting this Λ2 data to 2-dimensional Gaussian surfaces revealed that steam antigen retrieval and serum application contribute minimally to autofluorescence and that the three treatments are disparately efficacious. Together, these studies provide a set of guidelines for diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. Additionally, these characterization techniques are transferable to similar questions in other tissue types, as demonstrated on frozen human liver tissue and paraffin-embedded mouse lung tissue fixed in different fixatives. PMID:24722432

  6. Proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies

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    Bjerg Bennike, Tue; Kastaniegaard, Kenneth; Padurariu, Simona

    2016-01-01

    Large repositories of well characterized RNAlater preserved samples and formalin-fixed, paraffin-embedded samples have been generated worldwide. However, the impact on the proteome of the preservation methods remain poorly described. Therefore, we analyzed the impact on the proteome of preserving...... samples in RNAlater, and by formalin-fixation, paraffin-embedding on human soft tissue, using directly frozen samples as a control ("Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples" [1]). We here report the data from the analysis...

  7. PrPSc detection in formalin-fixed paraffin-embedded tissue by ELISA

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    Nicholson Eric M

    2011-10-01

    Full Text Available Abstract Background Formalin-fixed paraffin-embedded tissue is regularly employed in the diagnosis of transmissible spongiform encephalopathies (TSE by immunohistochemistry (IHC, the standard by which all other TSE diagnostic protocols are judged. While IHC affords advantages over diagnostic approaches that typically utilize fresh or frozen tissue, such as Western blot and ELISA, the process of fixing, staining, and analyzing individual sections by hand does not allow for rapid or high throughput screening. However, preservation of tissues in formalin is not dependent upon the availability of refrigeration. Findings Formalin-fixed paraffin-embedded tissues from TSE transmission studies of scrapie in sheep, chronic wasting disease in white-tailed deer or transmissible mink encephalopathy in cattle were cut at 5 μm thickness. Samples containing the tissue equivalent of as little as one 5 μm section can be used to readily discriminate positive from negative samples. Conclusions This approach cannot replace IHC but may be used along with IHC as both a more rapid and readily high throughput screen where fresh or frozen tissues are not available or impractical.

  8. Investigation of archived formalin-fixed paraffin-embedded pancreatic tissue with whole-genome gene expression microarray

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    Michelsen, Nete Vinstrup; Brusgaard, Klaus; Tan, Qihua

    2011-01-01

    The use of formalin-fixed, paraffin-embedded (FFPE) tissue overcomes the most prominent issues related to research on relatively rare diseases: limited sample size, availability of control tissue, and time frame. The use of FFPE pancreatic tissue in GEM may be especially challenging due to its very...

  9. Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples

    DEFF Research Database (Denmark)

    Bennike, Tue Bjerg; Kastaniegaard, Kenneth; Padurariu, Simona

    2016-01-01

    Large biobanks exist worldwide containing formalin-fixed, paraffin-embedded samples and samples stored in RNAlater. However, the impact of tissue preservation on the result of a quantative proteome analysis remains poorly described.Human colon mucosal biopsies were extracted from the sigmoideum...

  10. Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples.

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    Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here we evaluated transcriptomic dose responses us...

  11. A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER) for immunohistochemistry in formalin fixed paraffin-embedded tissue

    DEFF Research Database (Denmark)

    Paulsen, I M S; Dimke, H; Frische, S

    2015-01-01

    Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin...... of dewaxing in xylene. In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin fixed paraffin-embedded tissue....

  12. Retrospective analysis of sheep scrapie by western blotting with formalin-fixed paraffin-embedded (FFPE) tissues.

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    Dorj, Gantsetseg; Okada, Hiroyuki; Miyazawa, Kohtaro; Masujin, Kentaro; Kimura, Kumiko; Mohri, Shirou; Yokoyama, Takashi

    2012-09-01

    An abnormal isoform of prion protein (PrP(Sc)) was extracted from formalin-fixed paraffin-embedded (FFPE) tissues from sheep and analyzed by western blotting. PrP(Sc) immunoreactivity against anti-PrP monoclonal antibody T2, which recognizes discontinuous PrP sequences, differed amongst individual scrapie sheep cases. This may reflect structural differences in PrP(Sc) that have been formalin-fixed prior to their extraction. This study indicates that western blotting by using FFPE tissues is useful for the retrospective analysis of transmissible spongiform encephalopathies in which only formalin-fixed samples are available and in conducting transmissible spongiform encephalopathies surveillance where freezing system is insufficient.

  13. Automated array-CGH optimized for archival formalin-fixed, paraffin-embedded tumor material

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    Nederlof Petra M

    2007-03-01

    Full Text Available Abstract Background Array Comparative Genomic Hybridization (aCGH is a rapidly evolving technology that still lacks complete standardization. Yet, it is of great importance to obtain robust and reproducible data to enable meaningful multiple hybridization comparisons. Special difficulties arise when aCGH is performed on archival formalin-fixed, paraffin-embedded (FFPE tissue due to its variable DNA quality. Recently, we have developed an effective DNA quality test that predicts suitability of archival samples for BAC aCGH. Methods In this report, we first used DNA from a cancer cell-line (SKBR3 to optimize the aCGH protocol for automated hybridization, and subsequently optimized and validated the procedure for FFPE breast cancer samples. We aimed for highest throughput, accuracy, and reproducibility applicable to FFPE samples, which can also be important in future diagnostic use. Results Our protocol of automated array-CGH on archival FFPE ULS-labeled DNA showed very similar results compared with published data and our previous manual hybridization method. Conclusion This report combines automated aCGH on unamplified archival FFPE DNA using non-enzymatic ULS labeling, and describes an optimized protocol for this combination resulting in improved quality and reproducibility.

  14. Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue.

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    Potluri, Keerti; Mahas, Ahmed; Kent, Michael N; Naik, Sameep; Markey, Michael

    2015-10-01

    As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and "amplifiability" for aCGH and other downstream applications. Phenol-chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. DNA extraction from fresh-frozen and formalin-fixed, paraffin-embedded human brain tissue.

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    Wang, Jian-Hua; Gouda-Vossos, Amany; Dzamko, Nicolas; Halliday, Glenda; Huang, Yue

    2013-10-01

    Both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases, especially neurodegenerative disorders. To identify the optimal method for DNA extraction from human brain tissue, we compared methods on differently-processed tissues. Fragments of LRRK2 and MAPT (257 bp and 483 bp/245 bp) were amplified for evaluation. We found that for FFPE samples, the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method (successful DNA extraction from 76% versus 33% of samples). PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples. In the fresh-frozen samples, the DNA extraction success rate was 100% using either a commercial kit (QIAamp DNA Micro) or a laboratory-based method (sample boiling in 0.1 mol/L NaOH, followed by proteinase K digestion, and then DNA extraction using Chelex-100) regardless of PCR amplicon length or tissue storage time. Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples, fresh brain tissue is recommended for DNA extraction in future neuropathological studies.

  16. Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas.

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    Fu, Zongming; Yan, Kun; Rosenberg, Avraham; Jin, Zhicheng; Crain, Barbara; Athas, Grace; Heide, Richard S Vander; Howard, Timothy; Everett, Allen D; Herrington, David; Van Eyk, Jennifer E

    2013-04-01

    Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics. Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with MS. Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5-fold) or at RT (8.3-fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long-term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta. Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies.

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    Shi, Shan-Rong; Taylor, Clive R; Fowler, Carol B; Mason, Jeffrey T

    2013-04-01

    Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Molecular Markers for Prostate Cancer in Formalin-Fixed Paraffin-Embedded Tissues

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    Tamara Sequeiros

    2013-01-01

    Full Text Available Prostate cancer (PCa is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.

  19. Detection of alpha human papillomaviruses in archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens.

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    Kocjan, Boštjan J; Hošnjak, Lea; Poljak, Mario

    2016-03-01

    Formalin-fixed, paraffin-embedded (FFPE) tissue specimens stored in pathology departments worldwide are an invaluable source for diagnostic purposes when fresh clinical material is unavailable as well as for retrospective molecular and epidemiological studies, especially when dealing with rare clinical conditions for which prospective collection is not feasible. Accurate detection of HPV infection in these specimens is particularly challenging because nucleic acids are often degraded and therefore, not suitable for amplification of larger fragments of the viral genome or viral gene transcripts. This review provides a brief summary of molecular methods for detecting alpha-HPV DNA/RNA in FFPE tissue specimens. We specifically address the key procedural and environmental factors that have the greatest impact on the quality of nucleic acids extracted from FFPE tissue specimens, and describe some solutions that can be used to increase their integrity and/or amplifiability. Moreover, commonly used methods for HPV DNA/RNA detection in FFPE tissue specimens are presented and discussed, focusing on studies using polymerase chain reaction as an HPV detection method and published after 1999. Finally, we briefly summarize our 22 years of experience with HPV detection in FFPE tissue specimens. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks.

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    Guyard, Alice; Boyez, Alice; Pujals, Anaïs; Robe, Cyrielle; Tran Van Nhieu, Jeanne; Allory, Yves; Moroch, Julien; Georges, Odette; Fournet, Jean-Christophe; Zafrani, Elie-Serge; Leroy, Karen

    2017-10-01

    Formalin-fixed paraffin-embedded (FFPE) tissue blocks are widely used to identify clinically actionable molecular alterations or perform retrospective molecular studies. Our goal was to quantify degradation of DNA occurring during mid to long-term storage of samples in usual conditions. We selected 46 FFPE samples of surgically resected carcinomas of lung, colon, and urothelial tract, of which DNA had been previously extracted. We performed a second DNA extraction on the same blocks under identical conditions after a median period of storage of 5.5 years. Quantitation of DNA by fluorimetry showed a 53% decrease in DNA quantity after storage. Quantitative PCR (qPCR) targeting KRAS exon 2 showed delayed amplification of DNA extracted after storage in all samples but one. The qPCR/fluorimetry quantification ratio decreased from 56 to 15% after storage (p DNA analyzable by qPCR represented only 11% of the amount obtained at first extraction. Maximal length of amplifiable DNA fragments assessed with a multiplex PCR was reduced in DNA extracted from stored tissue, indicating that DNA fragmentation had increased in the paraffin blocks during storage. Next-generation sequencing was performed on 12 samples and showed a mean 3.3-fold decrease in library yield and a mean 4.5-fold increase in the number of single-nucleotide variants detected after storage. In conclusion, we observed significant degradation of DNA extracted from the same FFPE block after 4 to 6 years of storage. Better preservation strategies should be considered for storage of FFPE biopsy specimens.

  1. Analytical validation of a melanoma diagnostic gene signature using formalin-fixed paraffin-embedded melanocytic lesions.

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    Warf, M Bryan; Flake, Darl D; Adams, Doug; Gutin, Alexander; Kolquist, Kathryn A; Wenstrup, Richard J; Roa, Benjamin B

    2015-01-01

    These studies were to validate the analytical performance of a gene expression signature that differentiates melanoma and nevi, using RNA expression from 14 signature genes and nine normalization genes that generates a melanoma diagnostic score (MDS). Formalin-fixed paraffin-embedded melanocytic lesions were evaluated in these studies. The overall SD of the assay was determined to be 0.69 MDS units. Individual amplicons within the signature had an average amplification efficiency of 92% and a SD less than 0.5 CT. The MDS was reproducible across a 2000-fold dilution range of input RNA. Melanin, an inhibitor of PCR, does not interfere with the signature. These studies indicate this signature is robust and reproducible and is analytically validated on formalin-fixed paraffin-embedded melanocytic lesions.

  2. Enrichment of PrPSc in formalin-fixed, paraffin-embedded tissues prior to analysis by Western blot.

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    Nicholson, Eric M

    2011-07-01

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis, with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past, these approaches required formalin-fixed, paraffin-embedded tissue and fresh or frozen tissue, respectively; however, methods have been developed that allow for use of fixed tissue for Western blot. The present study describes a method of enriching PrP(Sc) in formalin-fixed, paraffin-embedded tissues prior to Western blot analysis for the detection of PrP(Sc). With this modified procedure, 5 times the previously reported sample size may be used for analysis, greatly enhancing the sensitivity of this procedure.

  3. Plasma cell lesions of the head and neck: immunofluorescent determination of clonality from formalin-fixed, paraffin-embedded tissue.

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    Regezi, J A; Zarbo, R J; Keren, D F

    1983-12-01

    An immunofluorescent technique was used on formalin-fixed, paraffin-embedded tissue to characterize and diagnose plasma cell lesions of the head and neck. Infiltrates were classified as either monoclonal or polyclonal with rhodamine and fluorescein staining of light chains and heavy chains. In the cases in which histopathologic diagnoses were relatively certain, immunofluorescence provided good correlation. In those cases in which histopathologic diagnoses were equivocal, immunofluorescence distinguished between reactive and neoplastic infiltrates through the determination of clonality of the infiltrates.

  4. Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing

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    Marosy, Beth A.; Craig, Brian D.; Hetrick, Kurt N.; Witmer, P. Dane; Ling, Hua; Griffith, Sean M.; Myers, Ben; Ostrander, Elaine A.; Stanford, Janet L.; Brody, Lawrence C.; Doheny, Kimberly F.

    2016-01-01

    This unit describes a protocol for generating exome enriched sequencing libraries using DNA extracted from Formalin Fixed Paraffin Embedded (FFPE) samples. Utilizing commercially available kits, we present a low input FFPE workflow starting with 50ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution targeted selection for exons, followed by sequencing using the Illumina next generation short read sequencing platform. PMID:28075488

  5. Elevated Pressure Improves the Extraction and Identification of Proteins Recovered from Formalin-Fixed, Paraffin-Embedded Tissue Surrogates

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    Fowler, Carol B.; Chesnick, Ingrid E.; Moore, Cedric D.; O'Leary, Timothy J.; Mason, Jeffrey T.

    2010-01-01

    Background Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozy...

  6. An Optimized Method of Metabolite Extraction from Formalin-Fixed Paraffin-Embedded Tissue for GC/MS Analysis.

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    Wojakowska, Anna; Marczak, Łukasz; Jelonek, Karol; Polanski, Krzysztof; Widlak, Piotr; Pietrowska, Monika

    2015-01-01

    Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffin embedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material.

  7. Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue.

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    Danielle Mercatante Carrick

    Full Text Available Next Generation Sequencing (NGS technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER registries Residual Tissue Repositories (RTR was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES. Following DNA extraction, 58 of 59 specimens (98% yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10% and lower average read depth (40x lower per 10 years, 95% CI: 18-60, although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59 of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.

  8. Buffered Romanowsky-Giemsa method for formalin fixed, paraffin embedded sections: taming a traditional stain.

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    Stefanović, D; Samardžija, G; Redžek, A; Arnaut, M; Nikin, Z; Stefanović, M

    2017-01-01

    Romanowsky-Giemsa (RG) stains were devised during the 19th century for identifying plasmodia parasites in blood smears. Later, RG stains became standard procedures for hematology and cytology. Numerous attempts have been made to apply RG staining to formalin-fixed paraffin-embedded tissue sections, with varied success. Most published work on this topic described RG staining methods in which sections were overstained, then subjected to acid differentiation; unfortunately, the differentiation step often caused inconsistent staining outcomes. If staining is performed under optimal conditions with control of dye concentration, pH, solution temperature and staining time, no differentiation is required. We used RG and 0.002 M buffer, pH 42, for staining and washing sections. All steps were performed at room temperature. After staining and air drying, sections were washed in 96-100% ethanol to remove extraneous stain. Finally, sections were washed in xylene and mounted using DPX. Staining results were similar to routine hemalum and eosin (H & E) staining. Nuclei were blue; intensity depended largely on chromatin density. RNA-rich sites were purple. Collagen fibers, keratin, muscle cells, erythrocytes and white matter of the central nervous system were stained pinkish and reddish hues. Cartilage matrix, mast cell granules and areas of myxomatous degeneration were purple. Sulfate-rich mucins were stained pale blue, while those lacking sulfate groups were unstained. Deposits of hemosiderin, lipofuscin and melanin were greenish, and calcium deposits were blue. Helicobacter pylori bacteria were violet to purple. The advantages of the method are its close similarity to H & E staining and technical simplicity. Hemosiderin, H. pylori, mast cell granules, melanin and specific granules of different hematopoietic cells, which are invisible or barely distinguishable by H & E staining, are visualized. Other advantages over previous RG stains include shorter staining time and avoidance

  9. Whole-genome gene expression profiling of formalin-fixed, paraffin-embedded tissue samples.

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    Craig April

    2009-12-01

    Full Text Available We have developed a gene expression assay (Whole-Genome DASL, capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE specimens.We demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2 approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2 approximately 0.86 and R(2 approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2>0.98 with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2 approximately 0.92, with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2 approximately 0.80, while still maintaining a correlation of R(2 approximately 0.75 with standard FFPE inputs (200 ng.Taken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material or when minimally invasive procedures are performed (e.g. biopsied specimens.

  10. Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next-Generation Sequencing.

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    Marosy, Beth A; Craig, Brian D; Hetrick, Kurt N; Witmer, P Dane; Ling, Hua; Griffith, Sean M; Myers, Benjamin; Ostrander, Elaine A; Stanford, Janet L; Brody, Lawrence C; Doheny, Kimberly F

    2017-01-11

    This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  11. Biomedical analysis of formalin-fixed, paraffin-embedded tissue samples: The Holy Grail for molecular diagnostics.

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    Donczo, Boglarka; Guttman, Andras

    2018-04-02

    More than a century ago in 1893, a revolutionary idea about fixing biological tissue specimens was introduced by Ferdinand Blum, a German physician. Since then, a plethora of fixation methods have been investigated and used. Formalin fixation with paraffin embedment became the most widely used types of fixation and preservation method, due to its proper architectural conservation of tissue structures and cellular shape. The huge collection of formalin-fixed, paraffin-embedded (FFPE) sample archives worldwide holds a large amount of unearthed information about diseases that could be the Holy Grail in contemporary biomarker research utilizing analytical omics based molecular diagnostics. The aim of this review is to critically evaluate the omics options for FFPE tissue sample analysis in the molecular diagnostics field. Copyright © 2018. Published by Elsevier B.V.

  12. Multicenter Evaluation of a Novel Automated Rapid Detection System of BRAF Status in Formalin-Fixed, Paraffin-Embedded Tissues.

    Science.gov (United States)

    Schiefer, Ana-Iris; Parlow, Laura; Gabler, Lisa; Mesteri, Ildiko; Koperek, Oskar; von Deimling, Andreas; Streubel, Berthold; Preusser, Matthias; Lehmann, Annika; Kellner, Udo; Pauwels, Patrick; Lambin, Suzan; Dietel, Manfred; Hummel, Michael; Klauschen, Frederick; Birner, Peter; Möbs, Markus

    2016-05-01

    The mutated BRAF oncogene represents a therapeutic target in malignant melanoma. Because BRAF mutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAF mutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAF V600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAF V600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  13. Progesterone receptor isoform analysis by quantitative real-time polymerase chain reaction in formalin-fixed, paraffin-embedded canine mammary dysplasias and tumors

    DEFF Research Database (Denmark)

    Guil-Luna, S.; Stenvang, Jan; Brünner, Nils

    2014-01-01

    and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign...... in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors...

  14. The effects of age-in-block on RNA-seq analysis of archival formalin-fixed paraffin-embedded (FFPE) samples

    Science.gov (United States)

    Archival samples represent a vast resource for identification of chemical and pharmaceutical targets. Previous use of formalin-fixed paraffin-embedded (FFPE) samples has been limited due to changes in RNA introduced by fixation and embedding procedures. Recent advances in RNA-seq...

  15. Molecular identification of Coccidioides immitis in formalin-fixed, paraffin-embedded (FFPE) tissues from a Colombian patient.

    Science.gov (United States)

    Canteros, Cristina E; Vélez H, Alejandro; Toranzo, Adriana I; Suárez-Alvarez, Roberto; Tobón O, Ángela; Jimenez A, María del Pilar; Restrepo M, Ángela

    2015-06-01

    Coccidioides immitis and C. posadasii are the etiologic agents of coccidioidomycosis, an endemic fungal disease of the Americas. In Colombia, this mycosis is uncommon, and only five cases, two of them imported, have been documented.By means of DNA sequencing, C. immitis was identified in formalin-fixed, paraffin-embedded archival tissues samples from the 5th Colombian patient diagnosed in 1997. The patient was born in Pinto, Department of Magdalena, and had never visited other geographic regions, a reason to consider that the mycosis had been acquired locally.This species is primarily found in California although it has been occasionally reported in other geographic areas such as Mexico and Brazil. This is the first indigenous report of C. immitis-associated coccidioidomycosis in a Colombian patient. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.

  16. Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

    Science.gov (United States)

    Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P

    2016-02-01

    Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies. © 2016 The Histochemical Society.

  17. Development and independent validation of a prognostic assay for stage II colon cancer using formalin-fixed paraffin-embedded tissue.

    LENUS (Irish Health Repository)

    Kennedy, Richard D

    2011-12-10

    Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples.

  18. MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling

    DEFF Research Database (Denmark)

    Glud, M.; Klausen, M.; Gniadecki, R.

    2009-01-01

    surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we...

  19. Detection of Streptococcus suis by in situ hybridization, indirect immunofluorescence, and peroxidase-antiperoxidase assays in formalin-fixed, paraffin-embedded tissue sections from pigs

    DEFF Research Database (Denmark)

    Boye, Mette; Feenstra, Anne Avlund; Tegtmeier, Conny

    2000-01-01

    and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from...

  20. Comparative investigations of T cell receptor gamma gene rearrangements in frozen and formalin-fixed paraffin wax-embedded tissues by capillary electrophoresis

    DEFF Research Database (Denmark)

    Christensen, M; Funder, A D; Bendix, K

    2006-01-01

    AIM: To compare clonal T cell receptor gamma (TCRgamma) gene rearrangements in frozen and formalin-fixed paraffin wax-embedded (FFPE) tissue, using capillary electrophoresis for use in diagnostics, as T cell lymphomas may be difficult to diagnose by conventional methods. METHODS: The DNA for PCR...

  1. Implementation of formalin-fixed, paraffin-embedded cell line pellets as high-quality process controls in quality assessment programs for KRAS mutation analysis

    DEFF Research Database (Denmark)

    Dijkstra, Jeroen R; Opdam, Frank J M; Boonyaratanakornkit, Jerry

    2013-01-01

    . We assessed a novel synthetic control for formalin-fixed, paraffin-embedded (FFPE) tumor samples in a blind study conducted within nine laboratories across Europe. We show that FFPE material can, at least in part, mimic clinical samples and we demonstrate this control to be a valuable tool...

  2. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

    DEFF Research Database (Denmark)

    Tramm, Trine; Hennig, Guido; Kyndi, Marianne

    2013-01-01

    Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal...

  3. Mining the archives: a cross-platform analysis of gene expression profiles in archival formalin-fixed paraffin-embedded (FFPE) tissue.

    Science.gov (United States)

    Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for genomic research into the molecular basis of disease. However, use of FFPE samples in gene expression studies has been limited by technical challenges resulting from degradation...

  4. Matrix-comparative genomic hybridization from multicenter formalin-fixed paraffin-embedded colorectal cancer tissue blocks

    Directory of Open Access Journals (Sweden)

    Köhne Claus-Henning

    2007-04-01

    Full Text Available Abstract Background The identification of genomic signatures of colorectal cancer for risk stratification requires the study of large series of cancer patients with an extensive clinical follow-up. Multicentric clinical studies represent an ideal source of well documented archived material for this type of analyses. Methods To verify if this material is technically suitable to perform matrix-CGH, we performed a pilot study using macrodissected 29 formalin-fixed, paraffin-embedded tissue samples collected within the framework of the EORTC-GI/PETACC-2 trial for colorectal cancer. The scientific aim was to identify prognostic genomic signatures differentiating locally restricted (UICC stages II-III from systemically advanced (UICC stage IV colorectal tumours. Results The majority of archived tissue samples collected in the different centers was suitable to perform matrix-CGH. 5/7 advanced tumours displayed 13q-gain and 18q-loss. In locally restricted tumours, only 6/12 tumours showed a gain on 13q and 7/12 tumours showed a loss on 18q. Interphase-FISH and high-resolution array-mapping of the gain on 13q confirmed the validity of the array-data and narrowed the chromosomal interval containing potential oncogenes. Conclusion Archival, paraffin-embedded tissue samples collected in multicentric clinical trials are suitable for matrix-CGH analyses and allow the identification of prognostic signatures and aberrations harbouring potential new oncogenes.

  5. Detection of Mycobacterium tuberculosis complex in formalin-fixed, paraffin-embedded tissue specimens with necrotizing granulomatous inflammation by strand displacement amplification

    DEFF Research Database (Denmark)

    Johansen, Isik Somuncu; Thomsen, Vibeke Østergaard; Forsgren, Arne

    2004-01-01

    Rapid, reliable diagnosis of tuberculosis is essential to initiate correct treatment, avoid severe complications, and prevent transmission. Conventional microbiological methods may not be an option if samples are formalin-fixed and paraffin-embedded (FFPE) for histopathological examination....... With the demonstration of necrotizing granulomatous inflammation, tuberculosis becomes an important differential diagnosis, although it was not initially suspected. Following paraffin extraction, BDProbeTec ET strand displacement amplification for detection of Mycobacterium tuberculosis complex (MTC) was applied to 47...

  6. Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics.

    Science.gov (United States)

    Luebker, Stephen A; Koepsell, Scott A

    2016-01-01

    Urea based protein extraction of formalin-fixed paraffin-embedded (FFPE) tissue provides the most efficient workflow for proteomics due to its compatibility with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This study optimizes the use of urea for proteomic analysis of clinical FFPE tissue. A series of protein extraction conditions manipulating temperature and buffer composition were compared to reduce carbamylation introduced by urea and increase protein detection. Each extraction was performed on a randomized pair of serial sections of homogenous FFPE tissue and analyzed with LC-ESI-MS/MS. Results were compared in terms of yield, missed cleavages, and peptide carbamylation. Lowering extraction temperature to 60°C decreased carbamylation at the cost of decreased protein detection and yield. Protein extraction for at least 20 minutes at 95°C followed by 60°C for 2 hours maximized total protein yield while maintaining protein detection and reducing carbamylation by 7.9%. When accounting for carbamylation during analysis, this modified extraction temperature provides equivalent peptide and protein detection relative to the commercially available Qproteome® FFPE Tissue Kit. No changes to buffer composition containing 7 M urea, 2 M thiourea, and 1 M ammonium bicarbonate resulted in improvements to control conditions. Optimized urea in-solution digestion provides an efficient workflow with maximized yields for proteomic analysis of clinically relevant FFPE tissue.

  7. Histology-guided protein digestion/extraction from formalin-fixed and paraffin-embedded pressure ulcer biopsies.

    Science.gov (United States)

    Taverna, Domenico; Pollins, Alonda C; Nanney, Lillian B; Sindona, Giovanni; Caprioli, Richard M

    2016-02-01

    Herein we present a simple, reproducible and versatile approach for in situ protein digestion and identification on formalin-fixed and paraffin-embedded (FFPE) tissues. This adaptation is based on the use of an enzyme delivery platform (hydrogel discs) that can be positioned on the surface of a tissue section. By simultaneous deposition of multiple hydrogels over select regions of interest within the same tissue section, multiple peptide extracts can be obtained from discrete histological areas. After enzymatic digestion, the hydrogel extracts are submitted for LC-MS/MS analysis followed by database inquiry for protein identification. Further, imaging mass spectrometry (IMS) is used to reveal the spatial distribution of the identified peptides within a serial tissue section. Optimization was achieved using cutaneous tissue from surgically excised pressure ulcers that were subdivided into two prime regions of interest: the wound bed and the adjacent dermal area. The robust display of tryptic peptides within these spectral analyses of histologically defined tissue regions suggests that LC-MS/MS in combination with IMS can serve as useful exploratory tools. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Impact of pre-analytical factors on the proteomic analysis of formalin-fixed paraffin-embedded tissue.

    Science.gov (United States)

    Thompson, Seonaid M; Craven, Rachel A; Nirmalan, Niroshini J; Harnden, Patricia; Selby, Peter J; Banks, Rosamonde E

    2013-04-01

    Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a tremendous potential resource for biomarker discovery, with large numbers of samples in hospital pathology departments and links to clinical information. However, the cross-linking of proteins and nucleic acids by formalin fixation has hampered analysis and proteomic studies have been restricted to using frozen tissue, which is more limited in availability as it needs to be collected specifically for research. This means that rare disease subtypes cannot be studied easily. Recently, improved extraction techniques have enabled analysis of FFPE tissue by a number of proteomic techniques. As with all clinical samples, pre-analytical factors are likely to impact on the results obtained, although overlooked in many studies. The aim of this review is to discuss the various pre-analytical factors, which include warm and cold ischaemic time, size of sample, fixation duration and temperature, tissue processing conditions, length of storage of archival tissue and storage conditions, and to review the studies that have considered these factors in more detail. In those areas where investigations are few or non-existent, illustrative examples of the possible importance of specific factors have been drawn from studies using frozen tissue or from immunohistochemical studies of FFPE tissue. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples.

    Science.gov (United States)

    Moens, Lotte N J; Falk-Sörqvist, Elin; Ljungström, Viktor; Mattsson, Johanna; Sundström, Magnus; La Fleur, Linnéa; Mathot, Lucy; Micke, Patrick; Nilsson, Mats; Botling, Johan

    2015-11-01

    In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  10. A new classification method for MALDI imaging mass spectrometry data acquired on formalin-fixed paraffin-embedded tissue samples.

    Science.gov (United States)

    Boskamp, Tobias; Lachmund, Delf; Oetjen, Janina; Cordero Hernandez, Yovany; Trede, Dennis; Maass, Peter; Casadonte, Rita; Kriegsmann, Jörg; Warth, Arne; Dienemann, Hendrik; Weichert, Wilko; Kriegsmann, Mark

    2017-07-01

    Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) shows a high potential for applications in histopathological diagnosis, and in particular for supporting tumor typing and subtyping. The development of such applications requires the extraction of spectral fingerprints that are relevant for the given tissue and the identification of biomarkers associated with these spectral patterns. We propose a novel data analysis method based on the extraction of characteristic spectral patterns (CSPs) that allow automated generation of classification models for spectral data. Formalin-fixed paraffin embedded (FFPE) tissue samples from N=445 patients assembled on 12 tissue microarrays were analyzed. The method was applied to discriminate primary lung and pancreatic cancer, as well as adenocarcinoma and squamous cell carcinoma of the lung. A classification accuracy of 100% and 82.8%, resp., could be achieved on core level, assessed by cross-validation. The method outperformed the more conventional classification method based on the extraction of individual m/z values in the first application, while achieving a comparable accuracy in the second. LC-MS/MS peptide identification demonstrated that the spectral features present in selected CSPs correspond to peptides relevant for the respective classification. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Molecular genotyping of Echinococcus granulosus using formalin-fixed paraffin-embedded preparations from human isolates in unusual tissue sites.

    Science.gov (United States)

    Hizem, A; M'rad, S; Oudni-M'rad, M; Mestiri, S; Hammedi, F; Mezhoud, H; Zakhama, A; Mokni, M; Babba, H

    2016-07-01

    Cystic echinococcosis (CE) caused by Echinococcus granulosus remains a serious problem worldwide for issues relating to public health and the economy. The most predominantly affected sites are the liver and the lungs, but other organs such as the heart, the spleen and the peritoneum can also be infected. Access to cysts from uncommon sites has limited genomic and molecular investigations. In the present study, genotypes of E. granulosus sensu lato were identified from formalin-fixed paraffin-embedded tissues (FF-PETs) implicated in human CE. Tissue samples were obtained from 57 patients with histologically confirmed CE. DNA samples were analysed using Egss 1 polymerase chain reaction (PCR) specific to the mitochondrial 12S rRNA gene of E. granulosus sensu stricto. All cysts were typed as E. granulosus sensu stricto with up to 35% of the liver and 16.6% of lungs being the most frequently infected, and up to 48.4% of samples being from rare sites. No correlation was found between cyst site and either the gender or the age of patients. This study demonstrates the possibility of exploiting atypical cysts using FF-PET samples and highlights the predominance of E. granulosus sensu stricto species in the Tunisian population, even in unusual infection sites.

  12. Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine.

    Science.gov (United States)

    Van Allen, Eliezer M; Wagle, Nikhil; Stojanov, Petar; Perrin, Danielle L; Cibulskis, Kristian; Marlow, Sara; Jane-Valbuena, Judit; Friedrich, Dennis C; Kryukov, Gregory; Carter, Scott L; McKenna, Aaron; Sivachenko, Andrey; Rosenberg, Mara; Kiezun, Adam; Voet, Douglas; Lawrence, Michael; Lichtenstein, Lee T; Gentry, Jeff G; Huang, Franklin W; Fostel, Jennifer; Farlow, Deborah; Barbie, David; Gandhi, Leena; Lander, Eric S; Gray, Stacy W; Joffe, Steven; Janne, Pasi; Garber, Judy; MacConaill, Laura; Lindeman, Neal; Rollins, Barrett; Kantoff, Philip; Fisher, Sheila A; Gabriel, Stacey; Getz, Gad; Garraway, Levi A

    2014-06-01

    Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.

  13. Applying a Real-Time PCR Assay for Histoplasma capsulatum to Clinically Relevant Formalin-Fixed Paraffin-Embedded Human Tissue

    Science.gov (United States)

    Koepsell, Scott A.; Hinrichs, Steven H.

    2012-01-01

    A real-time PCR assay to detect Histoplasma capsulatum in formalin-fixed, paraffin-embedded (FFPE) tissue is described. The assay had an analytical sensitivity of 6 pg/μl of fungal DNA, analytical specificity of 100%, and clinical sensitivity of 88.9%. This proof-of-concept study may aid in the diagnosis of histoplasmosis from FFPE tissue. PMID:22855519

  14. Clinical relevance of Ki67 gene expression analysis using formalin-fixed paraffin-embedded breast cancer specimens.

    Science.gov (United States)

    Yamamoto, Satoko; Ibusuki, Mutsuko; Yamamoto, Yutaka; Fu, Peifen; Fujiwara, Saori; Murakami, Keiichi; Iwase, Hirotaka

    2013-07-01

    Ki67 is a protein associated with cell cycle activity and shows a good correlation with the growth fraction, which has been proposed as a prognostic or predictive marker in breast cancer. In this study, we aimed to analyze the expression levels of Ki67 (MKI67) messenger RNA (mRNA) derived from formalin-fixed paraffin-embedded (FFPE) tissues for comparison with the immunohistochemical Ki67 labeling index, and investigate the correlation coefficients with clinical outcomes. We analyzed the data of Ki67 mRNA from FFPE and matched fresh-frozen (FF) tissues based on a real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay system in 203 cases of primary invasive breast cancer. The correlation between Ki67 mRNA expression of either FFPE or FF specimens and Ki67 labeling index was positive, as was the correlation between the FFPE and FF results (P Ki67 mRNA expression of FFPE specimens was significantly associated with clinicopathological characteristics: tumor size, lymph node status, nuclear grade, hormone receptors, human epidermal growth factor receptor 2 (Her2) status, and tumor subtype. In prognostic results, Ki67 gene expression in the FFPE specimens revealed almost similar patterns of significance in Kaplan-Meier curves and univariate and multivariate relapse-free survival results as the Ki67 labeling index. Gene expression analysis of Ki67 of FFPE specimens could be successfully performed using RT-qPCR, closely resembling the significant clinical characteristics of Ki67 labeling index. These results confirm that Ki67 gene expression of FFPE specimens has potential for evaluation of cell cycle activity of breast cancer specimens.

  15. An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays

    Directory of Open Access Journals (Sweden)

    Guo Huifang

    2012-09-01

    Full Text Available Abstract Introduction Protein extraction from formalin-fixed paraffin-embedded (FFPE tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA. Results Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples. The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p Conclusions With optimized protein extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type.

  16. An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays.

    Science.gov (United States)

    Guo, Huifang; Liu, Wenbin; Ju, Zhenlin; Tamboli, Pheroze; Jonasch, Eric; Mills, Gordon B; Lu, Yiling; Hennessy, Bryan T; Tsavachidou, Dimitra

    2012-09-24

    Protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA) is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA. Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples.The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type.

  17. Real-time PCR assays for diagnosing brucellar spondylitis using formalin-fixed paraffin-embedded tissues.

    Science.gov (United States)

    Li, Man; Zhou, Xingang; Li, Jingjing; Sun, Lei; Chen, Xiangmei; Wang, Peng

    2018-03-01

    It is difficult to diagnose brucellar spondylitis because of its nonspecific clinical, radiological, and histological characteristics. This study aimed to determine whether real-time polymerase chain reaction (PCR) using formalin-fixed paraffin-embedded (FFPE) tissues was superior to conventional serum-based methods for diagnosing brucellar spondylitis.This retrospective study included 31 patients with brucellosis and a control group of 20 people with no history of brucellosis or exposure to Brucella spp. Samples from all patients with brucellar spondylitis were evaluated using Giemsa staining, the standard tube agglutination (STA) test, blood culture, and real-time PCR.The brucellar spondylitis was acute in 7 patients (22.6%), subacute in 15 patients (48.4%), and chronic in 9 patients (29%). Serological assays provided positive results for 25 patients (80.1%), real-time PCR provided positive results for 29 patients (93.5%), and blood cultures provided positive results for 11 patients (35.5%). The real-time PCR provided sensitivity of 93.5%, specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 100%. The corresponding values for the STA test were 80.1%, 100%, 100%, and 76.9%, respectively. Real-time PCR provided better sensitivity than Giemsa staining, the STA test, and blood culture, although the difference between PCR and STA was not statistically significant (P = .22). B melitensis was the only pathogen that was detected in patient with brucellar spondylitis using real-time PCR.These results suggest that real-time PCR provides a high sensitivity for diagnosing brucellar spondylitis. Furthermore, the real-time PCR results indicate that B melitensis was the causative pathogen in these cases.

  18. MicroRNA expression profiles of multiple system atrophy from formalin-fixed paraffin-embedded samples.

    Science.gov (United States)

    Wakabayashi, Koichi; Mori, Fumiaki; Kakita, Akiyoshi; Takahashi, Hitoshi; Tanaka, Shinya; Utsumi, Jun; Sasaki, Hidenao

    2016-12-02

    MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. Recently, we have shown that informative miRNA data can be derived from archived formalin-fixed paraffin-embedded (FFPE) samples from postmortem cases of amyotrophic lateral sclerosis and normal controls. miRNA analysis has now been performed on FFPE samples from affected brain regions in patients with multiple system atrophy (MSA) and the same areas in neurologically normal controls. We evaluated 50 samples from patients with MSA (n=13) and controls (n=13). Twenty-six samples were selected for miRNA analysis on the basis of the criteria reported previously: (i) a formalin fixation time of less than 4 weeks, (ii) a total RNA yield per sample of more than 500ng, and (iii) sufficient quality of the RNA electrophoresis pattern. These included 11 cases of MSA and 5 controls. Thus, the success rate for analysis of RNA from FFPE samples was 52% (26 of 50). For MSA, a total of 395 and 383 miRNAs were identified in the pons and cerebellum, respectively; 5 were up-regulated and 33 were down-regulated in the pons and 5 were up-regulated and 18 were down-regulated in the cerebellum. Several miRNAs down-regulated in the pons (miR-129-2-3p and miR-129-5p) and cerebellum (miR-129-2-3p, miR-129-5p and miR-132-3p) had already been identified in frozen cerebellum from MSA patients. These findings suggest that archived FFPE postmortem samples can be a valuable source for miRNA profiling in MSA. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples.

    Science.gov (United States)

    Bak, Stine T; Staunstrup, Nicklas H; Starnawska, Anna; Daugaard, Tina F; Nyengaard, Jens R; Nyegaard, Mette; Børglum, Anders; Mors, Ole; Dorph-Petersen, Karl-Anton; Nielsen, Anders L

    2018-01-01

    We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques. Overall, we found that DNA quality was inversely correlated with storage time and DNA quality was insufficient for Illumina methylation arrays; data from methylated DNA immunoprecipitation, clonal bisulfite sequencing, and pyrosequencing of BDNF and ST6GALNAC1 suggested that the original methylation pattern is indeed preserved. Proof-of-principle experiments predicting sex based on the methylation status of the X-inactivated SLC9A7 gene, or genotype differences of the Y and X chromosomes, showed consistency between predicted and actual sex for a subset of FFPE samples. In conclusion, even though DNA from FFPE samples is of low quality and technically challenging, it is likely that a subset of samples can provide reliable data given that the methodology used is designed for small DNA fragments. We propose that simple PCR-based quality control experiments at the genetic and DNA methylation level, carried out at the beginning of any given project, can be used to enrich for the best-performing FFPE samples. The apparent preservation of genetic and DNA methylation patterns in archival FFPE samples may bring along new perspectives for the identification of genetic and epigenetic changes associated with brain-manifested diseases.

  20. Molecular markers associated with nonepithelial ovarian cancer in formalin-fixed, paraffin-embedded specimens by genome wide expression profiling

    Directory of Open Access Journals (Sweden)

    Koon Vui-Kee

    2012-05-01

    Full Text Available Nonepithelial ovarian cancer (NEOC is a rare cancer that is often misdiagnosed as other malignant tumors. Research on this cancer using fresh tissues is nearly impossible because of its limited number of samples within a limited time provided. The study is to identify potential genes and their molecular pathways related to NEOC using formalin-fixed paraffin embedded samples. Total RNA was extracted from eight archived NEOCs and seven normal ovaries. The RNA samples with RNA integrity number >2.0, purity >1.7 and cycle count value <28 cycles were hybridized to the Illumina Whole-Genome DASL assay (cDNA-mediated annealing, selection, extension, and ligation. We analyzed the results using the GeneSpring GX11.0 and FlexArray software to determine the differentially expressed genes. Microarray results were validated using an immunohistochemistry method. Statistical analysis identified 804 differentially expressed genes with 443 and 361 genes as overexpressed and underexpressed in cancer, respectively. Consistent findings were documented for the overexpression of eukaryotic translation elongation factor 1 alpha 1, E2F transcription factor 2, and fibroblast growth factor receptor 3, except for the down-regulated gene, early growth response 1 (EGR1. The immunopositivity staining for EGR1 was found in the majority of cancer tissues. This finding suggested that the mRNA level of a transcript did not always match with the protein expression in tissues. The current gene profile can be the platform for further exploration of the molecular mechanism of NEOC.

  1. Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples.

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    Ensel Oh

    Full Text Available Formalin fixing with paraffin embedding (FFPE has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.

  2. Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition.

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    Dimo Dietrich

    Full Text Available Formalin-fixed and paraffin-embedded (FFPE tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.

  3. Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.

    Science.gov (United States)

    Fowler, Carol B; Chesnick, Ingrid E; Moore, Cedric D; O'Leary, Timothy J; Mason, Jeffrey T

    2010-12-08

    Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.

  4. Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas.

    Science.gov (United States)

    Vera-Lozada, Gabriela; Scholl, Vanesa; Barros, Mário Henrique M; Sisti, Davide; Guescini, Michele; Stocchi, Vilberto; Stefanoff, Claudio Gustavo; Hassan, Rocio

    2014-12-01

    Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy0 quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy0 method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Elevated Pressure Improves the Extraction and Identification of Proteins Recovered from Formalin-Fixed, Paraffin-Embedded Tissue Surrogates

    Science.gov (United States)

    Fowler, Carol B.; Chesnick, Ingrid E.; Moore, Cedric D.; O'Leary, Timothy J.; Mason, Jeffrey T.

    2010-01-01

    Background Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. Principal Findings In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. Conclusions These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form

  6. Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples.

    Science.gov (United States)

    Kalmár, Alexandra; Péterfia, Bálint; Hollósi, Péter; Wichmann, Barnabás; Bodor, András; Patai, Árpád V; Schöller, Andrea; Krenács, Tibor; Tulassay, Zsolt; Molnár, Béla

    2015-09-01

    We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits.

  7. Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.

    Directory of Open Access Journals (Sweden)

    Carol B Fowler

    2010-12-01

    Full Text Available Proteomic studies of formalin-fixed paraffin-embedded (FFPE tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%. Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates.These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.

  8. Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples.

    Science.gov (United States)

    Zhu, Yazhen; Lu, Dan; Lira, Maruja E; Xu, Qing; Du, Yunzhi; Xiong, Jianghong; Mao, Mao; Chung, Hyun Cheol; Zheng, Guangjuan

    2016-04-01

    Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels. Our objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples. Genomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR. Based on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22). ddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Influence of parasite density and sample storage time on the reliability of Entamoeba histolytica-specific PCR from formalin-fixed and paraffin-embedded tissues.

    Science.gov (United States)

    Frickmann, Hagen; Tenner-Racz, Klara; Eggert, Petra; Schwarz, Norbert G; Poppert, Sven; Tannich, Egbert; Hagen, Ralf M

    2013-12-01

    We report on the reliability of polymerase chain reaction (PCR) for the detection of Entamoeba histolytica from formalin-fixed, paraffin-embedded tissue in comparison with microscopy and have determined predictors that may influence PCR results. E. histolytica-specific and Entamoeba dispar-specific real-time PCR and microscopy from adjacent histologic sections were performed using a collection of formalin-fixed, paraffin-embedded tissue specimens obtained from patients with invasive amebiasis. Specimens had been collected during the previous 4 decades. Association of sample age, parasite density, and reliability of PCR was analyzed. E. histolytica PCR was positive in 20 of 34 biopsies (58.8%); 2 of these 20 were microscopically negative for amebae in neighboring tissue sections. PCR was negative in 9 samples with visible amebae in neighboring sections and in 5 samples without visible parasites in neighboring sections. PCR was negative in all specimens that were older than 3 decades. Low parasite counts and sample ages older than 20 years were predictors for false-negative PCR results. All samples were negative for E. dispar DNA. PCR is suitable for the detection of E. histolytica in formalin-fixed, paraffin-embedded tissue samples that are younger than 2 decades and that contain intermediate to high parasite numbers. Negative results in older samples were due to progressive degradation of DNA over time as indicated by control PCRs targeting the human 18S rRNA gene. Moreover, our findings support previous suggestions that only E. histolytica but not E. dispar is responsible for invasive amebiasis.

  10. Detection of West Nile virus using formalin fixed paraffin embedded tissues in crows and horses: quantification of viral transcripts by real-time RT-PCR.

    Science.gov (United States)

    Tewari, Deepanker; Kim, Hyun; Feria, Willard; Russo, Brigite; Acland, Helen

    2004-08-01

    West Nile virus (WNV) RNA was quantified in WNV infected crows and horses with the help of a real-time reverse transcriptase-PCR assay. A 5' nuclease assay, based on NS5 gene detection with a fluorescent probe was used for quantifying WNV RNA using formalin fixed paraffin embedded tissue specimens. Quantitative detection of WNV RNA showed the presence of a higher amount of the viral RNA in crow tissues compared to equine tissues and these results correlated well with the detection of WNV antigen by immunostaining. In crows, the highest amount of virus was seen in the intestine and in horses in the brain.

  11. Novel protein extraction approach using micro-sized chamber for evaluation of proteins eluted from formalin-fixed paraffin-embedded tissue sections

    Directory of Open Access Journals (Sweden)

    Hatakeyama Keiichi

    2012-03-01

    Full Text Available Abstract We describe a novel antigen-retrieval method using a micro-sized chamber for mass spectrometry (MS analysis to identify proteins that are preferentially eluted from formalin-fixed paraffin-embedded (FFPE samples. This approach revealed that heat-induced antigen retrieval (HIAR from an FFPE sample fixed on a glass slide not only improves protein identification, but also facilitates preferential elution of protein subsets corresponding to the properties of antigen-retrieval buffers. Our approach may contribute to an understanding of the mechanism of HIAR.

  12. Multiplex polymerase chain reaction for the detection of high-risk-human papillomavirus types in formalin-fixed paraffin-embedded cervical tissues

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    Mini P Singh

    2017-01-01

    Full Text Available Detecting high-risk-human papillomavirus (HPV types has become an integral part of the cervical cancer screening programmes. This study aimed to develop a multiplex polymerase chain reaction (PCR for identification of HPV types 16 and 18 along with the beta globin gene in formalin-fixed and paraffin-embedded cervical biopsy specimens. A total of 59 samples from patients with cervical abnormalities were tested. HPV 16 positivity was 50% in cervical cancers and 52.9% in cervical intraepithelial neoplasia. Our multiplex PCR protocol can be used as a simple and cost-effective tool for high-risk-HPV detection in cervical cancer screening programmes.

  13. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

    DEFF Research Database (Denmark)

    Tramm, Trine; Hennig, Guido; Kyndi, Marianne

    2013-01-01

    Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal...... tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor...... receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133...

  14. Detection of PrP(Sc) in formalin-fixed, paraffin-embedded tissue by Western blot differentiates classical scrapie, Nor98 scrapie, and bovine spongiform encephalopathy.

    Science.gov (United States)

    Loiacono, Christina M; Beckwith, Nadine; Kunkle, Robert A; Orcutt, Dennis; Hall, S Mark

    2010-09-01

    Transmissible, spongiform encephalopathies including bovine spongiform encephalopathy (BSE) and scrapie are fatal neurodegenerative disorders associated with the presence of an infectious abnormal isoform of normal mammalian proteins called prions. Identification of the prion protein associated with scrapie (PrP(Sc)) in the central nervous system is typically based upon immunoassays including immunohistochemistry (IHC) using formalin-fixed tissues or Western blot (WB) assays using fresh and/or frozen, non-formalin-fixed tissues. Each assay can discriminate between BSE, classical scrapie, and a previously reported strain of scrapie recently identified in the United States named Nor98 scrapie. Different tissue samples are required from the same animal to run these 2 different immunoassays. This may result in inconsistent test results for the same animal. Sampling problems such as collecting insufficient volumes of fresh tissue or less than optimal anatomic location of brainstem for IHC can affect the ability of the test procedures to offer definitive and discriminatory results. Recently, a WB method using formalin-fixed, paraffin-embedded (FFPE) tissue to identify PrP(Sc) was developed that successfully identified PrP(Sc) in sheep affected by classical scrapie. In the current study, the use of this technique to produce discriminatory results identifying classical BSE in bovine tissue and both classical and Nor98 scrapie in ovine tissue using paraffin-embedded brain samples is described. Protein-banding patterns from WB using FFPE tissue were similar to protein-banding patterns produced by WB assays utilizing fresh tissues from the same animals, and results correlated well with the IHC PrP(Sc)-positive staining present in the cerebellum and obex regions of brain samples from these animals.

  15. Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins.

    Science.gov (United States)

    Wiśniewski, Jacek R; Duś, Kamila; Mann, Matthias

    2013-04-01

    Archival formalin-fixed and paraffin-embedded clinical samples represent a very diverse source of material for proteomic investigation of diseases, often with follow-up patient information. Here, we describe an analytical workflow for analysis of laser-capture microdissected formalin-fixed and paraffin-embedded samples that allows studying proteomes to a depth of 10 000 proteins per sample. The workflow involves lysis of tissue in SDS-containing buffer, detergent removal, and consecutive digestion of the proteins with two enzymes by the multienzyme digestion filter-aided sample preparation method. Resulting peptides are fractionated by pipette-tip based strong anion exchange into six fractions and analyzed by LC-MS/MS on a bench top quadrupole Orbitrap mass spectrometer. Analysis of the data using the MaxQuant software resulted in the identification of 9502 ± 28 protein groups per a 110 nL sample of microdissected cells from human colonic adenoma. This depth of proteome analysis enables systemic insights into the organization of the adenoma cells and an estimation of the abundances of known biomarkers. It also allows the identification of proteins expressed from tumor suppressors, oncogenes, and other key players in the development and progression of the colorectal cancer. Our proteomic platform can be used for quantitative comparisons between samples representing different stages of diseases and thus can be applied to the discovery of biomarkers or drug targets. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Genetic Characterization of Echinococcus granulosus from a Large Number of Formalin-Fixed, Paraffin-Embedded Tissue Samples of Human Isolates in Iran

    Science.gov (United States)

    Rostami, Sima; Torbaghan, Shams Shariat; Dabiri, Shahriar; Babaei, Zahra; Mohammadi, Mohammad Ali; Sharbatkhori, Mitra; Harandi, Majid Fasihi

    2015-01-01

    Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, presents an important medical and veterinary problem globally, including that in Iran. Different genotypes of E. granulosus have been reported from human isolates worldwide. This study identifies the genotype of the parasite responsible for human hydatidosis in three provinces of Iran using formalin-fixed paraffin-embedded tissue samples. In this study, 200 formalin-fixed paraffin-embedded tissue samples from human CE cases were collected from Alborz, Tehran, and Kerman provinces. Polymerase chain reaction amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit 1 gene were performed for genetic characterization of the samples. Phylogenetic analysis of the isolates from this study and reference sequences of different genotypes was done using a maximum likelihood method. In total, 54.4%, 0.8%, 1%, and 40.8% of the samples were identified as the G1, G2, G3, and G6 genotypes, respectively. The findings of the current study confirm the G1 genotype (sheep strain) to be the most prevalent genotype involved in human CE cases in Iran and indicates the high prevalence of the G6 genotype with a high infectivity for humans. Furthermore, this study illustrates the first documented human CE case in Iran infected with the G2 genotype. PMID:25535316

  17. Hydrolysis Profiles of Formalin Fixed Paraffin-Embedded Tumors Based on IOD (Integrated Optical Density and Nuclear Texture Feature Measurements

    Directory of Open Access Journals (Sweden)

    Margareta Fležar

    1999-01-01

    Full Text Available The aim of the study was to determine optimal hydrolysis time for the Feulgen DNA staining of archival formalin fixed paraffin‐embedded surgical samples, prepared as single cell suspensions for image cytometric measurements. The nuclear texture features along with the IOD (integrated optical density of the tumor nuclei were analysed by an automated high resolution image cytometer as a function of duration of hydrolysis treatment (in 5 N HCl at room temperature. Tissue blocks of breast carcinoma, ovarian serous carcinoma, ovarian serous tumor of borderline malignancy and leiomyosarcoma were included in the study. IOD hydrolysis profiles showed plateau between 30 and 60 min in the breast carcinoma and leiomyosarcoma, and between 40 and 60 min in the ovarian serous carcinoma and ovarian serous tumor of borderline malignancy. Most of the nuclear texture features remained stable after 20 min of hydrolysis treatment. Our results indicate that the optimal hydrolysis time for IOD and for nuclear texture feature measurements, was between 40 and 60 min in the cell preparations from tissue blocks of three epithelial and one soft tissue tumor.

  18. MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.

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    Thomas W Powers

    Full Text Available A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

  19. MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.

    Science.gov (United States)

    Powers, Thomas W; Neely, Benjamin A; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A; Mehta, Anand S; Haab, Brian B; Drake, Richard R

    2014-01-01

    A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

  20. A novel xylene-free deparaffinization method for the extraction of proteins from human derived formalin-fixed paraffin embedded (FFPE) archival tissue blocks.

    Science.gov (United States)

    Mansour, Anthony; Chatila, Rajaa; Bejjani, Noha; Dagher, Carole; Faour, Wissam H

    2014-01-01

    Protein detection methods in formalin-fixed paraffin embedded (FFPE) tissue blocks are widely used in research and clinical setting in order to diagnose or to confirm a diagnosis of various types of diseases. Therefore, multiple protein extraction methods from FFPE tissue sections have been developed in this regard. However, the yield and the quality of proteins extracted from FFPE tissues are significantly reduced in blocks stored for longer periods of time. Regardless the protein extraction method used, tissue sections must be first deparaffinized with xylene, and then washed in serial dilutions of ethanol in order to remove the toxic organic solvent "xylene" and rehydrate the tissue. The objective of this study was first to develop a method to deparaffinize FFPE blocks that excludes the use of toxic solvent "xylene". Second minimize the time required to perform the extraction. Here we describe a method where:•The entire paraffin embedded blocks are deparaffinized and rehydrated using only hot distilled water as a substitute for both xylene and ethanol•The entire procedure takes about 15 min•Deparaffinized blocks are immediately homogenized in lysis buffer, and the obtained lysate analyzed by Western blot. With this new modified technique, we were able to successfully detect actin and AKT proteins in lysates from blocks embedded in paraffin for up to 9 years.

  1. Clinical performance evaluation of the Idylla NRAS-BRAF mutation test on retrospectively collected formalin-fixed paraffin-embedded colorectal cancer tissue

    DEFF Research Database (Denmark)

    Johnston, Louise; Power, Michael; Sloan, Philip

    2017-01-01

    system. The Idylla NRAS-BRAF mutation test has been developed for the qualitative detection of mutations in NRAS and BRAF oncogenes, facilitating genetic profiling of patients with cancer. The aim of this study was to carry out a formal clinical performance evaluation. METHODS: Two-hundred and forty......-two formalin-fixed paraffin-embedded (FFPE) human malignant colorectal cancer (CRC) tissue samples were identified in departmental archives and tested with both the Idylla NRAS-BRAF mutation test and the Agena Bioscience MassARRAY test. RESULTS: The overall concordance between the Idylla NRAS-BRAF mutation...... reference test. Reanalysis of this sample by droplet digital PCR confirmed that the mutation was present, but at an allelic frequency below the stated sensitivity level of the MassARRAY system. CONCLUSION: These results confirm that the Idylla NRAS-BRAF mutation test has high concordance with a widely used...

  2. MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling

    DEFF Research Database (Denmark)

    Glud, Martin; Klausen, Mikkel; Gniadecki, Robert

    2009-01-01

    surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we...... identified 84 miRNAs that were expressed in both types of samples and represented an miRNA profile of melanocytic nevi. Our results showed a high correlation in miRNA expression (Spearman r-value of 0.80) between paired FFPE and fresh frozen material. The data were further validated by quantitative RT......-PCR. In conclusion, FFPE specimens of melanocytic lesions are suitable as a source for miRNA microarray profiling....

  3. Antigen retrieval prior to on-tissue digestion of formalin-fixed paraffin-embedded tumour tissue sections yields oxidation of proline residues.

    Science.gov (United States)

    Djidja, Marie-Claude; Claude, Emmanuelle; Scriven, Peter; Allen, David W; Carolan, Vikki A; Clench, Malcolm R

    2017-07-01

    MALDI-mass spectrometry imaging (MALDI-MSI) has been shown to allow the study of protein distribution and identification directly within formalin-fixed paraffin-embedded (FFPE) tissue sections. However, direct protein identification from tissue sections remains challenging due to signal interferences and/or existing post-translational or other chemical modifications. The use of antigen retrieval (AR) has been demonstrated for unlocking proteins prior to in situ enzymatic digestion and MALDI-MSI analysis of FFPE tissue sections. In the work reported here, the identification of proline oxidation, which may occur when performing the AR protocol, is described. This facilitated and considerably increased the number of identified peptides when adding proline oxidation as a variable modification to the MASCOT search criteria. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Comparative investigations of T cell receptor gamma gene rearrangements in frozen and formalin-fixed paraffin wax-embedded tissues by capillary electrophoresis

    DEFF Research Database (Denmark)

    Christensen, M; Funder, A D; Bendix, K

    2006-01-01

    AIM: To compare clonal T cell receptor gamma (TCRgamma) gene rearrangements in frozen and formalin-fixed paraffin wax-embedded (FFPE) tissue, using capillary electrophoresis for use in diagnostics, as T cell lymphomas may be difficult to diagnose by conventional methods.METHODS: The DNA for PCR...... was extracted from frozen and FFPE tissue, cell lines and blood. PCR primers Vgamma1-8, Vgamma9, Vgamma10 or Vgamma11 (5' end labelled) combined with a mixture of JgammaP1/JgammaP/JgammaP2/Jgamma2 (unlabelled) were used. Monoclonal cases were sequenced and clonality, reproducibility, sensitivity and specificity......% for patient specimens and the specificity 100%. The junctional region between the Vgamma and Jgamma segments was specific for each patient.CONCLUSIONS: Capillary electrophoresis of PCR products from frozen and FFPE tissue is suitable for detecting clonal TCRgamma gene rearrangements. It is important, however...

  5. Detection of fowl adenovirus DNA from formalin-fixed and paraffin-embedded sections by PCR and classification of serotypes by sequencing of PCR products.

    Science.gov (United States)

    Ohizumi, Takuya; Nakamura, Kikuyasu; Yamamoto, Yu; Mase, Masaji; Yamada, Manabu

    2012-12-01

    Detection of fowl adenovirus (FAV) DNA from formalin-fixed and paraffin-embedded (FFPE) sections was attempted by PCR. Serotypes of FAV were classified by sequencing the PCR products. In trials of PCR using a positive control infected with serotype 2 FAV, the best primer set was 57F forward primer (5'-CAARTTCAGRCAGACGGT-3') and 26R reverse primer (5'-GGCTTGACGTACGCTCCGTA-3'). A second PCR with the same primer set revealed a clearer band in the electrophoresis of generated PCR products. Generated PCR products were confirmed to be derived from infected FAV. In addition, PCR and sequencing of PCR products of the liver FFPE sections, from two natural inclusion body hepatitis cases that were not examined for virologic isolation, suggested that the detected FAV was serotype 8a. The PCR of FFPE sections, and serotyping by the sequencing of PCR products, are useful for diagnosis and epidemiologic analysis of FAV infections.

  6. Evaluation of two methods DNA extraction from formalin-fixed, paraffin-embedded tissues on non-optimal conditions

    International Nuclear Information System (INIS)

    Bustamante, Javier Andres; Astudillo, Miryam; Pazos, Alvaro Jairo; Bravo, Luis Eduardo

    2011-01-01

    Paraffin wax embedded tissues are an invaluable material for retrospective studies requiring the application of molecular analysis. Multiple methods are available to extract DNA from these kinds of samples. However, the most common methods are slow and the reagents often contribute to the fragmentation of genetic material. In order to optimize the procedure, two methods for DNA extraction from paraffin embedded tissue non-optimal conditions were used. 47 blocks containing paraffin-embedded biopsies of pleura, lung and pericardium from 24 patients (66.6% males) older than 18 years, with biopsy proven chronic granulomatous inflammation referred to the department of pathology at University Hospital of Valle between 2002 and 2007 were selected. Each sample was subjected to 10 cuts and was to two methods of DNA extraction: 1. conventional and 2. QIAamp - DNA mini kit. The efficiency of the extracted DNA was assessed by spectrophotometry and PCR amplification of a fragment of the housekeeping gene GAPDH. The concentration of DNA samples extracted by the conventional method was of 65.52 ng/Mu l ± 11.47 (mean ± SE) and the 260/280 absorbance ratio ranged between 0.52 and 2.30 the average concentration of DNA of the samples extracted by the commercial method was 60.89 ng/Mu l ± 6.02 (mean ± SE), with an absorbance that fluctuated between 0 and 2.64. The DNA obtained was amplified by PCR, of 47 samples extracted by methods, 25 and 23 respectively the GAPDH gene amplified successfully. The methods used to obtain DNA showed similar performance, highlighting the potential utility of both extraction methods for the retrospective studies from paraffin embedded tissues in unsuitable conditions.

  7. Differential N-glycan patterns identified in lung adenocarcinoma by N-glycan profiling of formalin-fixed paraffin-embedded (FFPE) tissue sections.

    Science.gov (United States)

    Wang, Xiaoning; Deng, Zaian; Huang, Chuncui; Zhu, Tong; Lou, Jiatao; Wang, Lin; Li, Yan

    2018-02-10

    N-glycan profiling is a powerful approach for analyzing the functional relationship between N-glycosylation and cancer. Current methods rely on either serum or fresh tissue samples; however, N-glycan patterns may differ between serum and tissue, as the proteins of serum originate from a variety of tissues. Furthermore, fresh tissue samples are difficult to ship and store. Here, we used a profiling method based on formalin-fixed paraffin-embedded (FFPE) tissue sections from lung adenocarcinoma patients. We found that our method was highly reproducible. We identified 58 N-glycan compositions from lung adenocarcinoma FFPE samples, 51 of which were further used for MS n -based structure prediction. We show that high mannose type N-glycans are upregulated, while sialylated N-glycans are downregulated in our FFPE lung adenocarcinoma samples, compared to the control samples. Our receiver operating characteristic (ROC) curve analysis shows that high mannose type and sialylated N-glycans are useful discriminators to distinguish between lung adenocarcinoma and control tissue. Together, our results indicate that expression levels of specific N-glycans correlate well with lung adenocarcinoma, and strongly suggest that our FFPE-based method will be useful for N-glycan profiling of cancer tissues. Glycosylation is one of the most important post-translational protein modifications, and is associated with several physiopathological processes, including carcinogenesis. In this study, we tested the feasibility of using formalin-fixed paraffin-embedded (FFPE) tissue sections to identify changes in N-glycan patterns and identified the differentially expressed N-glycans of lung adenocarcinoma. Our study shows that the FFPE-based N-glycan profiling method is useful for clinical diagnosis as well as identification of potential biomarkers, and our data expand current knowledge of differential N-glycan patterns of lung adenocarcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.

    Science.gov (United States)

    Zhang, Liangxuan; Chen, Liangjing; Sah, Sachin; Latham, Gary J; Patel, Rajesh; Song, Qinghua; Koeppen, Hartmut; Tam, Rachel; Schleifman, Erica; Mashhedi, Haider; Chalasani, Sreedevi; Fu, Ling; Sumiyoshi, Teiko; Raja, Rajiv; Forrest, William; Hampton, Garret M; Lackner, Mark R; Hegde, Priti; Jia, Shidong

    2014-04-01

    The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

  9. Sensitive detection of human growth hormone mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues by non-isotopic in situ hybridization.

    Science.gov (United States)

    Ehrlein, J; Wanke, R; Weis, S; Brem, G; Hermanns, W

    1994-08-01

    A sensitive technique of non-isotopic in situ hybridization (NISH) is presented, which permits the detection of human growth hormone (hGH) mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues and human post mortem pituitaries; the latter were used as positive tissue controls in this study. In addition, a double staining procedure combining NISH and immunohistochemistry for the visualization of both hGH and hGH mRNA in the same paraffin section is described. Digoxigenin-labelled antisense hGH RNA was used for NISH of hGH mRNA. The NISH protocol was based upon an established radioactive method. Alkaline phosphatase and horseradish peroxidase-based immunoenzymatic procedures for the detection of digoxigenin-labelled RNA probes using different chromogens [4-nitro blue tetrazolium chloride (NBT), Fast Blue BB, New Fuchsin, and 3,3'-diaminobenzidine tetrahydrochloride (DAB) with or without intensification of the DAB staining] were compared. The proteolytic tissue pretreatment and the detection procedure were found to be the most critical steps for successful visualization of hGH mRNA. After optimization of the permeabilization conditions, hGH mRNA could be visualized in each case studied when alkaline phosphatase/NBT-based detection was employed. The NISH technique presented here, performed either separately or in combination with immunohistochemistry, permits retrospective analyses, of hGH (trans)gene expression in archival, paraffin-embedded specimens.

  10. Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer

    DEFF Research Database (Denmark)

    Solassol, Jérôme; Vendrell, Julie; Märkl, Bruno

    2016-01-01

    , was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE...

  11. Assessing HER2 amplification by IHC, FISH, and real-time polymerase chain reaction analysis (real-time PCR) following LCM in formalin-fixed paraffin embedded tissue from 40 women with ovarian cancer

    DEFF Research Database (Denmark)

    Hillig, Thore; Thode, Jørgen; Breinholt, Ellen Marie

    2012-01-01

    We compare HER2 receptor amplification analysis by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and real-time polymerase chain reaction (real-time PCR) DNA copy-number assay following laser capture microdissection (LCM) in formalin-fixed paraffin embedded tissue from 40...

  12. The effect of aging of formalin-fixed paraffin-embedded tissues on the in situ hybridization and immunohistochemistry signals in cervical lesions.

    Science.gov (United States)

    Nuovo, Allison J; Garofalo, Michela; Mikhail, Alexandria; Nicol, Alcina F; Vianna-Andrade, Cecilia; Nuovo, Gerard J

    2013-09-01

    Formalin-fixed, paraffin-embedded tissues are widely used in biomedical research but little is known about the effect of the age of the block or unstained slides on the in situ hybridization or immunohistochemistry signal. We compared the in situ-based and immunohistochemistry-based signals for cervical intraepithelial neoplasia samples that ranged from 0 to 15 years of age. There was a progressive and statistically significant decrease in the strength of the p16 signal when comparing tissues prepared from recent unstained slides (0 to 1 y old, mean score of 92%) to those of intermediate age (5 to 7 y old, mean score of 49%) to old unstained slides (cut 13 to 15 y ago, mean score of 10%). Equivalent, progressive, and significant decreases in the intensity of the signals for microRNAs, CD45, and human papillomavirus DNA were seen in tissues stored on slides from 5 to 7 years and 13 to 15 years, respectively. However, the diminution of signal was much less, although still statistically significant, if the sections from the 13- to 15-year-old paraffin blocks were prepared in 2012. The data likely does not represent degradation of the targets as extraction of several microRNA from the old blocks showed no detectable degradation, despite the markedly weakened in situ hybridization signal. It is concluded that in situ-based signal for DNA, microRNAs, and proteins in paraffin-embedded tissues are significantly reduced over time, especially when stored long term on glass slides which, in turn, can lead to a significant underestimation of the amount and presence of the nucleic acid or protein target.

  13. Development of a monoclonal antibody that specifically detects tissue inhibitor of metalloproteinase-4 (TIMP-4) in formalin-fixed, paraffin-embedded human tissues.

    Science.gov (United States)

    Donover, P Scott; Wojciechowski, Brian S; Thirumaran, Rajesh; Zemba-Palko, Vlasta; Prendergast, George C; Wallon, U Margaretha

    2010-08-01

    Overexpression of the extracellular metalloproteinase inhibitor TIMP-4 in estrogen receptor-negative breast cancers was found recently to be associated with a poor prognosis for survival. To pursue exploration of the theranostic applications of TIMP-4, specific antibodies with favorable properties for immunohistochemical use and other clinical assays are needed. Here we report the characterization of a monoclonal antibody (clone 9:4-7) specific for full-length human TIMP-4 with suitable qualities. The antibody was determined to be an IgG(2b) immunoglobulin. In enzyme-linked immunosorbent assay (ELISA) and immunoblotting assays, it did not exhibit any detectable crossreactivity with recombinant forms of the other human TIMPs 1, 2, and 3. In contrast, the antibody displayed high specificity and sensitivity for TIMP-4 including in formalin-fixed and paraffin-embedded specimens of human breast specimens. An analysis of tissue microarrays of human cancer and corresponding normal tissues revealed specific staining patterns with excellent signal-to-noise ratios. This study documents TIMP-4 monoclonal antibody clone 9:4-7 as an effective tool for preclinical and clinical investigations. Published 2010 Wiley-Liss, Inc.

  14. Validation of tumor markers in central nervous system germ cell tumors by real-time reverse transcriptase polymerase chain reaction using formalin-fixed paraffin-embedded tissues.

    Science.gov (United States)

    Kim, Dowhan; Lee, Da Hye; Choi, Junjeong; Shim, Kyu Won; Kim, Se Hoon

    2013-01-01

    The therapeutic protocols for treatment of germinomas and non-germinomatous germ cell tumors (NGGCTs) are completely different, so it is important to distinguish pure germinomas from NGGCTs. As it can be difficult to diagnose by morphology alone, immunohisto-chemistry (IHC) has been widely used as an ancillary test to improve diagnostic accuracy. However, IHC has limitations due to the misinterpretation of results or the aberrant loss of immunoreactivity. However, real-time RT-PCR has certain advantages over IHC, including its quantitative nature. The aim of our study was to evaluate the usefulness of real-time RT-PCR on formalin-fixed paraffin-embedded (FFPE) tissue blocks for the diagnosis of germ cell tumors of the central nervous system. We selected eight markers of germ cell tumors using a literature search, and validated them using real-time RT-PCR. Among them, POU5F1, NANOG and TGFB2 were statistically significant (P=0.05) in multiple comparisons (MANOVA) of three groups (pure germinomas, mature teratomas and malignant germ cell tumors). Two-group (pure germinomas and NGGCTs) discriminant analysis achieved a 70.0% success rate in cross-validation. We concluded that real-time RT-PCR using FFPE tissue has adequate validating power comparable to IHC in the diagnosis of central nervous system germ cell tumors; therefore, when IHC is not available, not conclusive or not informative, RT-PCR is a potential alternative to a repeat biopsy.

  15. MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling.

    Science.gov (United States)

    Glud, Martin; Klausen, Mikkel; Gniadecki, Robert; Rossing, Maria; Hastrup, Nina; Nielsen, Finn C; Drzewiecki, Krzysztof T

    2009-05-01

    MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate cellular differentiation, proliferation, and apoptosis. MiRNAs are expressed in a developmentally regulated and tissue-specific manner. Aberrant expression may contribute to pathological processes such as cancer, and miRNA may therefore serve as biomarkers that may be useful in a clinical environment for diagnosis of various diseases. Most miRNA profiling studies have used fresh tissue samples. However, in some types of cancer, including malignant melanoma, fresh material is difficult to obtain from primary tumors, and most surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we identified 84 miRNAs that were expressed in both types of samples and represented an miRNA profile of melanocytic nevi. Our results showed a high correlation in miRNA expression (Spearman r-value of 0.80) between paired FFPE and fresh frozen material. The data were further validated by quantitative RT-PCR. In conclusion, FFPE specimens of melanocytic lesions are suitable as a source for miRNA microarray profiling.

  16. Detection of c-myc amplification in formalin-fixed paraffin-embedded tumor tissue by chromogenic in situ hybridization (CISH).

    Science.gov (United States)

    Todorović-Raković, Nataša

    2013-01-01

    In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40 × magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology and the slides can be stored for a long time.

  17. Scores for standardization of on-tissue digestion of formalin-fixed paraffin-embedded tissue in MALDI-MS imaging.

    Science.gov (United States)

    Erich, Katrin; Sammour, Denis A; Marx, Alexander; Hopf, Carsten

    2017-07-01

    On-slide digestion of formalin-fixed and paraffin-embedded human biopsy tissue followed by mass spectrometry imaging of resulting peptides may have the potential to become an additional analytical modality in future ePathology. Multiple workflows have been described for dewaxing, antigen retrieval, digestion and imaging in the past decade. However, little is known about suitable statistical scores for method comparison and systematic workflow standardization required for development of processes that would be robust enough to be compatible with clinical routine. To define scores for homogeneity of tissue processing and imaging as well as inter-day repeatability for five different processing methods, we used human liver and gastrointestinal stromal tumor tissue, both judged by an expert pathologist to be >98% histologically homogeneous. For mean spectra-based as well as pixel-wise data analysis, we propose the coefficient of determination R 2 , the natural fold-change (natFC) value and the digest efficiency DE% as readily accessible scores. Moreover, we introduce two scores derived from principal component analysis, the variance of the mean absolute deviation, MAD, and the interclass overlap, J overlap , as computational scores that may help to avoid user bias during future workflow development. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. [Use of archival formalin-fixed, paraffin-embedded (FFPE) tissue samples for molecular genetic analysis in diffuse large B-cell lymphoma (DLBCL)].

    Science.gov (United States)

    Jarošová, Marie; Kučerová, Jana; Flodr, Patrik; Mikešová, Michaela; Procházka, Vít; Papajík, Tomáš

    2014-04-01

    The currently valid molecular genetic subclassification of patients with diffuse large B-cell lymphoma (DLBCL) into three prognostic subgroups based on expression profiling has been the objective of numerous genetic studies. In routine clinical practice, however, expression profiling technology remains unavailable for the most of centers. Apart from the technology, in some cases molecular genetic laboratories have problems obtaining high-quality material, i.e. fresh tissues, for RNA isolation to determine gene expression. One possibility is to determine the gene expression from RNA obtained by isolation from formalin-fixed, paraffin-embedded (FFPE) tissue. This pilot study aimed at isolating RNA from FFPE in patients diagnosed with DLBCL and verifying the potential use of such RNA for the expression analysis of 7 selected genes. Although the study showed that it is possible to isolate RNA and determine the expression of the selected genes from archival material, the values of relative expression of some genes in the set were too variable to be used for unambiguous prognostic classification. It was confirmed that retrospective analyses of selected genes may be performed with sufficient material obtained, and that properly archived blocks may be used for molecular biology analyses even after 8 years.

  19. Evaluation of positive Rift Valley fever virus formalin-fixed paraffin embedded samples as a source of sequence data for retrospective phylogenetic analysis.

    Science.gov (United States)

    Mubemba, B; Thompson, P N; Odendaal, L; Coetzee, P; Venter, E H

    2017-05-01

    Rift Valley fever (RVF), caused by an arthropod borne Phlebovirus in the family Bunyaviridae, is a haemorrhagic disease that affects ruminants and humans. Due to the zoonotic nature of the virus, a biosafety level 3 laboratory is required for isolation of the virus. Fresh and frozen samples are the preferred sample type for isolation and acquisition of sequence data. However, these samples are scarce in addition to posing a health risk to laboratory personnel. Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are safe and readily available, however FFPE derived RNA is in most cases degraded and cross-linked in peptide bonds and it is unknown whether the sample type would be suitable as reference material for retrospective phylogenetic studies. A RT-PCR assay targeting a 490 nt portion of the structural G N glycoprotein encoding gene of the RVFV M-segment was applied to total RNA extracted from archived RVFV positive FFPE samples. Several attempts to obtain target amplicons were unsuccessful. FFPE samples were then analysed using next generation sequencing (NGS), i.e. Truseq ® (Illumina) and sequenced on the Miseq ® genome analyser (Illumina). Using reference mapping, gapped virus sequence data of varying degrees of shallow depth was aligned to a reference sequence. However, the NGS did not yield long enough contigs that consistently covered the same genome regions in all samples to allow phylogenetic analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

    Directory of Open Access Journals (Sweden)

    Reis Patricia P

    2010-06-01

    Full Text Available Abstract Background MicroRNAs (miRs are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE tissue. Results Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p 35, we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p Conclusion Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

  1. Integrated and convenient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for LC-MS/MS analysis.

    Science.gov (United States)

    Lai, Xianyin; Schneider, Bryan P

    2014-11-01

    Because fresh-frozen tissue samples associated with long-term clinical data and of rare diseases are often unobtainable at the present time, formalin-fixed paraffin-embedded (FFPE) tissue samples are considered a highly valuable resource for researchers. However, protein extraction from FFPE tissues faces challenges of deparaffinization and cross-link reversion. Current procedures for protein extraction from FFPE tissue require separate steps and toxic solvents, resulting in inconvenience in protein extraction. To overcome these limitations, an integrated method was developed using nontoxic solvents in four types of FFPE tissues. The average amount of proteins from three replicates of bladder, kidney, liver, and lung FFPE tissues were 442.6, 728.9, 736.4, and 694.7 μg with CVs of 7.5, 5.8, 2.4, and 4.5%, respectively. Proteomic analysis showed that 348, 417, 607, and 304 unique proteins were identified and quantified without specification of isoform by a least two peptides from bladder, kidney, liver, and lung FFPE tissue samples, respectively. The analysis of individual protein CV demonstrated that 97-99% of the proteins were quantified with a CV ≤ 30%, verifying the reproducibility of the integrated protein extraction method. In summary, the developed method is high-yield, reproducible, convenient, simple, low cost, nonvolatile, nonflammable, and nontoxic. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. A Comparison of Fresh Frozen vs. Formalin-Fixed, Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay

    Directory of Open Access Journals (Sweden)

    Florenza Lüder Ripoli

    2016-05-01

    Full Text Available Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE tissues are an invaluable source of archived biological material. Fresh frozen (FF tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16 target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2 were analyzed via branched-DNA assay (b-DNA. ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer.

  3. Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS: Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE Samples

    Directory of Open Access Journals (Sweden)

    Gladys Arreaza

    2016-09-01

    Full Text Available In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC, circulating tumor DNA (ctDNA, etc., tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC, in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.. Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research.

  4. Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue.

    Science.gov (United States)

    Adam, Benjamin; Afzali, Bahman; Dominy, Katherine M; Chapman, Erin; Gill, Reeda; Hidalgo, Luis G; Roufosse, Candice; Sis, Banu; Mengel, Michael

    2016-03-01

    Histopathologic diagnoses in transplantation can be improved with molecular testing. Preferably, molecular diagnostics should fit into standard-of-care workflows for transplant biopsies, that is, formalin-fixed paraffin-embedded (FFPE) processing. The NanoString(®) gene expression platform has recently been shown to work with FFPE samples. We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE renal allograft samples. A literature-derived antibody-mediated rejection (ABMR) 34-gene set, comprised of endothelial, NK cell, and inflammation transcripts, was analyzed in different retrospective biopsy cohorts and showed potential to molecularly discriminate ABMR cases, including FFPE samples. NanoString(®) results were reproducible across a range of RNA input quantities (r = 0.998), with different operators (r = 0.998), and between different reagent lots (r = 0.983). There was moderate correlation between NanoString(®) with FFPE tissue and quantitative reverse transcription polymerase chain reaction (qRT-PCR) with corresponding dedicated fresh-stabilized tissue (r = 0.487). Better overall correlation with histology was observed with NanoString(®) (r = 0.354) than with qRT-PCR (r = 0.146). Our results demonstrate the feasibility of multiplexed gene expression quantification from FFPE renal allograft tissue. This represents a method for prospective and retrospective validation of molecular diagnostics and its adoption in clinical transplantation pathology. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis.

    Science.gov (United States)

    Mansour, Anthony G; Khalil, Pamela Abou; Bejjani, Noha; Chatila, Rajaa; Dagher-Hamalian, Carole; Faour, Wissam H

    2017-03-01

    Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and β-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and β-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and β-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.

  6. Automated Extraction of Formalin-Fixed, Paraffin-Embedded Tissue for High-Risk Human Papillomavirus Testing of Head and Neck Squamous Cell Carcinomas Using the Roche Cobas 4800 System.

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    Kerr, Darcy A; Sweeney, Brenda; Arpin, Ronald N; Ring, Melissa; Pitman, Martha B; Wilbur, David C; Faquin, William C

    2016-08-01

    -Testing for high-risk human papillomavirus (HR-HPV) in head and neck squamous cell carcinomas (HNSCCs) is important for both prognostication and clinical management. Several testing platforms are available for HR-HPV; however, effective alternative automated approaches are needed. -To assess the performance of the automated Roche cobas 4800 HPV real-time polymerase chain reaction-based system on formalin-fixed, paraffin-embedded HNSCC specimens and compare results with standard methods of in situ hybridization (ISH) and p16 immunohistochemistry. -Formalin-fixed, paraffin-embedded samples of HNSCC were collected from archival specimens in the Department of Pathology, Massachusetts General Hospital (Boston), and prepared using the automated system by deparaffinization and dehydration followed by tissue lysis. Samples were integrated into routine cervical cytology testing runs by cobas. Corresponding formalin-fixed, paraffin-embedded samples were evaluated for HR-HPV by ISH and p16 by immunohistochemistry. Discrepant cases were adjudicated by polymerase chain reaction. -Sixty-two HNSCC samples were analyzed using the automated cobas system, ISH, and immunohistochemistry. Fifty-two percent (n = 32 of 62) of formalin-fixed, paraffin-embedded tumors were positive for HR-HPV by cobas. Eighty-eight percent (n = 28 of 32) of cases were the HPV 16 subtype and 12% (n = 4 of 32) were other HR-HPV subtypes. Corresponding testing with ISH was concordant in 92% (n = 57 of 62) of cases. Compared with the adjudication polymerase chain reaction standard, there were 3 false-positive cases by cobas. -Concordance in HNSCC HR-HPV status between cobas and ISH was more than 90%. The cobas demonstrated a sensitivity of 100% and a specificity of 91% for detection of HR-HPV. Advantages favoring cobas include its automation, cost efficiency, objective results, and ease of performance.

  7. Detection of a putative novel adenovirus by PCR amplification, sequencing and phylogenetic characterisation of two gene fragments from formalin-fixed paraffin-embedded tissues of a cat diagnosed with disseminated adenovirus disease.

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    Lakatos, Béla; Hornyák, Ákos; Demeter, Zoltán; Forgách, Petra; Kennedy, Frances; Rusvai, Miklós

    2017-12-01

    Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissue samples of a cat that had suffered from disseminated adenovirus infection. The identity of the amplified products from the hexon and DNA-dependent DNA polymerase genes was confirmed by DNA sequencing. The sequences were clearly distinguishable from corresponding hexon and polymerase sequences of other mastadenoviruses, including human adenoviruses. These results suggest the possible existence of a distinct feline adenovirus.

  8. Effect of Antigen Retrieval Methods on Nonspecific Binding of Antibody-Metal Nanoparticle Conjugates on Formalin-Fixed Paraffin-Embedded Tissue.

    Science.gov (United States)

    Zhang, Yuying; Wang, Xin-Ping; Perner, Sven; Bankfalvi, Agnes; Schlücker, Sebastian

    2018-01-02

    Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissues provides important diagnostic and prognostic information in pathology. Metal nanoparticles (NPs) and, in particular, surface-enhanced Raman scattering (SERS) nanotags as a new class of labeling reagents are promising to be used for multiplexed protein profiling on tissue sections. However, nonspecific binding of NPs onto the tissue specimens greatly hampers their clinical applications. In this study, we found that the antigen retrieval method strongly influences the extent of nonspecific binding of the antibody-SERS NP conjugates to the tissue. Our SERS labels comprised ca. 70 nm Au nanostars coated with ethylene glycol-modified Raman reporter molecules for hydrophilic stabilization and subsequent covalent bioconjugation to antibodies. We systematically investigated the influence of heat- and protease-induced epitope retrieval (HIER and PIER, respectively) on the immunostaining quality of prostate-specific antigen (PSA) on human prostate tissue sections. The best staining results were obtained with PIER. Pretreatment of the tissue sections by HIER led to selective but nonspecific adsorption of the antibody-Au nanostar conjugates onto epithelial cells, while enzymatic treatment within PIER did not. In addition to gold nanostars, also other types of metal NPs with different shapes and sizes (including ca. 20 nm quasi-spherical Au NPs and ca. 60 nm quasi-spherical Au/Ag nanoshells) as well as tissue sections from different organs (including prostate and breast) were tested; in each case the same tendency was observed, i.e., PIER yielded better results than HIER. Therefore, we recommend PIER for future NP-based tissue immunostaining such as immuno-SERS microscopy. Alternatively, for antigens that can only be unmasked by heating, PEGylation of the NPs is recommended to avoid nonspecific binding.

  9. Enhancement of Pathologist's Routine Practice: Reuse of DNA Extracted from Immunostained Formalin-fixed Paraffin-embedded (FFPE) Slides in Downstream Molecular Analysis of Cancer.

    Science.gov (United States)

    Al-Attas, Asmaa; Assidi, Mourad; Al-Maghrabi, Jaudah; Dallol, Ashraf; Schulten, Hans-Juergen; Abu-Elmagd, Muhammad; Chaudhary, Adeel; Abuzenadah, Adel; Budowle, Bruce; Buhmeida, Abdelbaset; Al-Qahtani, Mohammed

    To date, the conventional formalin-fixed, paraffin-embedded (FFPE) technique is the gold-standard for preserving histomorphology. Once FFPE tissues are stained, slides are routinely archived along with their blocks at biobanks/hospitals. However, the reuse of fixed and stained biospecimens as DNA source is not a common routine practice worldwide and, thus, indicates the need of studies to investigate the feasibility of extracting DNA from already immunohistochemistry (IHC) FFPE-stained slides and its possible reuse in subsequent downstream molecular analyses. FFPE IHC slides from colorectal cancer (CRC) patients were prepared and stored in the CEGMR Biobank. The workflow consists of digitalization of IHC stained slide's image, removing the slide cover-slip, crude dissection and DNA extraction. Following DNA quality assessment, mutation analysis of CTNNB1 and methylation profile of CDH1 were performed. High-quality DNA was obtained allowing 60% concordance between CDH1 methylation and membranous E-cadherin expression pattern. Clean CTNNB1 DNA chromatograms with evenly-spaced peaks were observed. This study is a proof of concept to recycle and reuse DNA from IHC stained slides with suitable concentration and integrity for further downstream molecular applications. These findings will enhance the pathologists' knowledge, attitudes and practices (KAP) towards the use of these biospecimens and support the implementation of this approach in clinical pathology practice. Therefore, the scientific community will benefit from the largest comprehensive database of human fully annotated FFPE biospecimens already available at their disposal in order to demystify the complexity and the heterogeneity of many challenging diseases and foster the transition towards precision medicine. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  10. Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

    Science.gov (United States)

    Seiler, Caroline; Sharpe, Alan; Barrett, J Carl; Harrington, Elizabeth A; Jones, Emma V; Marshall, Gayle B

    2016-01-01

    Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2-14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material.

  11. Proteomic analysis of formalin-fixed paraffin-embedded glomeruli suggests depletion of glomerular filtration barrier proteins in two-kidney, one-clip hypertensive rats.

    Science.gov (United States)

    Finne, Kenneth; Vethe, Heidrun; Skogstrand, Trude; Leh, Sabine; Dahl, Tone D; Tenstad, Olav; Berven, Frode S; Reed, Rolf K; Vikse, Bjørn Egil

    2014-12-01

    It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. In the present study, we used formalin-fixed paraffin-embedded (FFPE) tissue to investigate changes in the glomerular proteome in the non-clipped kidney of two-kidney one-clip (2K1C) hypertensive rats, with special emphasis on the glomerular filtration barrier. 2K1C hypertension was induced in 6-week-old Wistar Hannover rats (n = 6) that were sacrificed 23 weeks later and compared with age-matched sham-operated controls (n = 6). Tissue was stored in FFPE tissue blocks and later prepared on tissue slides for laser microdissection. Glomeruli without severe morphological damage were isolated, and the proteomes were analysed using liquid chromatography-tandem mass spectrometry. 2K1C glomeruli showed reduced abundance of proteins important for slit diaphragm complex, such as nephrin, podocin and neph1. The podocyte foot process had a pattern of reduced abundance of transmembrane proteins but unchanged abundances of the podocyte cytoskeletal proteins synaptopodin and α-actinin-4. Lower abundance of important glomerular basement membrane proteins was seen. Possible glomerular markers of damage with increased abundance in 2K1C were transgelin, desmin and acyl-coenzyme A thioesterase 1. Microdissection and tandem mass spectrometry could be used to investigate the proteome of isolated glomeruli from FFPE tissue. Glomerular filtration barrier proteins had reduced abundance in the non-clipped kidney of 2K1C hypertensive rats. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA.

  12. Chromosomal aberrations in bladder cancer: fresh versus formalin fixed paraffin embedded tissue and targeted FISH versus wide microarray-based CGH analysis.

    Directory of Open Access Journals (Sweden)

    Elena Panzeri

    Full Text Available Bladder carcinogenesis is believed to follow two alternative pathways driven by the loss of chromosome 9 and the gain of chromosome 7, albeit other nonrandom copy number alterations (CNAs were identified. However, confirmation studies are needed since many aspects of this model remain unclear and considerable heterogeneity among cases has emerged. One of the purposes of this study was to evaluate the performance of a targeted test (UroVysion assay widely used for the detection of Transitional Cell Carcinoma (TCC of the bladder, in two different types of material derived from the same tumor. We compared the results of UroVysion test performed on Freshly Isolated interphasic Nuclei (FIN and on Formalin Fixed Paraffin Embedded (FFPE tissues from 22 TCCs and we didn't find substantial differences. A second goal was to assess the concordance between array-CGH profiles and the targeted chromosomal profiles of UroVysion assay on an additional set of 10 TCCs, in order to evaluate whether UroVysion is an adequately sensitive method for the identification of selected aneuploidies and nonrandom CNAs in TCCs. Our results confirmed the importance of global genomic screening methods, that is array based CGH, to comprehensively determine the genomic profiles of large series of TCCs tumors. However, this technique has yet some limitations, such as not being able to detect low level mosaicism, or not detecting any change in the number of copies for a kind of compensatory effect due to the presence of high cellular heterogeneity. Thus, it is still advisable to use complementary techniques such as array-CGH and FISH, as the former is able to detect alterations at the genome level not excluding any chromosome, but the latter is able to maintain the individual data at the level of single cells, even if it focuses on few genomic regions.

  13. A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing.

    Science.gov (United States)

    Snow, Anthony N; Stence, Aaron A; Pruessner, Jonathan A; Bossler, Aaron D; Ma, Deqin

    2014-01-01

    Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column. THREE DNA EXTRACTION METHODS WERE COMPARED: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student's t-test. The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.

  14. High Quality Genomic Copy Number Data from Archival Formalin-Fixed Paraffin-Embedded Leiomyosarcoma: Optimisation of Universal Linkage System Labelling

    Science.gov (United States)

    Salawu, Abdulazeez; Ul-Hassan, Aliya; Hammond, David; Fernando, Malee; Reed, Malcolm; Sisley, Karen

    2012-01-01

    Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment. PMID:23209738

  15. Revealing the Molecular Portrait of Triple Negative Breast Tumors in an Understudied Population through Omics Analysis of Formalin-Fixed and Paraffin-Embedded Tissues.

    Science.gov (United States)

    Vaca-Paniagua, Felipe; Alvarez-Gomez, Rosa María; Maldonado-Martínez, Hector Aquiles; Pérez-Plasencia, Carlos; Fragoso-Ontiveros, Veronica; Lasa-Gonsebatt, Federico; Herrera, Luis Alonso; Cantú, David; Bargallo-Rocha, Enrique; Mohar, Alejandro; Durand, Geoffroy; Forey, Nathalie; Voegele, Catherine; Vallée, Maxime; Le Calvez-Kelm, Florence; McKay, James; Ardin, Maude; Villar, Stéphanie; Zavadil, Jiri; Olivier, Magali

    2015-01-01

    Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor, progesterone receptor and human epidermal receptor 2, is an aggressive form of breast cancer that is more prevalent in certain populations, in particular in low- and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffin embedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. Using dedicated reagents and analysis pipelines, we performed whole exome sequencing and miRNA and mRNA profiling of 12 FFPE tumor tissues collected from pathological archives in Mexico. Sequencing analyses of the tumor tissues and their blood pairs identified TP53 and RB1 genes as the most frequently mutated genes, with a somatic mutation load of 1.7 mutations/exome Mb on average. Transcriptional analyses revealed an overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), a repression of cell cycle control pathways (TP53, RB1), a deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the feasibility of using archived clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing retrospective studies on the search for treatment strategies or on the exploration of the geographic diversity of breast cancer.

  16. Detection of mucormycetes and other pathogenic fungi in formalin fixed paraffin embedded and fresh tissues using the extended region of 28S rDNA.

    Science.gov (United States)

    Gade, Lalitha; Hurst, Steven; Balajee, S Arunmozhi; Lockhart, Shawn R; Litvintseva, Anastasia P

    2017-06-01

    Molecular methods of detection based on DNA-sequencing of the internal transcribed spacer 1 and 2 (ITS1 and ITS2) or 5΄ end region of 28S (D1-D2 region) of ribosomal RNA gene (rDNA) have been used extensively for molecular identification and detection of fungal infections. However, these regions are not always informative for identification of mucormycetes and other rare fungal pathogens as they often contain large introns, heterogenic regions, and/or cannot be PCR-amplified using broad range fungal PCR primers. In addition, because of the difficulties of recovering intact fungal DNA from human specimens, smaller regions of DNA are more useful for the direct detection of fungal DNA in tissues and fluids. In this study, we investigated the utility of 12F/13R PCR primers targeting a 200-230 bp region of the extended 28S region of rDNA for molecular identification of fungal DNA in formalin fixed paraffin embedded tissues and other clinical specimens. We demonstrated that this region can be successfully used for identification of all genera and some species of clinically relevant mucormycetes, as well as other medically important fungi, such as Aspergillus, Fusarium, Coccidioides, and Cryptococcus. We also demonstrated that PCR amplification and direct sequencing of the extended 28S region of rDNA was more sensitive compared to targeting the ITS2 region, as we were able to detect and identify mucormycetes and other fungal pathogens in tissues from patients with histopathological and/or culture evidence of fungal infections that were negative with PCR using ITS-specific primers. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  17. Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

    Science.gov (United States)

    Zhang, Shenli; Tan, Iain B; Sapari, Nur S; Grabsch, Heike I; Okines, Alicia; Smyth, Elizabeth C; Aoyama, Toru; Hewitt, Lindsay C; Inam, Imran; Bottomley, Dan; Nankivell, Matthew; Stenning, Sally P; Cunningham, David; Wotherspoon, Andrew; Tsuburaya, Akira; Yoshikawa, Takaki; Soong, Richie; Tan, Patrick

    2015-05-01

    DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  18. Next-Generation Sequencing of RNA and DNA Isolated from Paired Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Samples of Human Cancer and Normal Tissue

    Science.gov (United States)

    Hedegaard, Jakob; Thorsen, Kasper; Lund, Mette Katrine; Hein, Anne-Mette K.; Hamilton-Dutoit, Stephen Jacques; Vang, Søren; Nordentoft, Iver; Birkenkamp-Demtröder, Karin; Kruhøffer, Mogens; Hager, Henrik; Knudsen, Bjarne; Andersen, Claus Lindbjerg; Sørensen, Karina Dalsgaard; Pedersen, Jakob Skou; Ørntoft, Torben Falck; Dyrskjøt, Lars

    2014-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70–80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available. PMID:24878701

  19. Fit for genomic and proteomic purposes: Sampling the fitness of nucleic acid and protein derivatives from formalin fixed paraffin embedded tissue.

    Directory of Open Access Journals (Sweden)

    Anna Yakovleva

    Full Text Available The demand for nucleic acid and protein derivatives from formalin-fixed paraffin-embedded (FFPE tissue has greatly increased due to advances in extraction and purification methods, making these derivatives available for numerous genomic and proteomic platforms. Previously, DNA, RNA, microRNA (miRNA, or protein derived from FFPE tissue blocks were considered "unfit" for such platforms, as the process of tissue immobilization by FFPE resulted in cross-linked, fragmented, and chemically modified macromolecules. We conducted a systematic examination of nucleic acids and proteins co-extracted from 118 FFPE blocks sampled from the AIDS and Cancer Specimen Resource (ACSR at The George Washington University after stratification by storage duration and the three most common tumor tissue types at the ACSR (adenocarcinoma, squamous cell carcinoma, and papillary carcinoma. DNA, RNA, miRNA, and protein could be co-extracted from 98% of the FFPE blocks sampled, with DNA and miRNA "fit" for diverse genomic purposes including sequencing. While RNA was the most labile of the FFPE derivatives, especially when assessed by RNA integrity number (RIN, it was still "fit" for genomic methods that use smaller sequence lengths, e.g., quantitative PCR. While more than half of the protein derivatives were fit for proteomic purposes, our analyses indicated a significant interaction effect on the absorbance values for proteins derived from FFPE, implying that storage duration may affect protein derivatives differently by tumor tissue type. The mean absorbance value for proteins derived from more recently stored FFPE was greater than protein derived from older FFPE, with the exception of adenocarcinoma tissue. Finally, the fitness of one type of derivative was weakly associated with the fitness of derivatives co-extracted from the same FFPE block. The current study used several novel quality assurance approaches and metrics to show that archival FFPE tissue blocks are a

  20. Early experience with formalin-fixed paraffin-embedded (FFPE) based commercial clinical genomic profiling of gliomas-robust and informative with caveats.

    Science.gov (United States)

    Movassaghi, Masoud; Shabihkhani, Maryam; Hojat, Seyed A; Williams, Ryan R; Chung, Lawrance K; Im, Kyuseok; Lucey, Gregory M; Wei, Bowen; Mareninov, Sergey; Wang, Michael W; Ng, Denise W; Tashjian, Randy S; Magaki, Shino; Perez-Rosendahl, Mari; Yang, Isaac; Khanlou, Negar; Vinters, Harry V; Liau, Linda M; Nghiemphu, Phioanh L; Lai, Albert; Cloughesy, Timothy F; Yong, William H

    2017-08-01

    Commercial targeted genomic profiling with next generation sequencing using formalin-fixed paraffin embedded (FFPE) tissue has recently entered into clinical use for diagnosis and for the guiding of therapy. However, there is limited independent data regarding the accuracy or robustness of commercial genomic profiling in gliomas. As part of patient care, FFPE samples of gliomas from 71 patients were submitted for targeted genomic profiling to one commonly used commercial vendor, Foundation Medicine. Genomic alterations were determined for the following grades or groups of gliomas; Grade I/II, Grade III, primary glioblastomas (GBMs), recurrent primary GBMs, and secondary GBMs. In addition, FFPE samples from the same patients were independently assessed with conventional methods such as immunohistochemistry (IHC), Quantitative real-time PCR (qRT-PCR), or Fluorescence in situ hybridization (FISH) for three genetic alterations: IDH1 mutations, EGFR amplification, and EGFRvIII expression. A total of 100 altered genes were detected by the aforementioned targeted genomic profiling assay. The number of different genomic alterations was significantly different between the five groups of gliomas and consistent with the literature. CDKN2A/B, TP53, and TERT were the most common genomic alterations seen in primary GBMs, whereas IDH1, TP53, and PIK3CA were the most common in secondary GBMs. Targeted genomic profiling demonstrated 92.3%-100% concordance with conventional methods. The targeted genomic profiling report provided an average of 5.5 drugs, and listed an average of 8.4 clinical trials for the 71 glioma patients studied but only a third of the trials were appropriate for glioma patients. In this limited comparison study, this commercial next generation sequencing based-targeted genomic profiling showed a high concordance rate with conventional methods for the 3 genetic alterations and identified mutations expected for the type of glioma. While it may not be feasible to

  1. Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process.

    Science.gov (United States)

    Wen, Xianyu; Jeong, Seorin; Kim, Younghoon; Bae, Jeong Mo; Cho, Nam Yun; Kim, Jung Ho; Kang, Gyeong Hoon

    2017-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively. Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95 °C for 30 min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer. Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the

  2. Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

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    Adam Kotorashvili

    Full Text Available BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. PRINCIPAL FINDINGS: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and -RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. SIGNIFICANCE: We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of

  3. A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds.

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    Dana A M Mustafa

    Full Text Available Gene expression profiling of tumors is a successful tool for the discovery of new cancer biomarkers and potential targets for the development of new therapeutic strategies. Reliable profiling is preferably performed on fresh frozen (FF tissues in which the quality of nucleic acids is better preserved than in formalin-fixed paraffin-embedded (FFPE material. However, since snap-freezing of biopsy materials is often not part of daily routine in pathology laboratories, one may have to rely on archival FFPE material. Procedures to retrieve the RNAs from FFPE materials have been developed and therefore, datasets obtained from FFPE and FF materials need to be made compatible to ensure reliable comparisons are possible.To develop an efficient method to compare gene expression profiles obtained from FFPE and FF samples using the same platform.Twenty-six FFPE-FF sample pairs of the same tumors representing various cancer types, and two FFPE-FF sample pairs of breast cancer cell lines, were included. Total RNA was extracted and gene expression profiling was carried out using Illumina's Whole-Genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL V3 arrays, enabling the simultaneous detection of 24,526 mRNA transcripts. A sample exclusion criterion was created based on the expression of 11 stably expressed reference genes. Pearson correlation at the probe level was calculated for paired FFPE-FF, and three cut-off values were chosen. Spearman correlation coefficients between the matched FFPE and FF samples were calculated for three probe lists with varying levels of significance and compared to the correlation based on all measured probes. Unsupervised hierarchical cluster analysis was performed to verify performance of the included probe lists to compare matched FPPE-FF samples.Twenty-seven FFPE-FF pairs passed the sample exclusion criterion. From the profiles of 27 FFPE and FF matched samples, the best correlating probes were identified

  4. Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

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    Liu, Christina; Lin, Juan; Ye, Kenny; Kim, Ryung; Hazan, Rachel; Rohan, Thomas; Fineberg, Susan; Loudig, Olivier

    2012-01-01

    Background Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. Significance We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which

  5. Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods.

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    Watanabe, Mototsugu; Hashida, Shinsuke; Yamamoto, Hiromasa; Matsubara, Takehiro; Ohtsuka, Tomoaki; Suzawa, Ken; Maki, Yuho; Soh, Junichi; Asano, Hiroaki; Tsukuda, Kazunori; Toyooka, Shinichi; Miyoshi, Shinichiro

    2017-09-01

    Techniques for the extraction and use of nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissues, preserved over long time periods in libraries, have been developed. However, DNA extracted from FFPE tissues is generally damaged, and long-term storage may affect DNA quality. Therefore, it is important to elucidate the effect of long-term storage on FFPE tissues and evaluate the techniques used to extract DNA from them. In the present study, the yield, purity, and integrity of DNA in FFPE tissue samples was evaluated. Two DNA extraction techniques were used: A silica-binding DNA collection method using QIAamp DNA FFPE Tissue kit (QIA) and a total tissue DNA collection method using a WaxFree DNA extraction kit (WAX). A total of 25 FFPE tissues from lung adenocarcinomas were studied, which had been surgically resected and fixed at Okayama University Hospital prior to examination and subsequent storage at room temperature for 0.5, 3, 6, 9 and 12 years. Extracted DNA was quantified using ultraviolet absorbance, fluorescent dye, and quantitative polymerase chain reaction (qPCR). The quality of the DNA was defined by the absorbance ratio of 260 to 280 nm (A260/280) and Q-score, which is the quantitative value of qPCR product size ratio. The results demonstrated that the yield of total DNA extracted using WAX was significantly greater than when QIA was used (PDNA extracted using WAX included more contaminants and was significantly more fragmented compared with DNA extracted using QIA (PDNA yield or DNA purity, although it did significantly contribute to increased DNA degradation for both QIA and WAX extraction (QIA P=0.02, WAX P=0.03; 0.5 years vs. 3 years, QIA Pextraction methods are viable depending on whether high yield or high quality of extracted DNA is required. However, due to the increased degradation with age, storage time limits the available DNA in FFPE tissues regardless of the extraction method.

  6. Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.

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    Vukmirovic, Milica; Herazo-Maya, Jose D; Blackmon, John; Skodric-Trifunovic, Vesna; Jovanovic, Dragana; Pavlovic, Sonja; Stojsic, Jelena; Zeljkovic, Vesna; Yan, Xiting; Homer, Robert; Stefanovic, Branko; Kaminski, Naftali

    2017-01-12

    Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues. We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four. Our results demonstrate that RNA sequencing of RNA

  7. Interpretation of genome-wide infinium methylation data from ligated DNA in formalin-fixed, paraffin-embedded paired tumor and normal tissue

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    Jasmine Farzana

    2012-02-01

    Full Text Available Abstract Background Formalin-fixed, paraffin-embedded (FFPE samples are a highly desirable resource for epigenetic studies, but there is no suitable platform to assay genome-wide methylation in these widely available resources. Recently, Thirlwell et al. (2010 have reported a modified ligation-based DNA repair protocol to prepare FFPE DNA for the Infinium methylation assay. In this study, we have tested the accuracy of methylation data obtained with this modification by comparing paired fresh-frozen (FF and FFPE colon tissue (normal and tumor from colorectal cancer patients. We report locus-specific correlation and concordance of tumor-specific differentially methylated loci (DML, both of which were not previously assessed. Methods We used Illumina's Infinium Methylation 27K chip for 12 pairs of FF and 12 pairs of FFPE tissue from tumor and surrounding healthy tissue from the resected colon of the same individual, after repairing the FFPE DNA using Thirlwell's modified protocol. Results For both tumor and normal tissue, overall correlation of β values between all loci in paired FF and FFPE was comparable to previous studies. Tissue storage type (FF or FFPE was found to be the most significant source of variation rather than tissue type (normal or tumor. We found a large number of DML between FF and FFPE DNA. Using ANOVA, we also identified DML in tumor compared to normal tissue in both FF and FFPE samples, and out of the top 50 loci in both groups only 7 were common, indicating poor concordance. Likewise, while looking at the correlation of individual loci between FFPE and FF across the patients, less than 10% of loci showed strong correlation (r ≥ 0.6. Finally, we checked the effect of the ligation-based modification on the Infinium chemistry for SNP genotyping on an independent set of samples, which also showed poor performance. Conclusion Ligation of FFPE DNA prior to the Infinium genome-wide methylation assay may detect a reasonable

  8. An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues.

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    Viegas, M S; Martins, T C; Seco, F; do Carmo, A

    2007-01-01

    Interference by autofluorescence is one of the major shortcomes of immunofluorescence analysis by confocal laser scanning microscopy (CLSM). CLSM requires minimal tissue autofluorescence and reduced unspecific fluorescence background, requisites that become more critical when direct immunofluorescence studies are concerned. To control autofluorescence, different reagents and treatments can be used. Until now, the efficacy of the processes described depended on the tissue type and on the processing technique, no general recipe for the control of autofluorescence being available. Using paraffin sections of archival formalin-fixed murine liver, kidney and pancreas, we have found that previously described techniques were not able to reduce autofluorescence to levels that allowed direct immunofluorescence labelling. In this work, we aimed at improving currently described methodologies so that they would allow reduction of the autofluorescent background without affecting tissue integrity or direct immunofluorescence labelling. We have found that the combination of short-duration, high-intensity UV irradiation and Sudan Black B was the best approach to reduce autofluorescence in highly vascularised, high lipofuscins' content tissues, such as murine liver and kidney, and poorly vascularised, low lipofuscins' content tissues such as the pancreas. In addition, we herein show that this methodology is highly effective in reducing autofluorescent background to levels that allow detection of specific signals by direct immunofluorescence.

  9. Real-time PCR assay based on the differential expression of microRNAs and protein-coding genes for molecular classification of formalin-fixed paraffin embedded medulloblastomas.

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    Kunder, Ratika; Jalali, Rakesh; Sridhar, Epari; Moiyadi, Aliasgar; Goel, Naina; Goel, Atul; Gupta, Tejpal; Krishnatry, Rahul; Kannan, Sadhana; Kurkure, Purna; Deopujari, Chandrashekhar; Shetty, Prakash; Biyani, Naresh; Korshunov, Andrey; Pfister, Stefan M; Northcott, Paul A; Shirsat, Neelam Vishwanath

    2013-12-01

    Medulloblastoma has recently been found to consist of 4 molecularly and clinically distinct subgroups: WNT, Sonce hedgehog (SHH), Group 3, and Group 4. Deregulated microRNA expression is known to contribute to pathogenesis and has been shown to have diagnostic and prognostic potential in the classification of various cancers. Molecular subgrouping and microRNA expression analysis of 44 frozen and 59 formalin-fixed paraffin embedded medulloblastomas from an Indian cohort were carried out by real-time RT-PCR assay. The differential expression of 9 microRNAs in the 4 molecular subgroups was validated in a set of 101 medulloblastomas. The tumors in the WNT subgroup showed significant (P classification of medulloblastomas into the 4 molecular subgroups was obtained using a set of 12 protein-coding genes and 9 microRNAs as markers in a real-time RT-PCR assay with an accuracy of 97% as judged by the Prediction Analysis of Microarrays. Age at diagnosis, histology, gender-related incidence, and the relative survival rates of the 4 molecular subgroups in the present Indian cohort were found to be similar to those reported for medulloblastomas from the American and European subcontinent. Non-WNT, non-SHH medulloblastomas underexpressing miR-592 or overexpressing miR-182 were found to have significantly inferior survival rates, indicating utility of these miRNAs as markers for risk stratification. The microRNA based real-time PCR assay is rapid, simple, inexpensive, and useful for molecular classification and risk stratification of medulloblastomas, in particular formalin-fixed paraffin embedded tissues, wherein the expression profile of protein-coding genes is often less reliable due to RNA fragmentation.

  10. Expression profiling of formalin-fixed paraffin-embedded primary breast tumors using cancer-specific and whole genome gene panels on the DASL® platform

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    Cunningham Julie M

    2010-12-01

    Full Text Available Abstract Background The cDNA-mediated Annealing, extension, Selection and Ligation (DASL assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panelv1 (1.5K and 24,526-gene panel (24K platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+ and 11 HER2-negative (HER2- paraffin-embedded breast tumors. Methods Bland-Altman plots and Spearman correlations evaluated intra/inter-panel agreement of normalized expression values. Unequal-variance t-statistics tested for differences in expression levels between HER2 + and HER2 - tumors. Regulatory network analysis was performed using Metacore (GeneGo Inc., St. Joseph, MI. Results Technical replicate correlations ranged between 0.815-0.956 and 0.986-0.997 for the 1.5K and 24K panels, respectively. Inter-panel correlations of expression values for the common 498 genes across the two panels ranged between 0.485-0.573. Inter-panel correlations of expression values of 17 probes with base-pair sequence matches between the 1.5K and 24K panels ranged between 0.652-0.899. In both panels, erythroblastic leukemia viral oncogene homolog 2 (ERBB2 was the most differentially expressed gene between the HER2 + and HER2 - tumors and seven additional genes had p-values |0.5| in expression between HER2 + and HER2 - tumors: topoisomerase II alpha (TOP2A, cyclin a2 (CCNA2, v-fos fbj murine osteosarcoma viral oncogene homolog (FOS, wingless-type mmtv integration site family, member 5a (WNT5A, growth factor receptor-bound protein 7 (GRB7, cell division cycle 2 (CDC2, and baculoviral iap repeat-containing protein 5 (BIRC5. The top 52 discriminating probes from the 24K panel are enriched with genes belonging to the regulatory networks centered around v-myc avian myelocytomatosis viral oncogene homolog (MYC, tumor protein p53 (TP53, and estrogen receptor

  11. Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.

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    Huang, Meggie Mo Chao; Leong, Sai Mun; Chua, Hui Wen; Tucker, Steven; Cheong, Wai Chye; Chiu, Lily; Li, Mo-Huang; Koay, Evelyn Siew-Chuan

    2014-08-01

    Among patients with colorectal cancer (CRC), KRAS mutations were reported to occur in 30-51 % of all cases. CRC patients with KRAS mutations were reported to be non-responsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatment in many clinical trials. Hence, accurate detection of KRAS mutations would be critical in guiding the use of anti-EGFR MoAb therapies in CRC. In this study, we carried out a detailed investigation of the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR), employing WT KRAS locked nucleic acid blockers, and Sanger sequencing, for KRAS mutation detection in rare cells. Analyses were first conducted on cell lines to optimize the assay protocol which was subsequently applied to peripheral blood and tissue samples from patients with CRC. The optimized assay provided a superior sensitivity enabling detection of as little as two cells with mutated KRAS in the background of 10(4) WT cells (0.02 %). The feasibility of this assay was further investigated to assess the KRAS status of 45 colorectal tissue samples, which had been tested previously, using a conventional PCR sequencing approach. The analysis showed a mutational discordance between these two methods in 4 of 18 WT cases. Our results present a simple, effective, and robust method for KRAS mutation detection in both paraffin embedded tissues and circulating tumour cells, at single-cell level. The method greatly enhances the detection sensitivity and alleviates the need of exhaustively removing co-enriched contaminating lymphocytes.

  12. RNA analysis of inner ear cells from formalin fixed paraffin embedded (FFPE) archival human temporal bone section using laser microdissection--a technical report.

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    Kimura, Yurika; Kubo, Sachiho; Koda, Hiroko; Shigemoto, Kazuhiro; Sawabe, Motoji; Kitamura, Ken

    2013-08-01

    Molecular analysis using archival human inner ear specimens is challenging because of the anatomical complexity, long-term fixation, and decalcification. However, this method may provide great benefit for elucidation of otological diseases. Here, we extracted mRNA for RT-PCR from tissues dissected from archival FFPE human inner ears by laser microdissection. Three human temporal bones obtained at autopsy were fixed in formalin, decalcified by EDTA, and embedded in paraffin. The samples were isolated into spiral ligaments, outer hair cells, spiral ganglion cells, and stria vascularis by laser microdissection. RNA was extracted and heat-treated in 10 mM citrate buffer to remove the formalin-derived modification. To identify the sites where COCH and SLC26A5 mRNA were expressed, semi-nested RT-PCR was performed. We also examined how long COCH mRNA could be amplified by semi-nested RT-PCR in archival temporal bone. COCH was expressed in the spiral ligament and stria vascularis. However, SLC26A5 was expressed only in outer hair cells. The maximum base length of COCH mRNA amplified by RT-PCR was 98 bp in 1 case and 123 bp in 2 cases. We detected COCH and SLC26A5 mRNA in specific structures and cells of the inner ear from archival human temporal bone. Our innovative method using laser microdissection and semi-nested RT-PCR should advance future RNA study of human inner ear diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Reference size-matching, whole-genome amplification, and fluorescent labeling as a method for chromosomal microarray analysis of clinically actionable copy number alterations in formalin-fixed, paraffin-embedded tumor tissue.

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    Gunn, Shelly R; Govender, Shailin; Sims, Cynthe L; Khurana, Aditi; Koo, Samuel; Scoggin, Jayne; Moore, Mathew W; Cotter, Philip D

    2018-02-19

    Cancer genome copy number alterations (CNAs) assist clinicians in selecting targeted therapeutics. Solid tumor CNAs are most commonly evaluated in formalin-fixed, paraffin-embedded (FFPE) tissue by fluorescence in situ hybridization. Although fluorescence in situ hybridization is a sensitive and specific assay for interrogating pre-selected genomic regions, it provides no information about co-existing clinically significant copy number changes. Chromosomal microarray analysis is an alternative DNA-based method for interrogating genome-wide CNAs in solid tumors. However, DNA extracted from FFPE tumor tissue produces an essential, yet problematic, sample type. The College of American Pathologists/American Society of Clinical Oncology guidelines for optimal tumor tissue handling published in 2007 for breast cancer, and in 2016 for gastroesophageal adenocarcinomas are lacking for other solid tumors. Thus, cold ischemia times are seldom monitored in non-breast cancer, non-gastroesophageal adenocarcinomas, and all tumor biospecimens are affected by chemical fixation. Although intended to preserve specimens for long-term storage, formalin fixation causes loss of genetic information through DNA damage. Here, we describe a reference size matching, whole-genome amplification, and fluorescent labeling method for FFPE-derived DNA designed to improve chromosomal microarray results from sub-optimal nucleic acids and salvage highly degraded samples. With this technological advance, whole-genome copy number analysis of tumor DNA can be reliably performed in the clinical laboratory for a wide variety of tissue conditions and tumor types. Copyright © 2018. Published by Elsevier Inc.

  14. Comparison of two methods to extract DNA from formalin-fixed, paraffin-embedded tissues and their impact on EGFR mutation detection in non-small cell lung carcinoma.

    Science.gov (United States)

    Hu, Yu-Chang; Zhang, Qian; Huang, Yan-Hua; Liu, Yu-Fei; Chen, Hong-Lei

    2014-01-01

    Molecular pathology tests are often carried for clinicopathological diagnosis and pathologists have established large collections of formalin-fixed, paraffin-embedded tissue (FFPE) banks. However, extraction of DNA from FFPE is a laborious and challenging for researchers in clinical laboratories. The aim of this study was to compare two widely used DNA extraction methods: using a QIAamp DNA FFPE kit from Qiagen and a Cobas Sample Preparation Kit from Roche, and evaluated the effect of the DNA quality on molecular diagnostics. DNA from FFPE non-small cell lung carcinoma tissues including biopsy and surgical specimens was extracted with both QIAamp DNA FFPE and Cobas Sample Preparation Kits and EGFR mutations of non-small cell lung carcinomas were detected by real-time quantitative PCR using the extracted DNA. Our results showed that DNA extracted by QIAamp and Cobas methods were both suitable to detect downstream EGFR mutation in surgical specimens. Howover, Cobas method could yield more DNA from biopsy specimens, and gain much better EGFR mutation results.

  15. Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation.

    Science.gov (United States)

    Einaga, Naoki; Yoshida, Akio; Noda, Hiroko; Suemitsu, Masaaki; Nakayama, Yuki; Sakurada, Akihisa; Kawaji, Yoshiko; Yamaguchi, Hiromi; Sasaki, Yasushi; Tokino, Takashi; Esumi, Mariko

    2017-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: 'microgenomics'. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.

  16. Evaluation of Mutational Testing of Preneoplastic Barrett's Mucosa by Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Endoscopic Samples for Detection of Concurrent Dysplasia and Adenocarcinoma in Barrett's Esophagus

    Science.gov (United States)

    Del Portillo, Armando; Lagana, Stephen M.; Yao, Yuan; Uehara, Takeshi; Jhala, Nirag; Ganguly, Tapan; Nagy, Peter; Gutierrez, Jorge; Luna, Aesis; Abrams, Julian; Liu, Yang; Brand, Randall; Sepulveda, Jorge L.; Falk, Gary W.; Sepulveda, Antonia R.

    2016-01-01

    Barrett's intestinal metaplasia (BIM) may harbor genomic mutations before the histologic appearance of dysplasia and cancer and requires frequent surveillance. We explored next-generation sequencing to detect mutations with the analytical sensitivity required to predict concurrent high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus by testing nonneoplastic BIM. Formalin-fixed, paraffin-embedded (FFPE) routine biopsy or endoscopic mucosal resection samples from 32 patients were tested: nonprogressors to HGD or EAC (BIM-NP) with BIM, who never had a diagnosis of dysplasia or EAC (N = 13); progressors to HGD or EAC (BIM-P) with BIM and a worse diagnosis of HGD or EAC (N = 15); and four BIM-negative samples. No mutations were detected in the BIM-NP (0 of 13) or BIM-negative samples, whereas the BIM-P samples had mutations in 6 (75%) of 8 cases in TP53, APC, and CDKN2A (P = 0.0005), detected in samples with as low as 20% BIM. We found that next-generation sequencing from routine FFPE nonneoplastic Barrett's esophagus samples can detect multiple mutations in minute areas of BIM with high analytical sensitivity. Next-generation sequencing panels for detection of TP53 and possibly combined mutations in other genes, such as APC and CDKN2A, may be useful in the clinical setting to improve dysplasia and cancer surveillance in patients with Barrett's esophagus. PMID:26068095

  17. Protocol for qRT-PCR analysis from formalin fixed paraffin embedded tissue sections from diffuse large b-cell lymphoma: Validation of the six-gene predictor score

    Science.gov (United States)

    Tekin, Nilgun; Conget, Paulette; Bruna, Flavia; Timar, Botond; Gagyi, Eva; Basak, Ranjan; Naik, Omkar; Auewarakul, Chirayu; Sritana, Narongrit; Levy, Debora; Cerci, Juliano Julio; Bydlowski, Sergio Paulo; Pereira, Juliana; Dimamay, Mark Pierre; Natividad, Filipinas; Chung, June-Key; Belder, Nevin; Kuzu, Isinsu; Paez, Diana; Dondi, Maurizio; Carr, Robert

    2016-01-01

    As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting. PMID:27825111

  18. Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

    Directory of Open Access Journals (Sweden)

    Paula R. Almeida

    2012-08-01

    Full Text Available The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC and polymerase chain reaction (PCR or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs or frozen (tissue pieces. M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

  19. High-risk Human Papillomavirus Determination in Formalin-fixed, Paraffin-embedded Cervical Tissue Using the Roche Cobas 4800 System: A Comparative Study With Liquid-based Cytology.

    Science.gov (United States)

    Tardío, Juan C; Cambero, Olivia; Sánchez-Estévez, Carolina; Sánchez-García, Ana B; Angulo, Fernando; Moreno, Amalia

    2017-11-14

    Roche cobas 4800 human papillomavirus (HPV) test is an automated real-time polymerase chain reaction-based system that allows the simultaneous detection of 14 human papillomavirus high-risk (HR-HPV) genotypes. This test is Food and Drug Administration approved since 2011 for HPV determination in liquid-based cytologic samples, but a clinically validated technique for formalin-fixed, paraffin-embedded (FFPE) tissue specimens is presently not commercially available. In our laboratory, we have developed an HPV detection procedure in FFPE tissue by cobas 4800 HPV test. In order to validate our method, we retrospectively studied 165 FFPE cervical biopsy and conization specimens with varied diagnoses from our files. In 50 of them, we contrasted the results with those obtained from simultaneous liquid-based cytologies from the same patients. Finally, seeking the possible complementary clinical usefulness of the procedure, we compared the HPV genotypes detected in cervical intraepithelial neoplasia grade 1 (CIN1)-diagnosed biopsies from 20 patients with a subsequent high-grade CIN (CIN2+) diagnosis with those from another group of 20 patients without a posterior CIN2+ diagnosis. Eighty-seven percent of the assays provided informative results. HR-HPV was detected in 28 of 32 (88%) invasive cervical squamous carcinomas. Coincidental HR-HPV genotypes were obtained in 32 of 50 (64%) cases with simultaneous cervical biopsy and liquid-based cytologic samples. A significant higher risk of progression to CIN2+ was found when HPV16 (P=0.022) or any HR-HPV genotype (P=0.037) was detected in CIN1 biopsies. The reported procedure provides an automated, technically time-saving, easy to integrate into laboratory routine, and reliable method of HR-HPV determination in FFPE specimens.

  20. Technical Advances for the Clinical Genomic Evaluation of Sudden Cardiac Death: Verification of Next-Generation Sequencing Panels for Hereditary Cardiovascular Conditions Using Formalin-Fixed Paraffin-Embedded Tissues and Dried Blood Spots.

    Science.gov (United States)

    Baudhuin, Linnea M; Leduc, Charles; Train, Laura J; Avula, Rajeswari; Kluge, Michelle L; Kotzer, Katrina E; Lin, Peter T; Ackerman, Michael J; Maleszewski, Joseph J

    2017-12-01

    Postmortem genetic testing for heritable cardiovascular (CV) disorders is often lacking because ideal specimens (ie, whole blood) are not retained routinely at autopsy. Formalin-fixed paraffin-embedded tissue (FFPET) is ubiquitously collected at autopsy, but DNA quality hampers its use with traditional sequencing methods. Targeted next-generation sequencing may offer the ability to circumvent such limitations, but a method has not been previously described. The primary aim of this study was to develop and evaluate the use of FFPET for heritable CV disorders via next-generation sequencing. Nineteen FFPET (heart) and blood (whole blood or dried blood spot) specimens underwent targeted next-generation sequencing using a custom panel of 101 CV-associated genes. Nucleic acid yield and quality metrics were evaluated in relation to FFPET specimen age (6 months to 15 years; n=14) and specimen type (FFPET versus whole blood and dried blood spot; n=12). Four FFPET cases with a clinical phenotype of heritable CV disorder were analyzed. Accuracy and precision were 100% concordant between all sample types, with read depths >100× for most regions tested. Lower read depth, as low as 40×, was occasionally observed with FFPET and dried blood spot. High-quality DNA was obtained from FFPET samples as old as 15 years. Genomic analysis of FFPET from the 4 phenotype-positive/genotype unknown cases all revealed putative disease-causing variants. Similar performance characteristics were observed for next-generation sequencing of FFPET, whole blood, and dried blood spot in the evaluation of inherited CV disorders. Although blood is preferable for genetic analyses, this study offers an alternative when only FFPET is available. © 2017 American Heart Association, Inc.

  1. Chromogenic In Situ Hybridization and p16/Ki67 Dual Staining on Formalin-Fixed Paraffin-Embedded Cervical Specimens: Correlation with HPV-DNA Test, E6/E7 mRNA Test, and Potential Clinical Applications

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    Roberta Zappacosta

    2013-01-01

    Full Text Available Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16INK4a/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+. p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16INK4a/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16INK4a/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

  2. Chromogenic In Situ Hybridization and p16/Ki67 Dual Staining on Formalin-Fixed Paraffin-Embedded Cervical Specimens: Correlation with HPV-DNA Test, E6/E7 mRNA Test, and Potential Clinical Applications

    Science.gov (United States)

    Zappacosta, Roberta; Colasante, Antonella; Viola, Patrizia; D'Antuono, Tommaso; Lattanzio, Giuseppe; Capanna, Serena; Gatta, Daniela Maria Pia; Rosini, Sandra

    2013-01-01

    Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16INK4a/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16INK4a/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16INK4a/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure. PMID:24369532

  3. The storage period of the formalin-fixed paraffin-embedded tumor blocks does not influence the concentration and purity of the isolated DNA in a series of 83 renal and thyroid carcinomas.

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    Nechifor-Boilă, Adela Corina; Loghin, Andrada; Vacariu, Victor; Halaţiu, Vasile Bogdan; Borda, Angela

    2015-01-01

    Optimal recovery of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues is highly dependent on a series of pre-extraction steps, mainly related (but not limited) to fixation. The aim of our study was to investigate if the storage period of the FFPE blocks had a significant effect on the isolated DNA. We examined the quantity and purity of the isolated DNA from 83 FFPE blocks, corresponding to malignant thyroid (n=28) and renal (n=55) carcinomas that had been stored in our department for up to eight years. The DNA extraction protocol was based on a precipitation method (MasterPure™ DNA Purification Kit, Epicentre), in accordance to the manufacturer instructions, optimized in our laboratory. A spectrophotometer was used to determine the yield (A260) and purity (A260/A280 ratio) of the isolated DNA. We successfully isolated good DNA quantity and purity from all our study cases (mean concentration: 223.4 ± 104.16 ng/μL; mean A260/A280 ratio: 1.68 ± 0.09). Moreover, no statistically significant differences were observed between tumor blocks stored for 2-3 years and 7-8 years, respectively, both in terms of DNA quantity (p=0.196) and purity (p=0.663). In conclusion, we successfully validated an efficient, reproducible DNA extraction technique that provided a good range of DNA concentrations and purity, regardless the type of tissue (thyroid or kidney). Moreover, we demonstrated that the storage period of the FFPE blocks does not have a significant influence on the DNA quantity and purity.

  4. Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples.

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    Annika Mohr

    Full Text Available Immunohistochemistry (IHC is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1, progesterone receptor (PGR, prolactin receptor (PRLR and growth hormone receptor (GHR gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.

  5. Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

    Science.gov (United States)

    Arvia, Rosaria; Sollai, Mauro; Pierucci, Federica; Urso, Carmelo; Massi, Daniela; Zakrzewska, Krystyna

    2017-08-01

    Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥10 5 copies per reaction, while the upper limit of ddPCR was 10 4 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. HPV16 detection by qPCR method in relation to quantity and quality of DNA extracted from archival formalin fixed and paraffin embedded head and neck cancer tissues by three commercially available kits.

    Science.gov (United States)

    Biesaga, Beata; Janecka, Anna; Mucha-Małecka, Anna; Adamczyk, Agnieszka; Szostek, Sława; Słonina, Dorota; Halaszka, Krzysztof; Przewoźnik, Marcin

    2016-10-01

    The aim of the present study was to compare HPV16 detection by quantitative polymerase chain reaction (qPCR) in relation to the quantity and quality of DNA isolated from 21 formalin fixed and paraffin embedded (FFPE) head and neck cancer tissues by three commercially available kits: EX-WAX™ DNA Extraction Kit (M) (Merck Millipore, Darmstadt, Germany), QIAamp(®) DNA FFPE Tissue (Q) (Qiagen, Hilden, Germany) and ReliaPrep™ FFPE gDNA Miniprep System (P) (Promega, Madison, USA). Quantity of extracted DNA was assessed spectrophometrically and fluorometrically. Its quality was analyzed using A260/280 and A260/230 ratios and the β-actin fragment amplifiability in qPCR. HPV16 presence was detected by qPCR, using specific primers and TaqMan probe. HPV infection was found in 8 DNA samples extracted with M kit (38.1%) and in 7 (33.3%) isolated with Q and P kits. Three samples from M and Q kits were characterized by HPV16 positivity and lack of β-actin amplifiability. They had significantly lower A260/280 ratio (M: 1.6±0.0, p=0.044 and Q: 1.7±0.0, p=0.016) compared to samples with both fragments amplification (M: 1.7±0.0 and Q: 1.9±0.0). Therefore, for HPV detection by qPCR in FFPE tissues we recommend ReliaPrep™ FFPE gDNA Miniprep System. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Nm23/nucleoside diphosphate kinase-A as a potent prognostic marker in invasive pancreatic ductal carcinoma identified by proteomic analysis of laser micro-dissected formalin-fixed paraffin-embedded tissue

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    Takadate Tatsuyuki

    2012-06-01

    Full Text Available Abstract Background Pancreatic cancer is among the most lethal malignancies worldwide. This study aimed to identify a novel prognostic biomarker, facilitating treatment selection, using mass spectrometry (MS-based proteomic analysis with formalin-fixed paraffin-embedded (FFPE tissue. Results The two groups with poor prognosis (n = 4 and with better prognosis (n = 4 had been carefully chosen among 96 resected cases of pancreatic cancer during 1998 to 2007 in Tohoku University Hospital. Although those 2 groups had adjusted background (UICC-Stage IIB, Grade2, R0, gemcitabine adjuvant, there was a significant difference in postoperative mean survival time (poor 21.0 months, better 58.1 months, P = 0.0067. Cancerous epithelial cells collected from FFPE tissue sections by laser micro-dissection (LMD were processed for liquid chromatography-tandem mass spectrometry (LC-MS/MS. In total, 1099 unique proteins were identified and 6 proteins showed different expressions in the 2 groups by semi-quantitative comparison. Among these 6 proteins, we focused on Nm23/Nucleoside Diphosphate Kinase A (NDPK-A and immunohistochemically confirmed its expression in the cohort of 96 cases. Kaplan-Meier analysis showed high Nm23/NDPK-A expression to correlate with significantly worse overall survival (P = 0.0103. Moreover, in the multivariate Cox regression model, Nm23/NDPK-A over-expression remained an independent predictor of poor survival with a hazard ratio of 1.97 (95% CI 1.16-3.56, P = 0.0110. Conclusions We identified 6 candidate prognostic markers for postoperative pancreatic cancer using FFPE tissues and immunohistochemically demonstrated high Nm23/NDPK-A expression to be a useful prognostic marker for pancreatic cancer.

  8. Improving the Prediction of Prostate Cancer Overall Survival by Supplementing Readily Available Clinical Data with Gene Expression Levels of IGFBP3 and F3 in Formalin-Fixed Paraffin Embedded Core Needle Biopsy Material.

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    Zhuochun Peng

    Full Text Available A previously reported expression signature of three genes (IGFBP3, F3 and VGLL3 was shown to have potential prognostic value in estimating overall and cancer-specific survivals at diagnosis of prostate cancer in a pilot cohort study using freshly frozen Fine Needle Aspiration (FNA samples.We carried out a new cohort study with 241 prostate cancer patients diagnosed from 2004-2007 with a follow-up exceeding 6 years in order to verify the prognostic value of gene expression signature in formalin fixed paraffin embedded (FFPE prostate core needle biopsy tissue samples. The cohort consisted of four patient groups with different survival times and death causes. A four multiplex one-step RT-qPCR test kit, designed and optimized for measuring the expression signature in FFPE core needle biopsy samples, was used. In archive FFPE biopsy samples the expression differences of two genes (IGFBP3 and F3 were measured. The survival time predictions using the current clinical parameters only, such as age at diagnosis, Gleason score, PSA value and tumor stage, and clinical parameters supplemented with the expression levels of IGFBP3 and F3, were compared.When combined with currently used clinical parameters, the gene expression levels of IGFBP3 and F3 are improving the prediction of survival time as compared to using clinical parameters alone.The assessment of IGFBP3 and F3 gene expression levels in FFPE prostate cancer tissue would provide an improved survival prediction for prostate cancer patients at the time of diagnosis.

  9. Application of automated mRNA in situ hybridization for formalin-fixed, paraffin-embedded mouse skin sections: effects of heat and enzyme pretreatment on mRNA signal detection.

    Science.gov (United States)

    Nitta, Hiroaki; Kishimoto, Jiro; Grogan, Thomas M

    2003-06-01

    Recently, an automated mRNA in situ hybridization application was introduced for the Ventana Discovery instrument. The application was designed so that all necessary steps from baking through signal detection were completed within 1 day on the instrument. We applied this technology for visualizing the expression site of versican in formalin-fixed mouse skin paraffin tissue sections. Our focus of this study was to demonstrate the effects of protease digestion or heating pretreatment, termed cell conditioning, on the hybridization signal using a well characterized versican antisense riboprobe. Paraffin sections were automatically deparaffinized, fixed, and acid-treated. Then, the tissue sections were subjected to protease digestion alone (3 strengths), cell conditioning alone, or the combination of cell conditioning and protease digestion. Hybridization was performed with digoxigenin-labeled versican antisense probe (20 ng/slide) for 6 hours, and the signal was detected using a Nitro blue Tetrazolium chloride 5-Bromo-4-cloro-3-indolyl phosphate toluidine salt (NBT/BCLIP) substrate solution for 3 hours on the instrument. Cell conditioning alone did not produce any signal, whereas the highest strength of protease digestion produced noticeable background staining. However, when cell conditioning and mild protease digestion were combined, the signal for versican mRNA was clearly demonstrated in the hair papilla region. Thus, we demonstrated the effects of the cell conditioning step followed by mild protease digestion for enhancing the mRNA target staining compared with protease digestion or the cell conditioning step alone. We verified that the automated in situ hybridization process was applicable for formalin-fixed mouse skin paraffin sections and that the automated 1-day protocol is simple and reproducible. The precise control of automation allows fine tuning of temperature and enzyme dose to find the optimized assay condition for the signal to noise ratio and

  10. Immunohistochemistry of sarcolemmal membrane-associated proteins in formalin-fixed and paraffin-embedded skeletal muscle tissue: a promising tool for the diagnostic evaluation of common muscular dystrophies.

    Science.gov (United States)

    Suriyonplengsaeng, Chinnawut; Dejthevaporn, Charungthai; Khongkhatithum, Chaiyos; Sanpapant, Suda; Tubthong, Nattha; Pinpradap, Koset; Srinark, Nippa; Waisayarat, Jariya

    2017-02-20

    The analysis of fresh frozen muscle specimens is standard following routine muscle biopsy, but this service is not widely available in countries with limited medical facilities, such as Thailand. Nevertheless, immunohistochemistry (IHC) analysis is essential for the diagnosis of patients with a strong clinical suspicion of muscular dystrophy, in the absence of mutations detected by molecular genetics. As the successful labelling of sarcolemmal membrane-associated proteins in formalin-fixed and paraffin-embedded (FFPE) muscle sections using IHC staining has rarely been described, this study aimed to develop a reproducible IHC method for such an analysis. Thirteen cases were studied from the files of the Department of Pathology, Mahidol University. Diagnoses included three Duchenne muscular dystrophy (DMD), one Becker muscular dystrophy (BMD), one dysferlinopathy, and several not-specified muscular dystrophies. IHC was performed on FFPE sections at different thicknesses (3 μm, 5 μm, and 8 μm) using the heat-mediated antigen retrieval method with citrate/EDTA buffer, followed by an overnight incubation with primary antibodies at room temperature. Antibodies against spectrin, dystrophin (rod domain, C-terminus, and N-terminus), dysferlin, sarcoglycans (α, β, and γ), and β-dystroglycan were used. Frozen sections were tested in parallel for comparative analysis. Antibodies labelling spectrin, dystrophin (rod domain and C-terminus), dysferlin, sarcoglycans (α, β, and γ), and β-dystroglycan clearly exhibited sarcolemmal staining in FFPE sections. However, staining of FFPE sections using the antibody directed against the N-terminus of dystrophin was unsuccessful. The absence of labeling for dystrophins and dysferlin in FFPE sections was documented in all three DMD patients and the dysferlinopathy patient. The BMD diagnosis could not be made using IHC in FFPE sections alone because of a lack of staining for the dystrophin N-terminus, indicating a limitation of

  11. Synthesis of SiO2-Coated Fe3O4 Nanoparticles Using Ultrasound and Its Application in DNA Extraction from Formalin-Fixed, Paraffin-Embedded Human Cancer Tissues

    Science.gov (United States)

    Hieu, Nguyen Minh; Nam, Nguyen Hoang; Huyen, Nguyen Thi; Van Anh, Nguyen Thi; Nghia, Phan Tuan; Khoa, Nguyen Ba; Toan, Nguyen Linh; Luong, Nguyen Hoang

    2017-06-01

    SiO2-coated Fe3O4 nanoparticles (Fe3O4@SiO2 NPs) were successfully synthesized using ultrasound in order to extract DNA from cancer tissues for application in diagnostics. The core 10.7-nm-diameter Fe3O4 nanoparticles were synthesized by co-precipitation of Fe3+ and Fe2+ as reaction substrates and NH4OH as precipitant, then coated with a thin layer of amorphous silica by a modified Stober method. Further SiO2 coating using alkaline hydrolysis of tetraethyl orthosilicate in ethanol and water mixture was accelerated in the presence of a 37-kHz ultrasound, resulting in the NPs having different sizes of 14.5 nm (version M1), 24.4 nm (version M2), and 34.9 nm (version M3) with saturation magnetization values of 50.2 emu/g, 18.6 emu/g, 10.3 emu/g, respectively. Among the three Fe3O4@SiO2 NPs versions, the M1 NPs allowed extraction of DNAs from 10 mg formalin-fixed and paraffin-embedded (FFPE) tissues of nasopharyngeal carcinoma patients with the highest recovery of about 100-500 ng/ μl and good purity (A260/A280: 1.8-1.9). The extracted DNAs could be used as templates for downstream amplification of 252-bp sequencing specifically for the Braf cancer biomarker gene using polymerase chain reaction (PCR), as well as detection of the pathogenic Epstein-Barr virus (EBV) and the human papilloma-virus (HPV) using real-time PCR. DNA extraction recoveries of both EBV and HPV using Fe3O4@SiO2 NPs M1 were significantly better that those using commercialized Fe3O4@SiO2 microbeads, as indicated by lower threshold cycles of all fluorescent signals including fluorescein amidite (FAM) dye representative for EBV infection, hexachlorofluorescein (HEX) dye representative for β-globin (internal control), and SYBR Green dye representative for HPV infection in tested clinical samples from patients with nasopharyngeal carcinoma (NPC).

  12. TruSeq-Based Gene Expression Analysis of Formalin-Fixed Paraffin-Embedded (FFPE Cutaneous T-Cell Lymphoma Samples: Subgroup Analysis Results and Elucidation of Biases from FFPE Sample Processing on the TruSeq Platform

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    Philippe Lefrançois

    2017-09-01

    Full Text Available Cutaneous T-cell lymphomas (CTCLs are a heterogeneous group of malignancies with courses ranging from indolent to potentially lethal. We recently studied in a 157 patient cohort gene expression profiles generated by the TruSeq targeted RNA gene expression sequencing. We observed that the sequencing library quality and depth from formalin-fixed paraffin-embedded (FFPE skin samples were significantly lower when biopsies were obtained prior to 2009. We also observed that the fresh CTCL samples clustered together, even though they included stage I–IV disease. In this study, we compared TruSeq gene expression patterns in older (≤2008 vs. more recent (≥2009 FFPE samples to determine whether these clustering analyses and earlier described differentially expressed gene findings are robust when analyzed based on the year of biopsy. We also explored biases found in FFPE samples when subjected to the TruSeq analysis of gene expression. Our results showed that ≤2008 and ≥2009 samples clustered equally well to the full data set and, importantly, both analyses produced nearly identical trends and findings. Specifically, both analyses enriched nearly identical DEGs when comparing benign vs. (1 stage I–IV and (2 stage IV (alone CTCL samples. Results obtained using either ≤2008 or ≥2009 samples were strongly correlated. Furthermore, by using subgroup analyses, we were able to identify additional novel differentially expressed genes (DEGs, which did not reach statistical significance in the prior full data set analysis. Those included CTCL-upregulated BCL11A, SELL, IRF1, SMAD1, CASP1, BIRC5, and MAX and CTCL-downregulated MDM4, SERPINB3, and THBS4 genes. With respect to sample biases, no matter if we performed subgroup analyses or full data set analysis, fresh samples tightly clustered together. While principal component analysis revealed that fresh samples were spatially closer together, indicating some preprocessing batch effect, they remained

  13. Identificação imuno-histoquímica de Listeria monocytogenes em placentas fixadas em formol e embebidas em parafina Immunohistochemical identification of Listeria monocytogenes in formalin-fixed and paraffin-embedded placentas

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    Jussara Pires Schwab

    2003-08-01

    pregnancy trimester, age of pregnant women, cases of abortion and premature delivery, and to the occurrence of habitual abortion. METHODS: a retrospective study was carried out at the pathology service of a teaching hospital in the city of Porto Alegre in 2000. The paraffin blocks of 254 placentas, obtained from abortion, premature delivery and full-term birth, were analyzed by conventional histology using hematoxylin and eosin (HE staining. The IHC assay consisted of a rabbit anti-listeria polyclonal antibody B65420R (Biodesign® diluted 1:1000, in addition to the avidin-biotin-streptavidin complex; 148 placentas revealed inflammatory disorders, hemorrhage, necrosis and thrombosis. The c² test was used for statistical analysis. RESULTS: Listeria monocytogenes was detected in 33.78% of the placentas analyzed by IHC. Chorioamnionitis and villitis showed significant statistical difference in the positive placentas. Lm occurred in the 1st, 2nd and 3rd trimester of pregnancy. The age of pregnant women, the cases of abortion and/or premature births were not statistically different as to the presence or absence of Lm in the placentas. Habitual abortions occurred in patients with or without Lm in the placental tissue. CONCLUSION: Immunohistochemistry may be used to confirm the histopathological diagnosis of listeriosis in all trimesters of pregnancy.

  14. Technical validation of an RT-qPCR in vitro diagnostic test system for the determination of breast cancer molecular subtypes by quantification of ERBB2, ESR1, PGR and MKI67 mRNA levels from formalin-fixed paraffin-embedded breast tumor specimens.

    Science.gov (United States)

    Laible, Mark; Schlombs, Kornelia; Kaiser, Katharina; Veltrup, Elke; Herlein, Stefanie; Lakis, Sotiris; Stöhr, Robert; Eidt, Sebastian; Hartmann, Arndt; Wirtz, Ralph M; Sahin, Ugur

    2016-07-07

    MammaTyper is a novel CE-marked in vitro diagnostic RT-qPCR assay which assigns routinely processed breast cancer specimens into the molecular subtypes Luminal A-like, Luminal B-like (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition. Until now and regarding formalin-fixed, paraffin-embedded material (FFPE), this has been a task mostly accomplished by immunohistochemistry (IHC). However the discrepancy rates of IHC for the four breast cancer biomarkers are frequently under debate, especially for Ki-67 which carries the highest degree of inter- and even intra-observer variability. Herein we describe a series of studies in FFPE specimens which aim to fully validate the analytical performance of the MammaTyper assay, including the site to site reproducibility of the individual marker measurements. Tumor RNA was extracted with the novel RNXtract RNA extraction kit. Synthetic RNA was used to assess the sensitivity of the RNXtract kit. DNA and RNA specific qPCR assays were used so as to determine analyte specificity of RNXtract. For the assessment of limit of blank, limit of detection, analytical measurement range and PCR efficiency of the MammaTyper kit serial dilutions of samples were used. Analytical precision studies of MammaTyper were built around two different real time PCR platforms and involved breast tumor samples belonging to different subtypes analyzed across multiple sites and under various stipulated conditions. The MammaTyper assay robustness was tested against RNA input variations, alternative extraction methods and tumor cell content. Individual assays were linear up to at least 32.33 and 33.56 Cqs (quantification cycles) for the two qPCR platforms tested. PCR efficiency ranged from 99 to 109 %. In qPCR platform 1, estimates for assay specific inter-site standard deviations (SD) were between 0.14 and

  15. Technical validation of an RT-qPCR in vitro diagnostic test system for the determination of breast cancer molecular subtypes by quantification of ERBB2, ESR1, PGR and MKI67 mRNA levels from formalin-fixed paraffin-embedded breast tumor specimens

    International Nuclear Information System (INIS)

    Laible, Mark; Schlombs, Kornelia; Kaiser, Katharina; Veltrup, Elke; Herlein, Stefanie; Lakis, Sotiris; Stöhr, Robert; Eidt, Sebastian; Hartmann, Arndt; Wirtz, Ralph M.; Sahin, Ugur

    2016-01-01

    MammaTyper is a novel CE-marked in vitro diagnostic RT-qPCR assay which assigns routinely processed breast cancer specimens into the molecular subtypes Luminal A-like, Luminal B-like (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition. Until now and regarding formalin-fixed, paraffin-embedded material (FFPE), this has been a task mostly accomplished by immunohistochemistry (IHC). However the discrepancy rates of IHC for the four breast cancer biomarkers are frequently under debate, especially for Ki-67 which carries the highest degree of inter- and even intra-observer variability. Herein we describe a series of studies in FFPE specimens which aim to fully validate the analytical performance of the MammaTyper assay, including the site to site reproducibility of the individual marker measurements. Tumor RNA was extracted with the novel RNXtract RNA extraction kit. Synthetic RNA was used to assess the sensitivity of the RNXtract kit. DNA and RNA specific qPCR assays were used so as to determine analyte specificity of RNXtract. For the assessment of limit of blank, limit of detection, analytical measurement range and PCR efficiency of the MammaTyper kit serial dilutions of samples were used. Analytical precision studies of MammaTyper were built around two different real time PCR platforms and involved breast tumor samples belonging to different subtypes analyzed across multiple sites and under various stipulated conditions. The MammaTyper assay robustness was tested against RNA input variations, alternative extraction methods and tumor cell content. Individual assays were linear up to at least 32.33 and 33.56 Cqs (quantification cycles) for the two qPCR platforms tested. PCR efficiency ranged from 99 to 109 %. In qPCR platform 1, estimates for assay specific inter-site standard deviations (SD) were between 0.14 and 0

  16. Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples Efeitos das etapas de tratamento e processamento do tecido na PCR para detecção de Mycobacterium tuberculosis em amostras fixadas em formalina e incluídas em parafina

    Directory of Open Access Journals (Sweden)

    Denise Barcelos

    2008-12-01

    Full Text Available Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.O desenvolvimento e a padronização de métodos confiáveis para a detecção de Mycobacterium tuberculosis em amostras clínicas é um objetivo importante nos laboratórios de todo o mundo. Neste trabalho, fragmentos de pulmão e baço de paciente que morreu com o diagnóstico de tuberculose miliar foram usados para avaliar a influência do tipo de fixador e dos protocolos de fixação e inclusão em parafina na performance da PCR. Fragmentos de tecido foram fixados por quatro h a 48 h, usando formalina não tamponada a 10% ou formalina tamponada a 10% e incluídos em parafina pura ou misturada a cera de abelha. As amostras foram submetidas a PCR para amplificação do gene da beta-actina humana e

  17. Proteomic Analysis of Stage-II Breast Cancer from Formalin-Fixed Paraffin-Embedded Tissues

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    Naif Abdullah Al-Dhabi

    2016-01-01

    Full Text Available Breast cancer is the most frequently occurring disease among women worldwide. The early stage of breast cancer identification is the key challenge in cancer control and prevention procedures. Although gene expression profiling helps to understand the molecular mechanism of diseases or disorder in the living system, gene expression pattern alone is not sufficient to predict the exact mechanisms. Current proteomics tools hold great application for analysis of cancerous conditions. Hence, the generation of differential protein expression profiles has been optimized for breast cancer and normal tissue samples in our organization. Normal and tumor tissues were collected from 20 people from a local hospital. Proteins from the diseased and normal tissues have been investigated by 2D gel electrophoresis and MALDI-TOF-MS. The peptide mass fingerprint data were fed into various public domains like Mascot, MS-Fit, and Pept-ident against Swiss-Prot protein database and the proteins of interest were identified. Some of the differentially expressed proteins identified were human annexin, glutathione S-transferase, vimentin, enolase-1, dihydrolipoamide dehydrogenase, glutamate dehydrogenase, Cyclin A1, hormone sensitive lipase, beta catenin, and so forth. Many types of proteins were identified as fundamental steps for developing molecular markers for diagnosis of human breast cancer as well as making a new proteomic database for future research.

  18. miRNA expression profiling of formalin-fixed paraffin-embedded (FFPE hereditary breast tumors

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    Miljana Tanić

    2015-03-01

    Full Text Available Hereditary breast cancer constitutes only 5–10% of all breast cancer cases and is characterized by strong family history of breast and/or other associated cancer types. Only ~25% of hereditary breast cancer cases carry a mutation in BRCA1 or BRCA2 gene, while mutations in other rare high and moderate-risk genes and common low penetrance variants may account for additional 20% of the cases. Thus the majority of cases are still unaccounted for and designated as BRCAX tumors. MicroRNAs are small non-coding RNAs that play important roles as regulators of gene expression and are deregulated in cancer. To characterize hereditary breast tumors based on their miRNA expression profiles we performed global microarray miRNA expression profiling on a retrospective cohort of 80 FFPE breast tissues, including 66 hereditary breast tumors (13 BRCA1, 10 BRCA2 and 43 BRCAX, 10 sporadic breast carcinomas and 4 normal breast tissues, using Exiqon miRCURY LNA™ microRNA Array v.11.0. Here we describe in detail the miRNA microarray expression data and tumor samples used for the study of BRCAX tumor heterogeneity (Tanic et al., 2013 and biomarkers associated with positive BRCA1/2 mutation status (Tanic et al., 2014. Additionally, we provide the R code for data preprocessing and quality control.

  19. Detection of hepatitis B virus covalently closed circular DNA in paraffin-embedded and cryo-preserved liver biopsies of chronic hepatitis B patients

    NARCIS (Netherlands)

    Takkenberg, R. Bart; Zaaijer, Hans L.; Menting, Sandra; Weegink, Christine J.; Terpstra, Valeska; Cornelissen, Marion; Dijkgraaf, Marcel G. W.; Jansen, Peter L. M.; Reesink, Hendrik W.; Beld, Marcel G. H. M.

    2010-01-01

    Background Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may become an important predictor for treatment outcome or long-term follow-up. Aim To detect cccDNA in formalin-fixed, paraffin embedded (FFPE) and to compare with cryo-preserved liver tissue. Methods Biopsies of 56 chronic

  20. Detection of hepatitis C viral RNA sequences in fresh and paraffin-embedded liver biopsy specimens of non-A, non-B hepatitis patients

    NARCIS (Netherlands)

    Bresters, D.; Cuypers, H. T.; Reesink, H. W.; Chamuleau, R. A.; Schipper, M. E.; Boeser-Nunnink, B. D.; Lelie, P. N.; Jansen, P. L.

    1992-01-01

    In this study methods of HCV-RNA detection in fresh frozen and formalin-fixed, paraffin-embedded liver biopsies are described. Of 22 untreated chronic non-A, non-B hepatitis patients and 6 control patients, a plasma sample and part of a liver biopsy were freshly frozen for hepatitis C virus (HCV)

  1. Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue

    DEFF Research Database (Denmark)

    Green, Tina Marie; de Stricker, Karin; Møller, Michael Boe

    2009-01-01

    , represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples for Q-RT-PCR studies by investigating gene expressions of relevant target genes...... in paired frozen and paraffin-embedded samples. Moreover, we studied the impact of amplicon sizes on the efficiency of Q-RT-PCR in paraffin-embedded tissues. Six putative reference genes were tested for stability of expression in 21 pairs of snap-frozen and formalin-fixed, paraffin-embedded lymph nodes...... and lymphomas. The genes were ranked according to their suitability as reference genes. According to both statistical approaches, beta-glucoronidase was the single most appropriate reference gene in both snap-frozen and paraffin-embedded samples. TATA box-binding protein gene and Abelson murine leukemia viral...

  2. Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

    DEFF Research Database (Denmark)

    Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

    2004-01-01

    We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods...... tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique...

  3. Evaluation of a fluorescence-labelled oligonucleotide tide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears

    DEFF Research Database (Denmark)

    Nordentoft, Steen; Christensen, H.; Wegener, Henrik Caspar

    1997-01-01

    A method for the detection of Salmonella based on fluorescence in situ hybridization (FISH) has been developed and applied for the direct detection of Salmonella in pure cultures and in formalin-fixed, paraffin-embedded tissue sections. On the basis of the 23S rRNA gene sequences representing all...... with the probe. The probe did not hybridize to serovars from subspecies IIIa (S. arizonae) or to S. bongori. No cross-reaction to 64 other strains of the family Enterobacteriaceae or 18 other bacterial strains outside this family was observed. The probe was tested with sections of formalin-fixed, paraffin...

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

    NARCIS (Netherlands)

    Adank, M. A.; Brogi, E.; Bogomolniy, F.; Wadsworth, E. A.; Lafaro, K. J.; Yee, C. J.; Kirchhoff, T.; Meijers-Heijboer, E. J.; Kauff, N. D.; Boyd, J.; Offit, K.

    2006-01-01

    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

  5. Antibody validation and scoring guidelines for ABCG2 immunohistochemical staining in formalin-fixed paraffin-embedded colon cancer tissue

    DEFF Research Database (Denmark)

    Cederbye, Camilla Natasha; Palshof, Jesper Andreas; Hansen, Tine Plato

    2016-01-01

    Overexpression of the ATP-dependent drug efflux pump ABCG2 is a major molecular mechanism of multidrug resistance in cancer and might be a predictive biomarker for drug response. Contradictory results have been reported for immunohistochemical studies of ABCG2 protein expression in colorectal...... cancer (CRC), probably because of the use of different antibodies and scoring approaches. In this study, we systematically studied six commercially available anti-ABCG2 antibodies, using cell lines with up-regulation of ABCG2, and selected one antibody for validation in CRC tissue. Furthermore, we...... established scoring guidelines for ABCG2 expression based on the clinically used guidelines for HER2 immunohistochemistry assessment in gastric cancer. The guidelines provide a semi-quantitative measure of the basolateral membrane staining of ABCG2 and disregard the apical membrane staining...

  6. RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining

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    Yan Guo

    2016-01-01

    Full Text Available Background. Proper rRNA depletion is crucial for the successful utilization of FFPE specimens when studying gene expression. We performed a study to evaluate two major rRNA depletion methods: Ribo-Zero and RNase H. RNAs extracted from 4 samples were treated with the two rRNA depletion methods in duplicate and sequenced (N=16. We evaluated their reducibility, ability to detect RNA, and ability to molecularly subtype these triple negative breast cancer specimens. Results. Both rRNA depletion methods produced consistent data between the technical replicates. We found that the RNase H method produced higher quality RNAseq data as compared to the Ribo-Zero method. In addition, we evaluated the RNAseq data generated from the FFPE tissue samples for noncoding RNA, including lncRNA, enhancer/super enhancer RNA, and single nucleotide variation (SNV. We found that the RNase H is more suitable for detecting high-quality, noncoding RNAs as compared to the Ribo-Zero and provided more consistent molecular subtype identification between replicates. Unfortunately, neither method produced reliable SNV data. Conclusions. In conclusion, for FFPE specimens, the RNase H rRNA depletion method performed better than the Ribo-Zero. Neither method generates data sufficient for SNV detection.

  7. Enrichment of PrPSc in Formalin Fixed Paraffin Embedded Tissues Prior to Analysis by Western Blot

    Science.gov (United States)

    Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past these app...

  8. Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

    DEFF Research Database (Denmark)

    Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

    2004-01-01

    We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods....... A single DCIS case was amplified in FISH, but not in IHC. Both HER-2 gene amplification and expression could be quantified in microdissected paraffin-embedded tumors using real-time PCR, DNA and RNA being successfully detected in 146 of 150 (97%) and 141 of 150 (94%) samples, respectively. PCR analysis...... for HER-2 DNA amplification using the LightCycler HER2/neu DNA Quantification kit (Roche Molecular Biochemicals, Mannheim, Germany) correlated fairly well with IHC and FISH. All IHC HER-2 3+ tumors were amplified according to the kit, as was the FISH-amplified DCIS case. DNA-PCR identified five additional...

  9. Successful protein extraction from over-fixed and long-term stored formalin-fixed tissues.

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    Claudia Wolff

    Full Text Available One of the major breakthroughs in molecular pathology during the last decade was the successful extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE clinical tissues. However, only limited data are available for the protein extraction efficiency of over-fixed tissues and FFPE blocks that had been stored for more than 15 years in pathology archives. In this study we evaluated the protein extraction efficiency of FFPE tissues which had been formalin-fixed for up to 144 hours and tissue blocks that were stored for 20 years, comparing an established and a new commercial buffer system. Although there is a decrease in protein yield with increasing fixation time, the new buffer system allows a protein recovery of 66% from 144 hours fixed tissues compared to tissues that were fixed for 6 hours. Using the established extraction procedure, less than 50% protein recovery was seen. Similarly, the protein extraction efficiency decreases with longer storage times of the paraffin blocks. Comparing the two buffer systems, we found that 50% more proteins can be extracted from FFPE blocks that were stored for 20 years when the new buffer system is used. Taken together, our data show that the new buffer system is superior compared to the established one. Because tissue fixation times vary in the routine clinical setting and pathology archives contain billions of FFPE tissues blocks, our data are highly relevant for research, diagnosis, and treatment of disease.

  10. Detection of African swine fever virus from formalin fixed and non-fixed tissues by polymerase chain reaction

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    P. D. Luka

    2014-10-01

    Full Text Available Aim: Formalin fixing and paraffin embedding of tissue samples is one of the techniques for preserving the structural integrity of cells for a very long time. However, extraction and analysis of genomic material from formalin fixed tissue (FFT remains a challenge despite numerous attempts to develop a more effective method. The success of polymerase chain reaction (PCR depends on the quality of DNA extract. Materials and Methods: Here we assessed the conventional method of DNA extraction from FFT for African swine fever virus (ASFV detection. The modified conventional method gave a higher quality DNA when compared with commercially available DNA extraction kits (QIAamp® DNA Mini Kit, DNeasy® Blood and Tissue Kit, and ZR Genomic DNA™ Tissue MiniPrep. Results: An average A260/A280 DNA purity of 0.86-1.68 and 3.22-5.32 μg DNA/mg for formalin fixed and non-fixed tissues, respectively using a conventional method. In a reproducible and three times repeat PCR, the ASFV DNA expected product size of 278 bp was obtained from the DNA extract of the conventional method but not from the DNA extract of the commercial kits. Conclusion: The present study has demonstrated that the conventional method extracts ASFV genome better than commercial kit. In summary, the commercial kit extraction appeared not suitable to purify ASFV DNA from FFT. We, therefore, recommend that the use of the conventional method be considered for African swine fever DNA extraction from FFT.

  11. Diagnosis of Nocardia paucivorans central nervous system infection by DNA sequencing from paraffin-embedded tissue.

    Science.gov (United States)

    Schiaroli, Elisabetta; Pasticci, Maria Bruna; De Carolis, Elena; Mello, Enrica; Pallotto, Carlo; Leli, Christian; De Socio, Giuseppe Vittorio; Baldelli, Franco; Sanguinetti, Maurizio; Mencacci, Antonella

    2016-06-01

    Infections by Nocardia spp. are generally regarded as opportunistic diseases in immunocompromised patients, but can also affect immunocompetent subjects. Such infections represent an important diagnostic challenge for clinicians and microbiologists, and diagnosis is frequently delayed or even conducted post mortem. A 54-year-old man was admitted to our hospital because of ventriculitis and relapsing brain abscess. Five months prior, this patient had undergone external ventricular drain and surgery for a cerebellar abscess. Histopathology demonstrated pyogenic inflammatory reaction, microbiologic investigations proved negative and empiric antimicrobial therapy was administered for a total of eight weeks. Six weeks later, the patient developed relapsing neurologic manifestations. On reviewing the patient's clinical history it emerged that the patient had suffered pneumonia two months prior to neurosurgery, treated with amoxicillin/clavulanate 3g a day and levofloxacin 500mg a day for three weeks. On the CNS relapsing manifestations, nocardiosis was suspected and DNA sequencing from the formalin-fixed paraffin-embedded cerebellar tissue collected during neurosurgery allowed diagnosis of Nocardia paucivorans infection. The patient received medical therapy for 11 months. At follow-up, eight months after treatment was discontinued, the patient was aymptomatic. Nocardia spp. infections need to be suspected not only in immunocompromised, but also in immunocompetent patients. Proper samples need to be collected for proper microbiologic investigations. Paraffin-embedded tissue genomic sequencing can be a useful tool for diagnosis of nocardiosis.

  12. Immunocytochemical characterization of a monoclonal antibody directed against mitochondria reactive in paraffin-embedded sections.

    Science.gov (United States)

    Weiss, L M; Gaffey, M J; Warhol, M J; Mehta, P; Bonsib, S M; Bruder, E; Santos, E; Mederios, L J

    1991-09-01

    The monoclonal antibody mES 13 was previously produced against bacterially expressed BALB ras p21 and was reported to have both membrane and cytoplasmic reactivity in formalin-fixed, paraffin-embedded tissue sections. In the current study, the cytoplasmic reactivity of mES 13 is investigated and demonstrated to be mitochondrial. Immunoelectron microscopic studies showed specific labeling of mitochondria without labeling of other organelles. In normal tissues, the antibody strongly labeled tissues known to have large amounts of mitochondria such as renal tubules, hepatocytes, and myocardium. The pattern of reactivity of tumors generally mimicked that of normal tissues, with carcinomas and melanomas usually showing stronger staining than sarcomas and lymphomas. Two granular cell tumors were negative. Among renal neoplasms, mES 13 strongly labeled renal oncocytomas and granular cell renal cell carcinomas and showed weaker staining of clear cell and chromophobe cell tumors. The mES 13 antibody should be useful in the characterization and diagnosis of tumors in which oncocytoma is in the differential diagnosis, especially when only paraffin-embedded tissue is available for study.

  13. Digital dewaxing of Raman signals: discrimination between nevi and melanoma spectra obtained from paraffin-embedded skin biopsies.

    Science.gov (United States)

    Tfayli, Ali; Gobinet, Cyril; Vrabie, Valeriu; Huez, Regis; Manfait, Michel; Piot, Olivier

    2009-05-01

    Malignant melanoma (MM) is the most severe tumor affecting the skin and accounts for three quarters of all skin cancer deaths. Raman spectroscopy is a promising nondestructive tool that has been increasingly used for characterization of the molecular features of cancerous tissues. Different multivariate statistical analysis techniques are used in order to extract relevant information that can be considered as functional spectroscopic descriptors of a particular pathology. Paraffin embedding (waxing) is a highly efficient process used to conserve biopsies in tumor banks for several years. However, the use of non-dewaxed formalin-fixed paraffin-embedded tissues for Raman spectroscopic investigations remains very restricted, limiting the development of the technique as a routine analytical tool for biomedical purposes. This is due to the highly intense signal of paraffin, which masks important vibrations of the biological tissues. In addition to being time consuming and chemical intensive, chemical dewaxing methods are not efficient and they leave traces of the paraffin in tissues, which affects the Raman signal. In the present study, we use independent component analysis (ICA) on Raman spectral images collected on melanoma and nevus samples. The sources obtained from these images are then used to eliminate, using non-negativity constrained least squares (NCLS), the paraffin contribution from each individual spectrum of the spectral images of nevi and melanomas. Corrected spectra of both types of lesion are then compared and classified into dendrograms using hierarchical cluster analysis (HCA).

  14. Factors affecting the yield of microRNAs from laser microdissectates of formalin-fixed tissue sections

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    Patnaik Santosh

    2012-01-01

    Full Text Available Abstract Background Quantification of microRNAs in specific cell populations microdissected from tissues can be used to define their biological roles, and to develop and deploy biomarker assays. In this study, a number of variables were examined for their effect on the yield of microRNAs in samples obtained from formalin-fixed paraffin-embedded tissues by laser microdissection. Results MicroRNA yield was improved by using cresyl violet instead of hematoxylin-eosin to stain tissue sections in preparation for microdissection, silicon carbide instead of glass fiber as matrix in RNA-binding columns, and overnight digestion of dissected samples with proteinase K. Storage of slides carrying stained tissue sections at room temperature for up to a week before microdissection, and storage of the microdissectates at room temperature for up to a day before RNA extraction did not adversely affect microRNA yield. Conclusions These observations should be of value for the efficient isolation of microRNAs from microdissected formalin-fixed tissues with a flexible workflow.

  15. Establishment of a Pcr Technique for Determination of Htlv-1 Infection in Paraffin-Embedded Tissues

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    M Rastin

    2007-04-01

    Full Text Available Introduction: HTLV-1 , the first known human retrovirus belongs to oncovirus subfamily of retroviruses. The major characteristic of HTLV-1 is its highly restricted geographic prevalence. Northern part of Khorasan is an endemic region of HTLV-1 infection. Epidemiological studies can help in designing preventive programs for HTLV-1 infection. The aim of this study was the establishment of a PCR technique for determination of HTLV-1 infection in paraffin-embedded tissues. Methods: In this experimental laboratory study for establishment of a technique, PCR was initially optimized using Beta-actin primers on various formalin fixed paraffin-embedded tissues from liver, spleen, skin and lymph nodes. The optimized concentration of Mgcl2 was 2mm, primer was 8 pmol. Optimized concentration of DNA was different according to the kind of tissue. HTLV-1 infection was determined by applying tax, pol, env and LTR primers on 50 paraffin-embedded lymph node tissues . The reporoducibility of this technique was shown for skin and lymph node tissues infected with HTLV-1. Resuls: In 50 lymph node tissues, one case with pathologic diagnosis of NHL was positive with all 5 sets of primers (tax, Pol, env and LTR primers and the other case was positive with only two sets of tax primers but was negative with pol, env and LTR primers. The prevalence of infection was 2% among lymph node specimens. (1 of 50 specimens and if the second case is considered, the prevalence would be 4%. Conclusion: Comparison of the results of this study with another study on blood specimens (seroprevalence2.3% was not statistically significant thus confirming the results of one another. (P=0.883

  16. Implementation of formalin-fixed, paraffin-embedded cell line pellets as high-quality process controls in quality assessment programs for KRAS mutation analysis.

    NARCIS (Netherlands)

    Dijkstra, J.R.; Opdam, F.J.M.; Boonyaratanakornkit, J.; Schonbrunner, E.R.; Shahbazian, M.; Edsjo, A.; Hoefler, G.; Jung, A.; Kotsinas, A.; Gorgoulis, V.G.; Lopez-Rios, F.; Stricker, K.; Rouleau, E.; Biesmans, B.; Krieken, J.H.J.M. van

    2012-01-01

    In recent years, the mutational status of the KRAS oncogene has become incorporated into standard medical care as a predictive marker for therapeutic decisions related to patients with metastasized colorectal cancer. This is necessary, because these patients benefit from epidermal growth factor

  17. C4d and C3d immunohistochemical evaluation on formalin-fixed paraffin-embedded tissue for the diagnosis of bullous pemphigoid: systematic review of the literatures

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    Mahsa Akbari Oryani

    2017-01-01

    Full Text Available Introduction: Several methods are available for the diagnosis of autoimmune bullous disease. Since the immunohistochemistry of complement component is easy and more accessible compared to other methods, it is thought that this technique as an efficient method can replace other difficult, and time-consuming procedures. Therefore, in this study we aimed to systematically review the literatures in which the diagnostic value of complement component 3d (C3d and C4d had been investigated in bullous pemphigoid. Methods: A systematic search was conducted in the PubMed, Google scholar, and Scopus using following search method (((C3d OR C4d OR complement component 3d OR complement component 4d immunohistochemistry OR (C3d OR C4d marker OR complement component 3d OR complement component 4d marker AND (bullous pemphigoid OR cutaneous pemphigoid to evaluate the diagnostic value of C3d and/or C4d for early and accurate detection of bullous pemphigoid on November 2015. Subsequently, the extracted data were described.Result: Total of 28 documents were collected and reviewed based on the purpose of this study. Of the collected articles, 21 documents were excluded in several steps of article selection process and only 7 relevant articles were included for data assessment. The results showed that the deposits of C3d and/or C4d in skin biopsies were found in 125 of 134 patients, indicating that immunohistochemistry is a reliable technique for the diagnosis of inflammatory skin diseases.Conclusion: The results of this review showed that C3d and/or C4d immunohistochemistry in skin biopsies is a reliable technique for the diagnosis of inflammatory skin diseases, particularly bullous pemphigoid.

  18. Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection

    Directory of Open Access Journals (Sweden)

    María Fernanda Loayza

    2015-01-01

    • The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.

  19. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

    Directory of Open Access Journals (Sweden)

    Altimari A

    2013-08-01

    Full Text Available Annalisa Altimari,1,* Dario de Biase,2,* Giovanna De Maglio,3 Elisa Gruppioni,1 Elisa Capizzi,1 Alessio Degiovanni,1 Antonia D'Errico,1 Annalisa Pession,2 Stefano Pizzolitto,3 Michelangelo Fiorentino,1,# Giovanni Tallini2,#1Laboratory of Molecular Oncologic and Transplantation Pathology, S. Orsola-Malpighi Hospital, Bologna, 2Laboratory of Molecular Pathology, Anatomic Pathology, Bellaria Hospital, Bologna, 3Department of Pathology, S. Maria della Misericordia Hospital, Udine, Italy*These authors contributed equally to this work #These authors share senior authorshipAbstract: Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen® real-time polymerase chain reaction (PCR, pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA, evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03, percentage of mutation for pyrosequencing (P = 0.001, ratio for chip array hybridization (P = 0.003, and percentage of mutation for 454 next-generation sequencing (P = 0.004. Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001. Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues.Keywords: colorectal cancer, targeted therapy, KRAS mutations, next-generation sequencing, real-time polymerase chain reaction, pyrosequencing

  20. Detection of reticuloendotheliosis virus by immunohistochemistry and in situ hybridization in experimentally infected Japanese quail embryos and archived formalin-fixed and paraffin-embedded tumours

    Science.gov (United States)

    Reticuloendotheliosis virus (REV) infection can result in immunosuppression, runting syndrome, high mortality, acute reticulum cell neoplasia, or T- and/or B-cell lymphomas, in a variety of domestic and wild birds. Histopathological changes in REV infection are not sufficient to differentiate it fro...

  1. Detection of Helicobacter pylori DNA in Formalin-Fixed Paraffin-Embedded Gastric Biopsies Using Laser Microdissection and qPCR.

    Science.gov (United States)

    Loayza Villa, María Fernanda; Herrera Sevilla, Valeria Liliana; Vivar-Diaz, Nicolás

    2017-01-01

    Molecular detection and analysis of virulence factors of Helicobacter pylori depends on the specificity of cell selection in the gastric biopsies. The laser microdissection (LM) instruments combine microscopy with laser cut sectioning. This combination allows one to choose only the bacteria that are in direct contact with epithelial cells in the gastric biopsy sample, avoiding those microorganisms attached to the mucus layer in the sample. The average concentration of DNA isolated from 25 cuts with selected bacteria is around 1.94 ng/μL, which is enough DNA to perform a qPCR protocol using real-time instruments to amplify 16sDNA or virulence factors like cagA or vacA. Consequently, the application of these technologies in the molecular analysis of Helicobacter pylori directly in contact with the surface of gastric epithelial cells is more precise and could yield better insights about the complex mechanisms of interactions between pathogen and host. Insights derived from research using the techniques described herein may in future facilitate prevention of infection or improved therapeutic options.

  2. Genotyping of Toxoplasma gondii: DNA extraction from formalin-fixed paraffin-embedded autopsy tissues from AIDS patients who died by severe disseminated toxoplasmosis.

    Science.gov (United States)

    Bastos da Silva, Inara; Batista, Tatiana Pimental de Andrade; Martines, Roosecelis Brasil; Kanamura, Cristina Takami; Ferreira, Isabelle Martins Ribeiro; Vidal, Jose Ernesto; Pereira-Chioccola, Vera Lucia

    2016-06-01

    This study investigated the genetic features of Toxoplasma gondii isolated directly in autopsies of HIV-infected patients who died with severe disseminated toxoplasmosis. This retrospective analysis was conducted in a cohort of 15 HIV-infected patients with clinical and laboratory data. They had previous cerebral toxoplasmosis at least 6 months before the disseminated toxoplasmosis episode. The hypothesis was that they were infected with highly virulent parasites due to the condition in which they died. T. gondii genotyping was done directly in DNA extracted from 30 autopsy brain and lung samples (2 per patient) and mutilocus PCR-RFLP genotyping was done using 12 molecular markers. The 30 clinical samples were genotyped successfully in 8 or more loci and six suggestive genotypes were identified. One of them was Toxo DB #11, previously identified in different domestic animals and virulent in experimental animals. The other five suggestive genotypes identified in 14 patients were not described. TgHuDis1 was the most frequent and was determined in 8 patients. TgHuDis3 and TgHuDis5 were identified in two patients each. TgHuDis2 and TgHuDis4 have been identified in one patient each. These suggestive genotypes could be considered as virulent, since they caused severe tissue damage and had similar characteristics as Toxo # DB 11. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Development of reliable techniques for the differential diagnosis of avian tumor viruses by immunohistochemistry and polymerase chain reaction from formalin-fixed paraffin-embedded tissue sections

    Science.gov (United States)

    In the past, several techniques have been developed as diagnostic tools for the differential diagnosis of tumours produced by Marek’s disease virus (MDV) from those induced by avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). However, most current techniques are unreliable using form...

  4. Detection of Chlamydia in postmortal formalin-fixed tissue

    DEFF Research Database (Denmark)

    Lundemose, A G; Banner, Jytte; Birkelund, Svend

    1989-01-01

    A procedure to detect Chlamydia in postmortal formalin-fixed tissue is described. Monoclonal antibodies against a genus specific chlamydia epitope were used in immunofluorescence to detect chlamydia inclusions in formalin-fixed tissue sections. Lung sections from chlamydia-infected mice were exam...

  5. HOPE technique enables Western blot analysis from paraffin-embedded tissues.

    Science.gov (United States)

    Uhlig, U; Uhlig, S; Branscheid, D; Zabel, P; Vollmer, E; Goldmann, T

    2004-01-01

    In contrast to the spectrum of biochemical analyses of fresh material, that of archived specimens is widely restricted. Fixation of specimens with formalin, the most commonly used fixative, usually prevents further molecular analysis, since it leads to degradation of nucleic acids and denaturation of the antigenic determinants of proteins. To overcome these problems, the Hepes-glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-fixation technique has been developed, which preserves nucleic acids and antigenic determinants of proteins, thus expanding the applicability of immunohistochemical methods. In this study, we investigated whether HOPE-fixed tissue can be analyzed by Western blotting. Furthermore, a comparison with conventionally fixed and frozen material was made. The specimens used were tumor-free and obtained from lobectomies for lung cancer. All four antibodies tested, i.e., antibodies specific for focal adhesion kinase, surfactant protein A, PI-3-kinase, and IKKalpha, worked well if used for immunoblotting of HOPE-fixed and frozen tissue. By contrast, these antibodies showed no or only very weak specific binding if formalin-fixed specimens were analyzed. Our findings show that HOPE fixation maintains the antigenicity of proteins better than formalin fixation. The possibility for performing Western blotting with archived paraffin-embedded specimens extends the options for diagnostic and scientific analyses of fixed tissues.

  6. Three dimensional imaging of paraffin embedded human lung tissue samples by micro-computed tomography.

    Directory of Open Access Journals (Sweden)

    Anna E Scott

    Full Text Available Understanding the three-dimensional (3-D micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data.FFPE human lung tissue samples (n = 4 were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging.The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15 mm x 7 mm. Resolution (voxel size 6.7 µm in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections.We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis.

  7. Rate of Clinical Complete Response for 1 Year or More in Bone-Metastatic Breast Cancer after Comprehensive Treatments including Autologous Formalin-Fixed Tumor Vaccine.

    Science.gov (United States)

    Kuranishi, Fumito; Imaoka, Yuki; Sumi, Yuusuke; Uemae, Yoji; Yasuda-Kurihara, Hiroko; Ishihara, Takeshi; Miyazaki, Tsubasa; Ohno, Tadao

    2018-01-01

    No effective treatment has been developed for bone-metastatic breast cancer. We found 3 cases with clinical complete response (cCR) of the bone metastasis and longer overall survival of the retrospectively examined cohort treated comprehensively including autologous formalin-fixed tumor vaccine (AFTV). AFTV was prepared individually for each patient from their own formalin-fixed and paraffin-embedded breast cancer tissues. Three patients maintained cCR status of the bone metastasis for 17 months or more. Rate of cCR for 1 year or more appeared to be 15% (3/20) after comprehensive treatments including AFTV. The median overall survival time (60.0 months) and the 3- to 8-year survival rates after diagnosis of bone metastasis were greater than those of historical control cohorts in Japan (1988-2002) and in the nationwide population-based cohort study of Denmark (1999-2007). Bone-metastatic breast cancer may be curable after comprehensive treatments including AFTV, although larger scale clinical trial is required.

  8. Rate of Clinical Complete Response for 1 Year or More in Bone-Metastatic Breast Cancer after Comprehensive Treatments including Autologous Formalin-Fixed Tumor Vaccine

    Directory of Open Access Journals (Sweden)

    Fumito Kuranishi

    2018-01-01

    Full Text Available Introduction. No effective treatment has been developed for bone-metastatic breast cancer. We found 3 cases with clinical complete response (cCR of the bone metastasis and longer overall survival of the retrospectively examined cohort treated comprehensively including autologous formalin-fixed tumor vaccine (AFTV. Patients and Methods. AFTV was prepared individually for each patient from their own formalin-fixed and paraffin-embedded breast cancer tissues. Results. Three patients maintained cCR status of the bone metastasis for 17 months or more. Rate of cCR for 1 year or more appeared to be 15% (3/20 after comprehensive treatments including AFTV. The median overall survival time (60.0 months and the 3- to 8-year survival rates after diagnosis of bone metastasis were greater than those of historical control cohorts in Japan (1988–2002 and in the nationwide population-based cohort study of Denmark (1999–2007. Conclusion. Bone-metastatic breast cancer may be curable after comprehensive treatments including AFTV, although larger scale clinical trial is required.

  9. Replacing xylene with n-heptane for paraffin embedding.

    Science.gov (United States)

    Stockert, J C; López-Arias, B; Del Castillo, P; Romero, A; Blázquez-Castro, A

    2012-10-01

    In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.

  10. Comparison of five protocols to extract DNA from paraffin-embedded tissues for the detection of human papillomavirus.

    Science.gov (United States)

    Alvarez-Aldana, Adalucy; Martínez, José William; Sepúlveda-Arias, Juan C

    2015-02-01

    Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable source of DNA with which to perform large retrospective studies on the epidemiology of HPV infection. Five different DNA extraction protocols were carried out to evaluate the DNA obtained from FFPE samples with polymerase chain reaction (PCR) using two primer sets to amplify a constitutive human gene, β-globin, and two primer sets to detect the L1 and E6 HPV genes. From the five DNA extraction protocols evaluated, the best results were obtained with protocol A, corresponding to a crude extract from the sample. With the procedures described herein, we were able to amplify DNA extracted from archival paraffin blocks stored for six years. However, the amplification products were more efficiently obtained with primers that amplified shorter fragments. This result indicates that a major factor limiting the extraction process in these samples is DNA fragmentation, a factor that will naturally vary between the different specimens evaluated. Also, depending upon the extraction method, PCR amplification of a human gene does not necessarily guarantee the successful extraction of viral DNA. In conclusion, different DNA and HPV detection methods can significantly influence the results. Therefore, the DNA extraction methods and primers used for DNA amplification in fixed tissues need to be chosen carefully, depending on the specific requirements of the study being carried out. Copyright © 2014 Elsevier GmbH. All rights reserved.

  11. Detection of Chlamydia in postmortal formalin-fixed tissue

    DEFF Research Database (Denmark)

    Lundemose, A G; Banner, Jytte; Birkelund, Svend

    1989-01-01

    A procedure to detect Chlamydia in postmortal formalin-fixed tissue is described. Monoclonal antibodies against a genus specific chlamydia epitope were used in immunofluorescence to detect chlamydia inclusions in formalin-fixed tissue sections. Lung sections from chlamydia-infected mice were....... Background and non-specific fluorescence were reduced by treating the tissue sections with trypsin, rabbit serum and Evans blue counterstain. Besides giving an exact diagnosis at autopsy, the method provides the possibility of determining the occurrence of chlamydia infections in various tissues, based...

  12. Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

    Directory of Open Access Journals (Sweden)

    O. M. Barth

    1988-06-01

    Full Text Available The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathology study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tisue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.A presença de antígeno viral em cortes de tecidos humanos fixados em formol e emblocados em parafina foi demonstrada pela digestão com tripsina foi demonstrada pela ingestão com tripsina seguida de imunofluorescência direta ou indireta. Os espécimens podem ser utilizados para diagnoses retrospectivas. A técnica da imunofluorescência deve ser adaptada à infecção viral suspeita segundo diagnosie histopatológica prévia. Os parâmetros para a digestão do tecido pela tripsina, relacionados à concentração, duração de atuação e temperatura, expõem diferentes antígenos virais e devem ser previamente testados para cada sistema a ser estabelecido. Uma digestão mais intensa deve ser aplicada para a detecção do vírus do sarampo em tecido pulmonar do que para adenovírus ou vírus respiratório sincicial no mesmo tecido. Por outro lado, o vírus da febre amarela em tecido de fígado necessita de uma digestão mais fraca.

  13. Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing.

    Science.gov (United States)

    Wong, Stephen Q; Li, Jason; Tan, Angela Y-C; Vedururu, Ravikiran; Pang, Jia-Min B; Do, Hongdo; Ellul, Jason; Doig, Ken; Bell, Anthony; MacArthur, Grant A; Fox, Stephen B; Thomas, David M; Fellowes, Andrew; Parisot, John P; Dobrovic, Alexander

    2014-05-13

    Clinical specimens undergoing diagnostic molecular pathology testing are fixed in formalin due to the necessity for detailed morphological assessment. However, formalin fixation can cause major issues with molecular testing, as it causes DNA damage such as fragmentation and non-reproducible sequencing artefacts after PCR amplification. In the context of massively parallel sequencing (MPS), distinguishing true low frequency variants from sequencing artefacts remains challenging. The prevalence of formalin-induced DNA damage and its impact on molecular testing and clinical genomics remains poorly understood. The Cancer 2015 study is a population-based cancer cohort used to assess the feasibility of mutational screening using MPS in cancer patients from Victoria, Australia. While blocks were formalin-fixed and paraffin-embedded in different anatomical pathology laboratories, they were centrally extracted for DNA utilising the same protocol, and run through the same MPS platform (Illumina TruSeq Amplicon Cancer Panel). The sequencing artefacts in the 1-10% and the 10-25% allele frequency ranges were assessed in 488 formalin-fixed tumours from the pilot phase of the Cancer 2015 cohort. All blocks were less than 2.5 years of age (mean 93 days). Consistent with the signature of DNA damage due to formalin fixation, many formalin-fixed samples displayed disproportionate levels of C>T/G>A changes in the 1-10% allele frequency range. Artefacts were less apparent in the 10-25% allele frequency range. Significantly, changes were inversely correlated with coverage indicating high levels of sequencing artefacts were associated with samples with low amounts of available amplifiable template due to fragmentation. The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories. In a limited validation of potentially actionable low frequency mutations, a NRAS G12D mutation in a melanoma was shown to be a false

  14. Enzymatic detection of formalin-fixed museum specimens for DNA analysis and enzymatic maceration of formalin-fixed specimens

    DEFF Research Database (Denmark)

    Sørensen, Margrethe; Redsted Rasmussen, Arne; Simonsen, Kim Pilkjær

    2016-01-01

    Abstract.—A simple enzymatic screening method has been developed to detect whether a tissue sample has been preserved with formalin or with ethanol only because such a method is a useful tool for predicting the quality of genetic test results. The method is based on enzymatic digestion at 55 C...... in museums where DNA analyses of archived specimens are performed. Wasted time and resources can be avoided through the detection of formalin-fixed specimens because these specimens yield low-quality, damaged DNA. In addition to the screening method, it is shown that formalin-preserved specimens can...

  15. Genomic characterization of pediatric B-lymphoblastic lymphoma and B-lymphoblastic leukemia using formalin-fixed tissues.

    Science.gov (United States)

    Meyer, Julia A; Zhou, Delu; Mason, Clinton C; Downie, Jonathan M; Rodic, Vladimir; Abromowitch, Minnie; Wistinghausen, Birte; Termuhlen, Amanda M; Angiolillo, Anne L; Perkins, Sherrie L; Lones, Mark A; Barnette, Phillip; Schiffman, Joshua D; Miles, Rodney R

    2017-07-01

    Recurrent genomic changes in B-lymphoblastic leukemia (B-ALL) identified by genome-wide single-nucleotide polymorphism (SNP) microarray analysis provide important prognostic information, but gene copy number analysis of its rare lymphoma counterpart, B-lymphoblastic lymphoma (B-LBL), is limited by the low incidence and lack of fresh tissue for genomic testing. We used molecular inversion probe (MIP) technology to analyze and compare copy number alterations (CNAs) in archival formalin-fixed paraffin-embedded pediatric B-LBL (n = 23) and B-ALL (n = 55). Similar to B-ALL, CDKN2A/B deletions were the most common alteration identified in 6/23 (26%) B-LBL cases. Eleven of 23 (48%) B-LBL patients were hyperdiploid, but none showed triple trisomies (chromosomes 4, 10, and 17) characteristic of B-ALL. IKZF1 and PAX5 deletions were observed in 13 and 17% of B-LBL, respectively, which was similar to the reported frequency in B-ALL. Immunoglobulin light chain lambda (IGL) locus deletions consistent with normal light chain rearrangement were observed in 5/23 (22%) B-LBL cases, compared with only 1% in B-ALL samples. None of the B-LBL cases showed abnormal, isolated VPREB1 deletion adjacent to IGL locus, which we identified in 25% of B-ALL. Our study demonstrates that the copy number profile of B-LBL is distinct from B-ALL, suggesting possible differences in pathogenesis between these closely related diseases. © 2016 Wiley Periodicals, Inc.

  16. M. tuberculosis in Lymph Node Biopsy Paraffin-Embedded Sections

    Directory of Open Access Journals (Sweden)

    Abdurehman Eshete

    2011-01-01

    Full Text Available Background. Tuberculosis lymphadenitis is one of the most common forms of all extrapulmonary tuberculosis. Objective. To evaluate the magnitude of M. tuberculosis from lymph node biopsy paraffin-embedded sections among suspected patients visiting the Jimma University Specialized Hospital. Method. A cross-sectional study design of histological examination among lymph node biopsy paraffin-embedded sections by Ziehl-Neelsen and hematoxylin/eosin staining technique was conducted from December, 2009, to October, 2010, at the Department of Medical Laboratory Science and Pathology. Result. Histopathological examination of the specimens by hematoxylin and eosin staining technique revealed the presence of granulomas. But for the caseation and necrosis they were present in 85% cases of nodal tissue biopsies. From those, 56.7% were from females. The presence of acid-fast bacilli was microscopically confirmed by ZN staining in 37 (61.7% of the nodal tissue biopsies. Conclusion and Recommendation. Tuberculosis lymphadenitis is significantly more common in females. Hence, attention should be given for control and prevention of extrapulmonary tuberculosis.

  17. Molecular Detection and Genotypic Characterization of Toxoplasma gondii in Paraffin-Embedded Fetoplacental Tissues of Women with Recurrent Spontaneous Abortion

    Directory of Open Access Journals (Sweden)

    Amir Abdoli

    2016-11-01

    Full Text Available Background: Congenital toxoplasmosis is an important cause of spontaneous abortion worldwide. However, there is limited information on detection and genotypic characterization of Toxoplasma gondii (T. gondii in women with recurrent spontaneous abortion (RSA. The aim of this study is the molecular detection and genotypic characterization of T. gondii in formalin-fixed, paraffin-embedded fetoplacental tissues (FFPTs of women with RSA that have referred to the Avicenna Research Institute in Tehran, Iran. Materials and Methods: This experimental research was undertaken on 210 FFPTs of women with RSA. The information of the patients was collected from the archives of Avicenna Research Institute in Tehran, Iran. After DNA extraction, the presence of T. gondii was examined by nested polymerase chain reaction targeting the GRA6 gene. Genotyping was performed on positive samples using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP that targeted the GRA6 and SAG3 genes. Sequencing was conducted on two GRA6 positive samples. Results: T. gondii DNA was detected in 3.8% (8/210 of the samples. Genotyping showed that all positive samples belonged to type III of the T. gondii genotype. Sequencing two genomic DNAs of the GRA6 gene revealed 99% similarity with each other and 99-100% similarity with T. gondii sequences deposited in GenBank. There were six patients with histories of more than three abortions; one patient had a healthy girl and another patient had two previous abortions. Abortions occurred in the first trimester of pregnancy in seven patients and in the second trimester of pregnancy in one patient. Conclusion: The results of this study have indicated that genotype III is the predominant type of T. gondii in women with RSA in Tehran, Iran. Also, our findings suggest that toxoplasmosis may play a role in the pathogenesis of RSA. However, further studies are needed to elucidate a clear relationship between T. gondii

  18. Molecular Detection and Genotypic Characterization of Toxoplasma gondii in Paraffin-Embedded Fetoplacental Tissues of Women with Recurrent Spontaneous Abortion

    Science.gov (United States)

    Abdoli, Amir; Dalimi, Abdolhossein; Soltanghoraee, Haleh; Ghaffarifar, Fatemeh

    2017-01-01

    Background Congenital toxoplasmosis is an important cause of spontaneous abortion worldwide. However, there is limited information on detection and genotypic characterization of Toxoplasma gondii (T. gondii) in women with recurrent spontaneous abortion (RSA). The aim of this study is the molecular detection and genotypic characterization of T. gondii in formalin-fixed, paraffin-embedded fetoplacental tissues (FFPTs) of women with RSA that have referred to the Avicenna Research Institute in Tehran, Iran. Materials and Methods This experimental research was undertaken on 210 FFPTs of women with RSA. The information of the patients was collected from the archives of Avicenna Research Institute in Tehran, Iran. After DNA extraction, the presence of T. gondii was examined by nested polymerase chain reaction targeting the GRA6 gene. Genotyping was performed on positive samples using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) that targeted the GRA6 and SAG3 genes. Sequencing was conducted on two GRA6 positive samples. Results T. gondii DNA was detected in 3.8% (8/210) of the samples. Genotyping showed that all positive samples belonged to type III of the T. gondii genotype. Sequencing two genomic DNAs of the GRA6 gene revealed 99% similarity with each other and 99-100% similarity with T. gondii sequences deposited in GenBank. There were six patients with histories of more than three abortions; one patient had a healthy girl and another patient had two previous abortions. Abortions occurred in the first trimester of pregnancy in seven patients and in the second trimester of pregnancy in one patient. Conclusion The results of this study have indicated that genotype III is the predominant type of T. gondii in women with RSA in Tehran, Iran. Also, our findings suggest that toxoplasmosis may play a role in the pathogenesis of RSA. However, further studies are needed to elucidate a clear relationship between T. gondii infection and RSA. PMID

  19. Equine herpesvirus 1 (EHV-1) nucleotide polymorphism determination using formalin fixed tissues in EHV-1 induced abortions and myelopathies with real-time PCR and pyrosequencing.

    Science.gov (United States)

    Tewari, Deepanker; Del Piero, Fabio; Cieply, Stephen; Feria, Willard; Acland, Helen

    2013-11-01

    Equine herpesvirus-1 (EHV-1) strains with a single point mutation at the 2254 nucleotide position with a G2254 constitution within the DNA polymerase gene are associated strongly with equine myeloencephalopathies. Infections with non-neuropathogenic EHV-1 strains without the G2254 nucleotide but with an A2254 nucleotide are associated less frequently with equine neurologic disease. A retrospective study utilizing DNA extracted from formalin fixed paraffin embedded tissues was conducted with real time PCR and pyrosequencing, to determine the infecting EHV-1 strains. Infection with EHV-1 A2254 and or G2254 strain was detected with real time PCR, and was confirmed with a rapid pyrosequencing technique. Pyrosequencing was useful in at least 2 cases where real time PCR was equivocal in determining the infecting EHV-1 strain type. The strain with G2254 mutation was detected in 9.4% of 21 studied abortion cases, and in 86.6% of 15 neurologic cases. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens

    Science.gov (United States)

    2014-01-01

    Background NanoString’s Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype (Luminal A/B, HER2-enriched, Basal-like). The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories. Methods Analytical precision was measured by testing five breast tumor RNA samples across 3 sites. Reproducibility was measured by testing replicate tissue sections from 43 FFPE breast tumor blocks across 3 sites following independent pathology review at each site. The RNA input range was validated by comparing assay results at the extremes of the specified range to the nominal RNA input level. Interference was evaluated by including non-tumor tissue into the test. Results The measured standard deviation (SD) was less than 1 ROR unit within the analytical precision study and the measured total SD was 2.9 ROR units within the reproducibility study. The ROR scores for RNA inputs at the extremes of the range were the same as those at the nominal input level. Assay results were stable in the presence of moderate amounts of surrounding non-tumor tissue (<70% by area). Conclusions The analytical performance of NanoString’s Prosigna assay has been validated using FFPE breast tumor specimens across multiple clinical testing laboratories. PMID:24625003

  1. Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens

    International Nuclear Information System (INIS)

    Nielsen, Torsten; Storhoff, James; Wallden, Brett; Schaper, Carl; Ferree, Sean; Liu, Shuzhen; Gao, Dongxia; Barry, Garrett; Dowidar, Naeem; Maysuria, Malini

    2014-01-01

    NanoString’s Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype (Luminal A/B, HER2-enriched, Basal-like). The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories. Analytical precision was measured by testing five breast tumor RNA samples across 3 sites. Reproducibility was measured by testing replicate tissue sections from 43 FFPE breast tumor blocks across 3 sites following independent pathology review at each site. The RNA input range was validated by comparing assay results at the extremes of the specified range to the nominal RNA input level. Interference was evaluated by including non-tumor tissue into the test. The measured standard deviation (SD) was less than 1 ROR unit within the analytical precision study and the measured total SD was 2.9 ROR units within the reproducibility study. The ROR scores for RNA inputs at the extremes of the range were the same as those at the nominal input level. Assay results were stable in the presence of moderate amounts of surrounding non-tumor tissue (<70% by area). The analytical performance of NanoString’s Prosigna assay has been validated using FFPE breast tumor specimens across multiple clinical testing laboratories

  2. In situ hybridization technique for the detection of swine enteric and respiratory coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in formalin-fixed paraffin-embedded tissues.

    Science.gov (United States)

    Sirinarumitr, T; Paul, P S; Kluge, J P; Halbur, P G

    1996-02-01

    The in situ hybridization (ISH) technique was developed to detect the swine coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in cell culture and tissue sections from TGEV-or PRCV-infected pigs. The 35S-labeled RNA probes were generated from two plasmids pPSP.FP1 and pPSP.FP2 containing part of the S gene of TGEV. The procedure was first standardized in cell cultures. The radiolabeled pPSP.FP2 probe detected both TGEV and PRCV in virus-inoculated cell cultures, whereas pPSP.FP1 probe detected TGEV but not PRCV. The probe was then used to detect TGEV or PRCV in tissues of pigs experimentally infected with TGEV or PRCV or naturally infected with TGEV. Again, the probes detected TGEV in intestines of experimentally and naturally infected pigs and PRCV in the lungs of experimentally infected pigs. TGEV RNA was detected mainly within the enterocytes at the tips of villi and, less often, within some crypt epithelial cells. PRCV was shown to replicate mainly in the bronchiolar epithelial cells and in lesser amount in type II pneumocytes, type I pneumocytes, alveolar macrophages and bronchial epithelial cells, respectively. ISH has potential applications as a diagnostic test for the detection and differentiation of TGEV and PRCV in tissues and in studies to gain a better understanding of the mechanism of pathogenesis of enteric and respiratory coronavirus infections.

  3. Risk of radiation-induced subcutaneous fibrosis in relation to single nucleotide polymorphisms in TGFB1, SOD2, XRCC1, XRCC3, APEX and ATM--a study based on DNA from formalin fixed paraffin embedded tissue samples

    DEFF Research Database (Denmark)

    Andreassen, C N; Alsner, J; Overgaard, M

    2006-01-01

    ) codon 148 polymorphisms were assessed based on archival histological material. Differences in fibrosis risk were quantified from dose-response assessments. RESULTS: For none of the investigated polymorphisms, significant associations with risk of subcutaneous fibrosis were observed. A detailed analysis...

  4. Evaluation of Human Epidermal Growth Factor Receptor 2 (HER2) Gene Status in Human Breast Cancer Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens by Fluorescence In Situ Hybridization (FISH).

    Science.gov (United States)

    Hwang, Harry C; Gown, Allen M

    2016-01-01

    Current standard of care requires that HER2 gene testing be performed on all newly diagnosed invasive breast cancers in order to determine eligibility for anti-HER2 antibody therapy and should be performed in accordance with current ASCO-CAP guidelines (Hammond et al., J Clin Oncol 29(15):e458, 2011; Wolff et al., J Clin Oncol 31(31):3997-4013, 2013). Here we describe a HER2 FISH methodology to evaluate HER2 gene status in FFPE breast tumor specimens.

  5. Evaluation of a panel of antibodies for the immunohistochemical identification of immune cells in paraffin-embedded lymphoid tissues of new- and old-world camelids.

    Science.gov (United States)

    Uhde, Ann-Kathrin; Lehmbecker, Annika; Baumgärtner, Wolfgang; Spitzbarth, Ingo

    2017-02-01

    Different species of camelids play an important role in the epidemiology of various emerging infectious diseases such as Middle East respiratory syndrome. For precise investigations of the immunopathogenesis in these host species, appropriate immunohistochemical markers are highly needed in order to phenotype distinct immune cells populations in camelids. So far, specific immunohistochemical markers for camelid immune cells are rarely commercially available, and cross-reactivity studies are restricted to the use of frozen dromedary tissues. To bridge this gap, 14 commercially available primary antibodies were tested for their suitability to demonstrate immune cell populations on formalin fixed paraffin-embedded (FFPE) tissue sections of dromedaries, Bactrian camels, llamas, and alpacas in the present study. Out of these, 9 antibodies directed against CD3, CD20, CD79α, HLA-DR, Iba-1, myeloid/histiocyte antigen, CD204, CD208, and CD68 antigen exhibited distinct immunoreaction patterns to certain camelid immune cell subsets. The distribution of these antigens was comparatively evaluated in different anatomical compartments of thymus, spleen, mesenteric, and tracheobronchial lymph nodes. The presented results will provide a basis for further investigations in camelids, especially with respect to the role of the immune response in certain infectious diseases, which harbor a considerable risk to spill over to other species. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens

    Directory of Open Access Journals (Sweden)

    Broukhanski George

    2008-09-01

    Full Text Available Abstract The Seeplex™ TB Detection-2 assay (Rockville, MD is a nested endpoint PCR for the Mycobacterium tuberculosis complex (MTBC targets IS6110 and MPB64 that utilizes dual priming oligonucleotide technology. When used to detect the presence of MTBC DNA in formalin-fixed paraffin-embedded tissue specimens, the sensitivity and specificity of this assay is equivalent to a labor-intensive traditional endpoint PCR assay and is more sensitive than a commercial real-time PCR assay.

  7. Copy number and loss of heterozygosity detected by SNP array of formalin-fixed tissues using whole-genome amplification.

    Directory of Open Access Journals (Sweden)

    Angela Stokes

    Full Text Available The requirement for large amounts of good quality DNA for whole-genome applications prohibits their use for small, laser capture micro-dissected (LCM, and/or rare clinical samples, which are also often formalin-fixed and paraffin-embedded (FFPE. Whole-genome amplification of DNA from these samples could, potentially, overcome these limitations. However, little is known about the artefacts introduced by amplification of FFPE-derived DNA with regard to genotyping, and subsequent copy number and loss of heterozygosity (LOH analyses. Using a ligation adaptor amplification method, we present data from a total of 22 Affymetrix SNP 6.0 experiments, using matched paired amplified and non-amplified DNA from 10 LCM FFPE normal and dysplastic oral epithelial tissues, and an internal method control. An average of 76.5% of SNPs were called in both matched amplified and non-amplified DNA samples, and concordance was a promising 82.4%. Paired analysis for copy number, LOH, and both combined, showed that copy number changes were reduced in amplified DNA, but were 99.5% concordant when detected, amplifications were the changes most likely to be 'missed', only 30% of non-amplified LOH changes were identified in amplified pairs, and when copy number and LOH are combined ∼50% of gene changes detected in the unamplified DNA were also detected in the amplified DNA and within these changes, 86.5% were concordant for both copy number and LOH status. However, there are also changes introduced as ∼20% of changes in the amplified DNA are not detected in the non-amplified DNA. An integrative network biology approach revealed that changes in amplified DNA of dysplastic oral epithelium localize to topologically critical regions of the human protein-protein interaction network, suggesting their functional implication in the pathobiology of this disease. Taken together, our results support the use of amplification of FFPE-derived DNA, provided sufficient samples are used

  8. Extraction and recovery technique for myxozoan parasites from the Piaractus mesopotamicus kidney embedded in paraffin

    Directory of Open Access Journals (Sweden)

    Wilson G. Manrique

    2013-11-01

    Full Text Available Tissues fixed and embedded in paraffin for histopathological studies keep their cell characteristics. There are several protocols for extracting genetic material from tissue embedded in paraffin, but there is no protocol for material aimed at the direct identification of parasites. The lack of techniques which describe the recovery of parasites from tissue embedded in paraffin has led us to test a technique for recovering myxosporean parasites found in Piaractus mesopotamicus kidney fragments embedded in paraffin, for a rapid, direct, and economic identification. Once the excess paraffin was removed from the kidney fragment, this was deparaffinized in xylene, hydrated in 70% alcohol, placed in an Eppendorf tube containing 70% alcohol, and left under vigorous and constant agitation in a vortex until the tissue was disintegrated. The precipitated material was mixed with the 70% alcohol and 20 μL were collected for preparing the smears, which were stained with Giemsa. Myxobolus sp. spores at many developmental stages were observed by light microscopy. The technique has proved to be useful for recovering myxosporean parasites from tissue embedded in paraffin and it constitutes an effective tool for prevalence studies when the myxosporean parasites are not detected in fresh mounts.

  9. Extraction and recovery technique for myxozoan parasites from the Piaractus mesopotamicus kidney embedded in paraffin

    Directory of Open Access Journals (Sweden)

    Wilson Gomez Manrique

    2013-09-01

    Full Text Available http://dx.doi.org/10.5007/2175-7925.2013v26n4p263 Tissues fixed and embedded in paraffin for histopathological studies keep their cell characteristics. There are several protocols for extracting genetic material from tissue embedded in paraffin, but there is no protocol for material aimed at the direct identification of parasites. The lack of techniques which describe the recovery of parasites from tissue embedded in paraffin has led us to test a technique for recovering myxosporean parasites found in Piaractus mesopotamicus kidney fragments embedded in paraffin, for a rapid, direct, and economic identification. Once the excess paraffin was removed from the kidney fragment, this was deparaffinized in xylene, hydrated in 70% alcohol, placed in an Eppendorf tube containing 70% alcohol, and left under vigorous and constant agitation in a vortex until the tissue was disintegrated. The precipitated material was mixed with the 70% alcohol and 20 µL were collected for preparing the smears, which were stained with Giemsa. Myxobolus sp. spores at many developmental stages were observed by light microscopy. The technique has proved to be useful for recovering myxosporean parasites from tissue embedded in paraffin and it constitutes an effective tool for prevalence studies when the myxosporean parasites are not detected in fresh mounts.

  10. Thiel embalming fluid--a new way to revive formalin-fixed cadaveric specimens.

    Science.gov (United States)

    Hunter, Amanda; Eisma, Roos; Lamb, Clare

    2014-09-01

    By soft fixing cadavers using the Thiel embalming method, our cadavers now exhibit a greater degree of flexibility and color retention compared to that of traditional formalin-fixed cadavers. The aim of this experiment was to discover whether Thiel embalming fluid could be used to revive and soften the muscles of formalin-fixed prosected specimens. Earlier this year, two severely dehydrated formalin-fixed forearm and hand specimens were fully submerged in a tank containing Thiel embalming fluid. After a period of six months the specimens were removed from the tank and noticeable changes were observed in flexibility, quality of the tissue, and color of the specimens. © 2014 Wiley Periodicals, Inc.

  11. Analysis of iron, zinc, selenium and cadmium in paraffin-embedded prostate tissue specimens using inductively coupled plasma mass-spectrometry

    Science.gov (United States)

    Sarafanov, A.G.; Todorov, T.I.; Kajdacsy-Balla, A.; Gray, Michael A.; MacIas, V.; Centeno, J.A.

    2008-01-01

    Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable and abundant resource of pathologic material for various biomedical studies. In the present study, we report the application of high-resolution inductively coupled mass-spectrometry (ICP-MS) for quantification of Fe, Zn, Se and Cd in FFPE prostate tissue. These elements have a possible role in the development of prostate diseases: while Zn and Se are needed for a healthy prostate, Cd shows multiple toxic and carcinogenic effects. Excessive accumulation of Fe induces the production of highly reactive hydroxyl radical species, which may play a role in cancer etiopathogenesis. To assess whether the levels of these metals in the FFPE prostate tissue represent their original content, we compared their levels with those in the fresh tissue (on dry weight basis) in samples obtained from 15 patients. We found that in FFPE tissue, the recoveries of Se, Fe, Cd and Zn were progressively decreased, 97??11% (r=0.88), 82??22% (r=0.86), 59??23% (r=0.69) and 24??11% (r=0.38), respectively. Thus, the use of correction factors, determined as k=0.16 for Se, k=0.20 for Fe, k=0.27 for Cd and k=0.67 for Zn, is required to estimate the retrospective levels of these elements in the parental non-processed fresh (wet) prostate tissue. The technique used in this study enables the analysis of archival FFPE prostate tissue for the concentrations of Fe, Zn, Se and Cd to study association between the levels of these metals and prostate disease. ?? 2008.

  12. A new method for real-time evaluation of pepsin digestion of paraffin-embedded tissue sections, prior to fluorescence in situ hybridisation.

    Science.gov (United States)

    Teng, Xiaojing; Zhang, Shuhong; Liu, Wei; Bi, Kuo; Zhang, Lei

    2017-05-01

    Fluorescence in situ hybridisation (FISH) is a molecular cytogenetic technique, which is regularly applied to formalin-fixed paraffin-embedded (FFPE) tissue sections of a variety of cancers to assess chromosomal aberrations. However, high-quality FISH requires optimal enzymatic digestion, and insufficient digestion is not noted until the hybridisation signals are evaluated in the fluorescence microscope. As a consequence, FISH results may be unreliable, and the experiment might have to be repeated. To solve this problem, we developed a new method for real-time evaluation of enzymatic tissue digestion. Termination of enzyme activity at the proper time facilitates successful hybridisation, and experiments do not have to be repeated. We first performed FISH on 20 FFPE samples, which had been pepsin digested for different times, and this revealed distinct morphological changes within the nucleus and perinuclear space that were detectable by light microscopy. These observations suggested that the presence of intact and clear bare nuclei, surrounded by a translucent perinuclear space, might serve as an indicator of adequate digestion. We developed a protocol for assessment of this indicator, based on morphological features, and applied this to a collection of 400 tissue samples, partly of breast cancer and partly of different types of lymphoma, prior to FISH. The FISH success rate was 99.5% (398/400), which was significantly higher than that of the conventional method. In all successful cases, morphological signs of adequate digestion were paralleled by easily interpretable FISH signals. This new method for the real-time assessment of digestion quality improved the success rate of FISH and in addition was simple and rapid.

  13. Targeted high throughput sequencing in clinical cancer Settings: formaldehyde fixed-paraffin embedded (FFPE tumor tissues, input amount and tumor heterogeneity

    Directory of Open Access Journals (Sweden)

    Schaefer Georg

    2011-09-01

    Full Text Available Abstract Background Massively parallel sequencing technologies have brought an enormous increase in sequencing throughput. However, these technologies need to be further improved with regard to reproducibility and applicability to clinical samples and settings. Methods Using identification of genetic variations in prostate cancer as an example we address three crucial challenges in the field of targeted re-sequencing: Small nucleotide variation (SNV detection in samples of formalin-fixed paraffin embedded (FFPE tissue material, minimal amount of input sample and sampling in view of tissue heterogeneity. Results We show that FFPE tissue material can supplement for fresh frozen tissues for the detection of SNVs and that solution-based enrichment experiments can be accomplished with small amounts of DNA with only minimal effects on enrichment uniformity and data variance. Finally, we address the question whether the heterogeneity of a tumor is reflected by different genetic alterations, e.g. different foci of a tumor display different genomic patterns. We show that the tumor heterogeneity plays an important role for the detection of copy number variations. Conclusions The application of high throughput sequencing technologies in cancer genomics opens up a new dimension for the identification of disease mechanisms. In particular the ability to use small amounts of FFPE samples available from surgical tumor resections and histopathological examinations facilitates the collection of precious tissue materials. However, care needs to be taken in regard to the locations of the biopsies, which can have an influence on the prediction of copy number variations. Bearing these technological challenges in mind will significantly improve many large-scale sequencing studies and will - in the long term - result in a more reliable prediction of individual cancer therapies.

  14. A simple quantitative diagnostic alternative for MGMT DNA-methylation testing on RCL2 fixed paraffin embedded tumors using restriction coupled qPCR.

    Science.gov (United States)

    Pulverer, Walter; Hofner, Manuela; Preusser, Matthias; Dirnberger, Elisabeth; Hainfellner, Johannes A; Weinhaeusel, Andreas

    2014-01-01

    MGMT promoter methylation is associated with favorable prognosis and chemosensitivity in glioblastoma multiforme (GBM), especially in elderly patients. We aimed to develop a simple methylation-sensitive restriction enzyme (MSRE)-based quantitative PCR (qPCR) assay, allowing the quantification of MGMT promoter methylation. DNA was extracted from non-neoplastic brain (n = 24) and GBM samples (n = 20) upon 3 different sample conservation conditions (-80 °C, formalin-fixed and paraffin-embedded (FFPE); RCL2-fixed). We evaluated the suitability of each fixation method with respect to the MSRE-coupled qPCR methylation analyses. Methylation data were validated by MALDITOF. qPCR was used for evaluation of alternative tissue conservation procedures. DNA from FFPE tissue failed reliable testing; DNA from both RCL2-fixed and fresh frozen tissues performed equally well and was further used for validation of the quantitative MGMT methylation assay (limit of detection (LOD): 19.58 pg), using individual's undigested sample DNA for calibration. MGMT methylation analysis in non-neoplastic brain identified a background methylation of 0.10 ± 11% which we used for defining a cut-off of 0.32% for patient stratification. Of GBM patients 9 were MGMT methylationpositive (range: 0.56 - 91.95%), and 11 tested negative. MALDI-TOF measurements resulted in a concordant classification of 94% of GBM samples in comparison to qPCR. The presented methodology allows quantitative MGMT promoter methylation analyses. An amount of 200 ng DNA is sufficient for triplicate analyses including control reactions and individual calibration curves, thus excluding any DNA qualityderived bias. The combination of RCL2-fixation and quantitative methylation analyses improves pathological routine examination when histological and molecular analyses on limited amounts of tumor samples are necessary for patient stratification.

  15. PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation.

    Science.gov (United States)

    Dowd, S E; Gerba, C P; Enriquez, F J; Pepper, I L

    1998-01-01

    The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.

  16. Paraffin-based immunohistochemistry in the evaluation of glomerular diseases in renal biopsies

    International Nuclear Information System (INIS)

    Rathore, M.U.; Khadim, M.T.; Atique, M.

    2012-01-01

    Objective: To determine sensitivity and specificity of paraffin-based immunohistochemistry in the evaluation of glomerular diseases in renal biopsies using immunofluorescence as gold standard. Study Design: Cross-sectional analytical study. Place and Duration of Study: Department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi, from August 2008 to August 2009. Methodology: Seventy renal biopsy specimens fulfilling the inclusion criteria for light microscopy and immuno-fluorescence during the study period were evaluated. Antibodies to immunoglobulins (IgG, IgA, and IgM) and components of complement system (C3) were applied on 70 formalin-fixed paraffin-embedded renal biopsy specimens previously classified by means of light microscopy and immunofluorescence (IF). Staining for these antibodies was recorded as positive and negative for immunohistochemistry (IHC) and IF in paired proportions presuming IF as gold standard test. The sensitivity, specificity, positive predictive value and negative predictive value of individual antibody were calculated. Results: Of 70 patients, mean age was 33 +- 18 years ranging from 2 to 80 years. Forty five (64%) were males and 25 (36%) were females. The sensitivity, specificity and predictive values of individual antibodies to IgG, IgA, IgM and C3 were very low and generally in the range of 40 - 60%. Conclusion: The sensitivity, specificity and predictive values of immunohistochemistry on formalin-fixed paraffin-embedded renal biopsy specimens were very low and therefore, not suitable for evaluation of renal biopsies in current circumstances. (author)

  17. Diagnosis of Marek's Disease From a Japanese Quail (Coturnix Japonica) Using Paraffin-embedded Liver

    Science.gov (United States)

    A single paraffin-embedded liver section was submitted from a research flock of Japanese quail that had revealed focal infiltrations of immature lymphocytes within multiple visceral organs. Tumor cells were characterized as T-cells positive for Marek's disease virus (MDV) pp38 antigen by IHC dual st...

  18. Detection of apoptosis in paraffin embedded tissues: the influence of tissue type and fixation

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Dubská, L.; Míšek, Ivan

    2002-01-01

    Roč. 71, č. 4 (2002), s. 529-533 ISSN 0001-7213 R&D Projects: GA ČR GP204/02/P112; GA AV ČR KSK6005114 Keywords : apoptosis * TUNEL test * paraffin embedded tissues Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.370, year: 2002

  19. The predictive value of Gen-Probe's amplified Mycobacterium tuberculosis direct test compared with culturing in paraffin-embedded lymph node tissue exhibiting granulomatous inflammation and negative acid fast stain.

    Science.gov (United States)

    Raslan, Wasim F; Rabaan, Ali; Al-Tawfiq, Jaffar A

    2014-01-01

    The diagnosis of granulomatous inflammation with possible tuberculosis (TB) infection in histopathology is often difficult. There is a need for a rapid and reliable diagnostic test. Thus, we evaluated the performance of the Mycobacterium tuberculosis direct (MTD) test in specimens with granulomatous lymphadenitis and negative acid fast stains. The M. tuberculosis direct (MTD) test by Gen-Probe was performed on 45 formalin-fixed paraffin-embedded tissue samples including 34 lymph nodes. We measured the predictive values of the MTD test in specimens with granulomatous lymphadenitis and negative acid fast stains. The overall test sensitivity was 73.9%, and specificity was 95.4%. The MTD test sensitivity and specificity for lymph node tissue were 72.7% and 91.67%, respectively. In the presence of granulomatous inflammation, the MTD test sensitivity and specificity were higher than those for all tissue samples, at 75% and 100%, respectively. Based on this study, the MTD test should be used as a supportive test in addition to conventional histochemical or immunological staining methods when evaluating lymph node tissue with a granulomatous inflammation to deliver stronger evidence to support clinical decisions at a much earlier time than a culture would allow. Copyright © 2014 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

  20. Extraction and Determination of Trace Copper (II Using Octadecyl Silica Membrane Disks Modified 1-(2-Pyridyl Azo 2-Naphtol(Pan in Water Samples and Paraffin-Embedded Tissues from Liver Loggerhead Turtles Specimens by FAAS

    Directory of Open Access Journals (Sweden)

    A. Moghimi

    2012-07-01

    Full Text Available A simple and reproducible method for the rapid extraction and determination of trace amounts of copper(II ions using octadecyl-bonded silica membrane disks modified by 1-(2-Pyridyl Azo2-Naphtol(PANand Atomic Absorption Spectrometry was presented. The method was based on complex formation on the surface of the ENVI-18 DISKTM disks followed by stripping of the retained species by minimum amounts of appropriate organic solvents. The elution was efficient and quantitative. The effect of potential interfering ions, pH, ligand amount, stripping solvent, and sample flow rate were also investigated. Under the optimal experimental conditions, the break-through volume was found to about 1000mL providing a preconcentration factor of 500. In the present study, we reported the application of preconcentration techniques still continues increasingly for trace metal determinations by flame atomic absorption spectrometry (FAAS for quantification of Cu in Formalin-fixed paraffin-embedded (FFPE tissues from Liver loggerhead turtles. The maximum capacity of the disks was found to be 389± 4 µg for Cu2+.The limit of detection of the proposed method was 5ng per 1000mL.The method was applied to the extraction and recovery of copper in different water samples.

  1. Proteomic MALDI-TOF/TOF-IMS examination of peptide expression in the formalin fixed brainstem and changes in sudden infant death syndrome infants.

    Science.gov (United States)

    Hunt, Nicholas J; Phillips, Leo; Waters, Karen A; Machaalani, Rita

    2016-04-14

    Matrix assisted laser desorption/ionisation imaging mass spectrometry (MALDI-IMS) has not previously been utilised to examine sudden infant death syndrome (SIDS). This study aimed to optimise MALDI IMS for use on archived formalin-fixed-paraffin-embedded human infant medulla tissue (n=6, controls; n=6, SIDS) to evaluate differences between multiple nuclei of the medulla by using high resolution IMS. Profiles were compared between SIDS and age/sex matched controls. LC-MALDI identified 55 proteins based on 321 peptides across all samples; 286 peaks were found using IMS, corresponding to these 55 proteins that were directly compared between controls and SIDS. Control samples were used to identify common peptides for neuronal/non-neuronal structures allowing identification of medullary regions. In SIDS, abnormal expression patterns of 41 peptides (p≤0.05) corresponding to 9 proteins were observed; these changes were confirmed with immunohistochemistry. The protein abnormalities varied amongst nuclei, with the majority of variations in the raphe nuclei, hypoglossal and pyramids. The abnormal proteins are not related to a previously identified neurological disease pathway but consist of developmental neuronal/glial/axonal growth, cell metabolism, cyto-architecture and apoptosis components. This suggests that SIDS infants have abnormal neurological development in the raphe nuclei, hypoglossal and pyramids of the brainstem, which may contribute to the pathogenesis of SIDS. This study is the first to perform an imaging mass spectrometry investigation in the human brainstem and also within sudden infant death syndrome (SIDS). LC MALDI and MALDI IMS identified 55 proteins based on 285 peptides in both control and SIDS tissue; with abnormal expression patterns present for 41/285 and 9/55 proteins in SIDS using IMS. The abnormal proteins are critical for neurological development; with the impairment supporting the hypothesis that SIDS may be due to delayed neurological

  2. Advantages of infrared transflection micro spectroscopy and paraffin-embedded sample preparation for biological studies

    Science.gov (United States)

    Yao, Jie; Li, Qian; Zhou, Bo; Wang, Dan; Wu, Rie

    2018-04-01

    Fourier-Transform Infrared micro-spectroscopy is an excellent method for biological analyses. In this paper, series metal coating films on ITO glass were prepared by the electrochemical method and the different thicknesses of paraffin embedding rat's brain tissue on the substrates were studied by IR micro-spetroscopy in attenuated total reflection (ATR) mode and transflection mode respectively. The Co-Ni-Cu alloy coating film with low cost is good reflection substrates for the IR analysis. The infrared microscopic transflection mode needs not to touch the sample at all and can get the IR spectra with higher signal to noise ratios. The Paraffin-embedding method allows tissues to be stored for a long time for re-analysis to ensure the traceability of the sample. Also it isolates the sample from the metal and avoids the interaction of biological tissue with the metals. The best thickness of the tissues is 4 μm.

  3. The frequency of p53, Ki67, CD99 and Fli-1 protein expression in paraffin-embedded tissue blocks in Ewing’s sarcoma

    Directory of Open Access Journals (Sweden)

    Bagheri Hossein-Abadi Z

    2011-06-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Ewing sarcoma family tumors (ESFTs are among the most malignant tumors in children and young adults. ESFTs include Ewing sarcoma (ES and peripheral primitive neuroectodermal tumors (pPNETs. As there seemed to be few studies on the molecular biology of ESFTs, we investigated the frequency of CD99, Ki67, p53 and Fli-1 protein expression in 15 Iranian patients with ESFTs. In addition, the correlation between expression rate of these proteins and various clinical factors, including age, sex and survival was computed."n"nMethods: The expression of the aforesaid proteins was studied by immunohisto-chemistry in formalin-fixed and paraffin-embedded blocks of 15 ESFTs specimens. Stained sections were classified according to the percentage of stained tumor cells."n"nResults: The results showed the membrane expression of CD99 protein in all of the specimens. The nuclear expression of Fli-1 protein was observed in 86.7% and the over-expression of p53 nuclear protein was seen in 53.3% of the specimens. The expression rate of Ki67 protein was 60%. Although a significant correlation was not shown between the expression levels of Ki67, p53 or Fli-1 proteins with age, sex or survival of the patients, there was a significant

  4. Identifying Corneal Infections in Formalin-Fixed Specimens Using Next Generation Sequencing.

    Science.gov (United States)

    Li, Zhigang; Breitwieser, Florian P; Lu, Jennifer; Jun, Albert S; Asnaghi, Laura; Salzberg, Steven L; Eberhart, Charles G

    2018-01-01

    We test the ability of next-generation sequencing, combined with computational analysis, to identify a range of organisms causing infectious keratitis. This retrospective study evaluated 16 cases of infectious keratitis and four control corneas in formalin-fixed tissues from the pathology laboratory. Infectious cases also were analyzed in the microbiology laboratory using culture, polymerase chain reaction, and direct staining. Classified sequence reads were analyzed with two different metagenomics classification engines, Kraken and Centrifuge, and visualized using the Pavian software tool. Sequencing generated 20 to 46 million reads per sample. On average, 96% of the reads were classified as human, 0.3% corresponded to known vectors or contaminant sequences, 1.7% represented microbial sequences, and 2.4% could not be classified. The two computational strategies successfully identified the fungal, bacterial, and amoebal pathogens in most patients, including all four bacterial and mycobacterial cases, five of six fungal cases, three of three Acanthamoeba cases, and one of three herpetic keratitis cases. In several cases, additional potential pathogens also were identified. In one case with cytomegalovirus identified by Kraken and Centrifuge, the virus was confirmed by direct testing, while two where Staphylococcus aureus or cytomegalovirus were identified by Centrifuge but not Kraken could not be confirmed. Confirmation was not attempted for an additional three potential pathogens identified by Kraken and 11 identified by Centrifuge. Next generation sequencing combined with computational analysis can identify a wide range of pathogens in formalin-fixed corneal specimens, with potential applications in clinical diagnostics and research.

  5. Toxicological analysis of formalin-fixed or embalmed tissues: a review.

    Science.gov (United States)

    Nikolaou, Panagiota; Papoutsis, Ioannis; Dona, Artemisia; Spiliopoulou, Chara; Athanaselis, Sotiris

    2013-12-10

    During the autopsy of forensic cases, when there is no suspicion of drug use or chemical exposure, biological fluids may not be obtained for toxicological analysis, while specimens of tissues may be collected and preserved in a formalin solution for histological examination. When specific questions arise after the burial, the only possible options are the exhumation of an embalmed body or the toxicological analysis of the formalin-fixed specimens. The drug concentrations in these specimens can be altered due to the extraction efficiency and/or the chemical activity of the formalin solutions used during chemical fixation or embalming process. The aim of this paper is to review the published studies about the determination of specific groups of drugs in formalin-fixed or embalmed specimens and their stability after chemical fixation or embalming process. The analytical aspects of this determination are also discussed. The stability of drugs in formalin environment and the possible reaction of the drugs with formaldehyde, which is a highly reactive chemical substance, should always be considered during post-mortem/post-embalming forensic analysis. The additional analysis of the formalin solution in which the tissue was preserved is considered necessary. The identification and the evaluation of the possible degradation products or chemical derivatives are extremely useful during the interpretation of the results. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  6. Light microscopic identification and semiquantification of polyethylene particles in methylmethacrylate and paraffin-embedded experimental bone implant specimens

    DEFF Research Database (Denmark)

    Rahbek, O; Kold, S; Overgaard, S

    2005-01-01

    The aim of this study was to evaluate the identification of polyethylene (PE) particles in relatively thick methylmethacrylate (MMA) sections widely used in bone implant research. The sensitivity and specificity were compared between decalcified paraffin-embedded oil red O (ORO) stained and MMA......-embedded sections using polarized light. Furthermore, we introduced a grading system to semiquantify the level of PE particles in peri-implant tissue. Paraffin-embedded and MMA-embedded sections were compared concerning intra-observer agreement of the grading system. Moreover, the semiquantitative assessment...... of particle level was compared between the two section types. We found a sensitivity and specificity of polarized light of 100% for both paraffin ORO-stained and MMA sections. The intra-observer agreement on both types was comparable and acceptable. The ratings of differently processed blocks (MMA...

  7. Relative shrinkage of adipocytes by paraffin in proportion to plastic embedding in human adipose tissue before and after weight loss.

    Science.gov (United States)

    Verhoef, Sanne P M; van Dijk, Paul; Westerterp, Klaas R

    2013-01-01

    Adipocyte size is a major modulator of endocrine functioning of adipose tissue and methods allowing accurate determination of adipocyte size are important to study energy metabolism. The aim of this study was to assess the relative shrinkage of adipocytes before and after weight loss by comparing adipose tissue from the same subjects embedded in paraffin and plastic. 18 healthy subjects (5 males and 13 females) aged 20-50 y with a BMI of 28-38 kg/m² followed a very low energy diet for 8 weeks. Adipose tissue biopsies were taken prior to and after weight loss and were processed for paraffin and plastic sections. Parameters of adipocyte size were determined with computer image analysis. Mean adipocyte size was smaller in paraffin compared to plastic embedded tissue both before (66 ± 4 vs. 103 ± 5 μm, P paraffin embedded tissue in proportion to plastic embedded tissue was not significantly different before and after weight loss (73 and 69%, respectively). Shrinkage due to the type of embedding of the adipose tissue can be ignored when comparing before and after weight loss. Plastic embedding of adipose tissue provides more accurate and sensitive results. © 2013 Asian Oceanian Association for the Study of Obesity . Published by Elsevier Ltd. All rights reserved.

  8. An inexpensive alternative equipment for the plant material embedding in the paraffin under the vacuum

    Directory of Open Access Journals (Sweden)

    Carlos André Espolador Leitão

    2008-10-01

    Full Text Available The present work describes an equipment constructed using inexpensive material for embedding the plant material in the paraffin under the vacuum, using an oven and a vacuum pump. The equipment was tested using the samples of Rodriguezia venusta (Orchidaceae buds embedded in paraffin, where half of the samples were submitted to the vacuum by the equipment during the embedding. The material was sectioned with a rotary microtome, obtaining full series of quality sections. The control was hard to section with the microtome, obtaining damaged sections due the air bubbles, making the ribbon formation difficult. These results proved the effectiveness of the equipment, making it a practical, inexpensive and more portable solution for newly established laboratories.O presente trabalho apresenta um equipamento feito com material barato, destinado à inclusão de material botânico em parafina sob vácuo, utilizando-se uma estufa e uma bomba de vácuo. O equipamento foi testado utilizando-se amostras de botão floral de Rodriguezia venusta (Orchidaceae incluídas em parafina, das quais metade foi submetida ao vácuo pelo equipamento durante a infiltração. O material foi seccionado em micrótomo rotativo, obtendo-se séries completas de cortes de boa qualidade das amostras submetidas ao vácuo. O controle foi de difícil microtomia, obtendo-se cortes danificados pela presença de bolhas de ar, dificultando assim a formação de fitas. Estes resultados comprovam a eficácia do equipamento proposto, sendo este uma solução prática, barata e portátil para laboratórios em início de estruturação.

  9. Metadata: JPST000084 [jPOST repository metadata[Archive

    Lifescience Database Archive (English)

    Full Text Available ded Tissue Sections Mounted on Microscope Slides FFPE slide; formalin-fixed and paraffin-embedded; phase-tra...alysis Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope

  10. Detection and typing of papillomavirus DNA in formaldehyde-fixed paraffin-embedded tissue.

    Science.gov (United States)

    Brandsma, J L; Lewis, A J; Abramson, A; Manos, M M

    1990-07-01

    Clinical specimens from nine patients with papillomatosis of the vocal cords and three patients with vocal cord polyps were evaluated for the presence of human papillomavirus (HPV) DNA using two complementary molecular hybridization techniques. In one method, involving polymerase chain reaction (PCR) amplification, HPV DNA sequences were replicated in vitro from tissue DNA extracted from paraffin sections prior to hybridization. Polymerase chain reaction amplification was compared with the standard method of Southern blot hybridization. Results of the two techniques for all nine laryngeal papillomas agreed completely: five patients harbored HPV type 6 and four HPV type 11. Both PCR amplification and Southern blot hybridization found two of the three polyps to be free of HPV infection, while PCR detected HPV type 18 in one polyp specimen that was reported negative by Southern blot hybridization, suggesting a greater sensitivity of PCR. Our results demonstrate that PCR amplification is as reliable and at least as sensitive as Southern blot hybridization. Moreover the PCR technique opens the way to the undertaking of a whole variety of retrospective studies using formaldehyde-fixed paraffin-embedded tissues.

  11. Selected Reaction Monitoring (SRM Analysis of Epidermal Growth Factor Receptor (EGFR in Formalin Fixed Tumor Tissue

    Directory of Open Access Journals (Sweden)

    Hembrough Todd

    2012-05-01

    Full Text Available Abstract Background Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR in clinical tissue samples is typically done by immunohistochemistry (IHC and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies. Methods A mass spectrometry-based Selected Reaction Monitoring (SRM assay for the EGFR protein (EGFR-SRM was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4 were analyzed by enzyme-linked immunosorbent assay (ELISA to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10 of non-small cell lung cancer (NSCLC origin and NSCLC patient tumor tissue samples (n = 23 were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel. Results The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991. The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and

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

    NARCIS (Netherlands)

    Brada, SJL; van de Loosdrecht, AA; Koudstaal, J; de Wolf, JTM; Vellenga, E

    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

  13. Combined in situ zymography, immunofluorescence, and staining of iron oxide particles in paraffin-embedded, zinc-fixed tissue sections.

    Science.gov (United States)

    Haeckel, Akvile; Schoenzart, Lena; Appler, Franziska; Schnorr, Joerg; Taupitz, Matthias; Hamm, Bernd; Schellenberger, Eyk

    2012-01-01

    Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.

  14. New comprehensive denaturing-gradient-gel-electrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours

    NARCIS (Netherlands)

    Hayes, VM; Westra, JL; Verlind, E; Bleeker, W; Plukker, JT; Hofstra, RMW; Buys, CHCM

    2000-01-01

    A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within

  15. Molecular detection of Coxiella burnetii from the formalin-fixed tissues of Q fever patients with acute hepatitis.

    Directory of Open Access Journals (Sweden)

    Young-Rock Jang

    Full Text Available Serologic diagnosis is one of the most widely used diagnostic methods for Q fever, but the window period in antibody response of 2 to 3 weeks after symptom onset results in significant diagnostic delay. We investigated the diagnostic utility of Q fever PCR from formalin-fixed liver tissues in Q fever patients with acute hepatitis.We reviewed the clinical and laboratory data in patients with Q fever hepatitis who underwent liver biopsy during a 17-year period, and whose biopsied tissues were available. We also selected patients who revealed granuloma in liver biopsy and with no Q fever diagnosis within the last 3 years as control. Acute Q fever hepatitis was diagnosed if two or more of the following clinical, serologic, or histopathologic criteria were met: (1 an infectious hepatitis-like clinical feature such as fever (≥ 38°C with elevated hepatic transaminase levels; (2 exhibition of a phase II immunoglobulin G (IgG antibodies titer by IFA of ≥ 1:128 in single determination, or a four-fold or greater rise between two separate samples obtained two or more weeks apart; (3 histologic finding of biopsy tissue showing characteristic fibrin ring granuloma.A total of 11 patients with acute Q fever hepatitis were selected and analyzed. Of the 11 patients, 3 (27% had exposure to zoonotic risk factors and 7 (63% met the serologic criteria. Granulomas with either circumferential or radiating fibrin deposition were observed in 10 cases on liver biopsy and in 1 case on bone marrow biopsy. 8 (73% revealed positive Coxiella burnetii PCR from their formalin-fixed liver tissues. In contrast, none of 10 patients with alternative diagnosis who had hepatic granuloma revealed positive C. burnetii PCR from their formalin-fixed liver tissues.Q fever PCR from formalin-fixed liver tissues appears to be a useful adjunct for diagnosing Q fever hepatitis.

  16. Multimodal Raman-fluorescence spectroscopy of formalin fixed samples is able to discriminate brain tumors from dysplastic tissue

    Science.gov (United States)

    Anand, Suresh; Cicchi, Riccardo; Giordano, Flavio; Buccoliero, Anna Maria; Pavone, Francesco Saverio

    2014-05-01

    In the recent years, there has been a considerable surge in the application of spectroscopy for disease diagnosis. Raman and fluorescence spectra provide characteristic spectral profile related to biochemical and morphological changes when tissues progress from normal state towards malignancy. Spectroscopic techniques offer the advantage of being minimally invasive compared to traditional histopathology, real time and quantitative. In biomedical optical diagnostics, freshly excised specimens are preferred for making ex-vivo spectroscopic measurements. With regard to fresh tissues, if the lab is located far away from the clinic it could pose a problem as spectral measurements have to be performed immediately after dissection. Tissue samples are usually placed in a fixative agent such as 4% formaldehyde to preserve the samples before processing them for routine histopathological studies. Fixation prevents the tissues from decomposition by arresting autolysis. In the present study, we intend to investigate the possibility of using formalin fixed samples for discrimination of brain tumours from dysplastic tissue using Raman and fluorescence spectroscopy. Formalin fixed samples were washed with phosphate buffered saline for about 5 minutes in order to remove the effects of formalin during spectroscopic measurements. In case of fluorescence spectroscopy, changes in spectral profile have been observed in the region between 550-670 nm between dysplastic and tumor samples. For Raman measurements, we found significant differences in the spectral profiles between dysplasia and tumor. In conclusion, formalin fixed samples can be potentially used for the spectroscopic discrimination of tumor against dysplastic tissue in brain samples.

  17. Detection of the sarin hydrolysis product in formalin-fixed brain tissues of victims of the Tokyo subway terrorist attack.

    Science.gov (United States)

    Matsuda, Y; Nagao, M; Takatori, T; Niijima, H; Nakajima, M; Iwase, H; Kobayashi, M; Iwadate, K

    1998-06-01

    One of the hydrolysis products of sarin (isopropyl methylphosphonofluoridate) was detected in formalin-fixed brain tissues of victims poisoned in the Tokyo subway terrorist attack. Part of this procedure, used for the detection of sarin hydrolysis products in erythrocytes of sarin victims, has been described previously. The test materials were four individual cerebellums, which had been stored in formalin fixative for about 2 years. Sarin-bound acetylcholinesterase (AChE) was solubilized from these cerebellums, purified by immunoaffinity chromatography, and digested with trypsin. Then the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion, subjected to trimethylsilyl derivatization (TMS), and detected by gas chromatography-mass spectrometry. Peaks at m/z 225 and m/z 240, which are indicative of TMS-methylphosphonic acid, were observed within the retention time range of authentic methylphosphonic acid. However, no isopropyl methylphosphonic acid was detected in the formalin-fixed cerebellums of these 4 sarin victims, probably because the isopropoxy group of isopropyl methylphosphonic acid underwent chemical hydrolysis during storage. This procedure will be useful for the forensic diagnosis of poisoning by protein-bound, highly toxic agents, such as sarin, which are easily hydrolysed. This appears to be the first time that intoxication by a nerve agent has been demonstrated by analyzing formalin-fixed brains obtained at autopsy.

  18. Multi-elemental imaging of paraffin-embedded human samples by laser-induced breakdown spectroscopy

    Science.gov (United States)

    Moncayo, S.; Trichard, F.; Busser, B.; Sabatier-Vincent, M.; Pelascini, F.; Pinel, N.; Templier, I.; Charles, J.; Sancey, L.; Motto-Ros, V.

    2017-07-01

    Chemical elements play central roles for physiological homeostasis in human cells, and their dysregulation might lead to a certain number of pathologies. Novel imaging techniques that improve the work of pathologists for tissue analysis and diagnostics are continuously sought. We report the use of Laser-Induced Breakdown Spectroscopy (LIBS) to perform multi-elemental images of human paraffin-embedded skin samples on the entire biopsy scale in a complementary and compatible way with microscope histopathological examination. A specific instrumental configuration is proposed in order to detect most of the elements of medical interest (i.e. P, Al, Mg, Na, Zn, Si, Fe, and Cu). As an example of medical application, we selected and analysed skin biopsies, including healthy skin tissue, cutaneous metastasis of melanoma, Merkel-cell carcinoma and squamous cell carcinoma. Clear distinctions in the distribution of chemical elements are observed from the different samples investigated. This study demonstrates the high complementarity of LIBS elemental imaging with conventional histopathology, opening new opportunities for any medical application involving metals.

  19. Polymerase chain reaction detection of retinoblastoma gene deletions in paraffin-embedded mouse lung adenocarcinomas

    International Nuclear Information System (INIS)

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1991-01-01

    A Polymerase chain reaction (PCR) technique was used to detect deletions in the mouse retinoblastoma (mRb) gene using microtomed sections from paraffin-embedded radiation-induced and spontaneous tumors as the DNA source. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. Absence of any of these fragments relative to control PCR products on a Southern blot indicated a deletion of that portion of the mRb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death. Spontaneous tumors as well as those from irradiated mice (569 cGy of 60 Co γ rays or 60 cGy of JANUS neutrons) were analyzed. Tumors in six neutron-irradiated mice also had no mRb deletions. However, one of six tumors from γ-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice showed a deletion in one or both mRb alleles. All deletions detected were in the 5' region of the mRb gene

  20. Comparison of Different Double Immunostaining Protocols for Paraffin Embedded Liver Tissue

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    Alexander Schütz

    1999-01-01

    Full Text Available Most of the double immunostaining protocols that have been introduced so far have been developed for application on fresh frozen material or based on different species antibodies. In liver tissue, general problems of double immunostaining techniques are further complicated by tissue‐specific difficulties, such as necrosis or high intracellular protein content. To assess a reliable double immunostaining protocol for archived, paraffin embedded liver tissue, different protocols based on the use of same species primary antibodies were evaluated in terms of sensitivity, specificity and non‐specific background staining in pathological liver specimens. We compared peroxidase–anti‐peroxidase, alkaline phosphatase–anti‐alkaline phosphatase (PAP/APAP, labelled‐avidin–biotin (LAB/LAB and digoxigenin–anti‐digoxigenin (dig–a‐dig/PAP techniques using different cytokeratin antibodies and an antibody against PCNA. Comparison of the double immunostaining techniques revealed a high sensitivity and specificity in all procedures. Sections, which were stained employing PAP/APAP‐technique, displayed a higher background staining compared to sections which were treated with the LAB/LAB or dig–a‐dig/PAP protocol. In contrast to the dig–a‐dig/PAP protocol, the LAB/LAB technique provides a better time/cost relationship. Therefore, we would like to recommend a modified LAB/LAB protocol for simultaneous detection of different antigens in archived liver tissue.

  1. In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections.

    Science.gov (United States)

    Ellison, Mitchell A; McMahon, Michael B; Bonde, Morris R; Palmer, Cristi L; Luster, Douglas G

    2016-01-01

    Rust fungi are obligate pathogens with multiple life stages often including different spore types and multiple plant hosts. While individual rust pathogens are often associated with specific plants, a wide range of plant species are infected with rust fungi. To study the interactions between these important pathogenic fungi and their host plants, one must be able to differentiate fungal tissue from plant tissue. This can be accomplished using the In situ hybridization (ISH) protocol described here. To validate reproducibility using the ISH protocol, samples of Chrysanthemum × morifolium infected with Puccinia horiana, Gladiolus × hortulanus infected with Uromyces transversalis and Glycine max infected with Phakopsora pachyrhizi were tested alongside uninfected leaf tissue samples. The results of these tests show that this technique clearly distinguishes between rust pathogens and their respective host plant tissues. This ISH protocol is applicable to rust fungi and potentially other plant pathogenic fungi as well. It has been shown here that this protocol can be applied to pathogens from different genera of rust fungi with no background staining of plant tissue. We encourage the use of this protocol for the study of plant pathogenic fungi in paraffin embedded sections of host plant tissue.

  2. Immunohistochemical diagnosis of Alport's syndrome in paraffin-embedded renal sections: antigen retrieval with autoclave heating.

    Science.gov (United States)

    Naito, Ichiro; Ninomiya, Yoshifumi; Nomura, Shinsuke

    2003-03-01

    Alport's syndrome (AS) is a hereditary renal disease caused by mutations in the genes encoding collagen type IV. Immunohistochemical analysis of the alpha chains of collagen type IV has been found to be useful for the diagnosis of this disease. The monoclonal antibodies (mAbs) generated by us recognize alpha 1(IV) through alpha 6(IV) chains of collagen type IV on fresh-frozen sections but not on paraffin-embedded sections. Antigen retrieval by autoclave heating has been found to restore the epitopes recognized by the mAbs; however the heating conditions had not been well established. In this study, the heating conditions were carefully examined using renal sections obtained from AS and non-AS patients. The heating was performed in an autoclave, at 105 degrees -127 degrees C for 6-8 min. During the heating, the sections were immersed in 0.2 N HCl solution (pH 0.9). Then, the mAbs were applied for 30 min, and the bound mAbs were detected using the LSAB kit. The optimal temperature for the antigen retrieval varied among specimens, and was dependent on the type of basement membrane examined. Thus, it was considered that heating at two or three different temperatures could be helpful for the precise diagnosis of AS. Adopting the antigen retrieval method could extend the possibility of immunohistochemical diagnosis of AS to cases without using fresh-frozen sections.

  3. HER-2 and INT-2 amplification estimated by quantitative PCR in paraffin-embedded ovarian cancer tissue samples.

    Science.gov (United States)

    Hruza, C; Dobianer, K; Beck, A; Czerwenka, K; Hanak, H; Klein, M; Leodolter, S; Medl, M; Müllauer-Ertl, S; Preiser, J

    1993-01-01

    Competitive polymerase chain reaction (PCR) systems were developed for rapid and quantitative estimation of HER-2 (c-erbB-2) and INT-2 oncogene amplification in paraffin-embedded ovarian cancer tissue samples. The beta-globin gene was used as reference and DNA from paraffin-embedded placenta tissue as single copy control. Reliability of the PCR method could be demonstrated by comparing dot blot data with PCR data of identical tumour samples. The PCR method was used to determine HER-2 and INT-2 copy numbers in 196 ovarian cancer samples. HER-2 and INT-2 were found to be amplified in 40 and 19%, respectively. In 8% HER-2 copy numbers were greater than five, but no high INT-2 copies were noted. Kaplan-Meier estimates did not reveal significant association with overall survival. Indirect correlation between HER-2 and INT-2 amplification was observed. The present PCR system is a valuable method for prospective and retrospective studies.

  4. Automated Analysis of Protein Expression and Gene Amplification within the Same Cells of Paraffin-Embedded Tumour Tissue

    Directory of Open Access Journals (Sweden)

    Timo Gaiser

    2010-01-01

    Full Text Available Background: The simultaneous detection of protein expression and gene copy number changes in patient samples, like paraffin-embedded tissue sections, is challenging since the procedures of immunohistochemistry (IHC and Fluorescence in situ Hybridization (FISH negatively influence each other which often results in suboptimal staining. Therefore, we developed a novel automated algorithm based on relocation which allows subsequent detection of protein content and gene copy number changes within the same cell.

  5. Single-Nucleotide Polymorphism-Microarray Ploidy Analysis of Paraffin-Embedded Products of Conception in Recurrent Pregnancy Loss Evaluations.

    Science.gov (United States)

    Maslow, Bat-Sheva L; Budinetz, Tara; Sueldo, Carolina; Anspach, Erica; Engmann, Lawrence; Benadiva, Claudio; Nulsen, John C

    2015-07-01

    To compare the analysis of chromosome number from paraffin-embedded products of conception using single-nucleotide polymorphism (SNP) microarray with the recommended screening for the evaluation of couples presenting with recurrent pregnancy loss who do not have previous fetal cytogenetic data. We performed a retrospective cohort study including all women who presented for a new evaluation of recurrent pregnancy loss over a 2-year period (January 1, 2012, to December 31, 2013). All participants had at least two documented first-trimester losses and both the recommended screening tests and SNP microarray performed on at least one paraffin-embedded products of conception sample. Single-nucleotide polymorphism microarray identifies all 24 chromosomes (22 autosomes, X, and Y). Forty-two women with a total of 178 losses were included in the study. Paraffin-embedded products of conception from 62 losses were sent for SNP microarray. Single-nucleotide polymorphism microarray successfully diagnosed fetal chromosome number in 71% (44/62) of samples, of which 43% (19/44) were euploid and 57% (25/44) were noneuploid. Seven of 42 (17%) participants had abnormalities on recurrent pregnancy loss screening. The per-person detection rate for a cause of pregnancy loss was significantly higher in the SNP microarray (0.50; 95% confidence interval [CI] 0.36-0.64) compared with recurrent pregnancy loss evaluation (0.17; 95% CI 0.08-0.31) (P=.002). Participants with one or more euploid loss identified on paraffin-embedded products of conception were significantly more likely to have an abnormality on recurrent pregnancy loss screening than those with only noneuploid results (P=.028). The significance remained when controlling for age, number of losses, number of samples, and total pregnancies. These results suggest that SNP microarray testing of paraffin-embedded products of conception is a valuable tool for the evaluation of recurrent pregnancy loss in patients without prior fetal

  6. [Comparison of two different real-time PCR systems in postmortem diagnosis of tuberculosis in paraffin-embedded tissues].

    Science.gov (United States)

    Yağmur, Gülhan; Albayrak, Nurhan; Daş, Taner; Yıldırım, Muzaffer; Ozgün, Ayşe; Büyük, Yalçın

    2014-10-01

    Tuberculosis (TB) is one of those infections with high morbidity and mortality in all around the world. Hundreds of people died from this disease without diagnosed or due to resistant strains in Turkey. Therefore, it is important to identify postmortem cases who have died from tuberculosis. Molecular methods have been widely used as well as conventional methods in the diagnosis of tuberculosis. The aim of this study was to compare the two different real-time polymerase chain reaction (Rt-PCR) system in the postmortem diagnosis of Mycobacterium tuberculosis infections in paraffin-embedded tissues. A total of 40 paraffin-embedded tissue samples [lung (n= 35), brain (n= 2), heart (n= 2), lymph node (n= 1)] in which histopathologic findings consistent with TB (necrotizing granulomatous inflammation, gelatinous caseous pneumonia, necrotic fibrous nodul) obtained from 37 autopsy cases (31 male, 6 female; age range: 25-85 yrs) were included in the study. Paraffin-embedded tissues were deparafinized with xylene and ethyl alcohol and then DNA isolation was done with QIAsymphony DSP Virus/Pathogen Midi kit in the QIAsymphony device. DNA amplification process was performed by Rt-PCR using the kit Artus® M. tuberculosis RG-PCR in the Rotor-Gene® Q device (Qiagen, Germany). Likewise, after deparafinization process, samples placed in the cartridge and isolation and Rt-PCR was performed by Xpert® MTB/RIF (Cepheid, USA) system, simultaneosly. Seventeen and 20 out of the 40 paraffin-embedded tissues yielded positive results with Qiagen and Xpert system, respectively. M.tuberculosis DNA was found positive in 13 (32.5%) and negative in 16 (40%) of the samples by both of the systems, exhibiting 72.5% (29/40) of concordance. On the other hand, seven (17.5%) samples that were positive with Xpert system yielded negative result with the Qiagen, while four (10%) samples that were positive with Qiagen yielded negative result with the Xpert system. Of the 20 positive cases detected with

  7. Microdissecção e captura a laser na investigação do gene TP53 em tecidos incluídos em parafina Laser-capture microdissection for TP53 gene analysis in paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    Shadia Muhammad Ihlaseh

    2007-02-01

    characterize its enormous potential for diagnosis and research. OBJECTIVE: This study describes the standardization of LCM and DNA extraction from formalin fixed and paraffin-embedded tissues. MATERIAL AND METHOD: The gene TP53 exon 8 and the cyclophilin gene were studied in normal and neoplastic liver and kidney samples from a chemical carcinogenesis model in rat. DNA extraction was confirmed by nested-PCR. RESULTS: Histological sections preparation for LCM and the nested-PCR procedures were standardized; 48.3% amplifications of the gene TP53 exon 8 and 51.7% of the cyclophilin gene samples were obtained. When at least one of the gene segments was considered, 79.3% samples presented amplification. DISCUSSION AND CONCLUSION: Procedures for DNA extraction from formalin fixed and paraffin-embedded tissues collected by LCM were standardized. They can be useful for DNA collection for molecular studies.

  8. Semi-Nested Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Successful Quantitation of Cytokeratin mRNA Expression Levels for the Subtyping of Non-Small-Cell Lung Carcinoma Using Paraffin-Embedded and Microdissected Lung Biopsy Specimens

    International Nuclear Information System (INIS)

    Nakanishi, Yoko; Shimizu, Tetsuo; Tsujino, Ichiro; Obana, Yukari; Seki, Toshimi; Fuchinoue, Fumi; Ohni, Sumie; Oinuma, Toshinori; Kusumi, Yoshiaki; Yamada, Tsutomu; Takahashi, Noriaki; Hashimoto, Shu; Nemoto, Norimichi

    2013-01-01

    In patients with inoperable advanced non-small cell lung carcinomas (NSCLCs), histological subtyping using small-mount biopsy specimens was often required to decide the indications for drug treatment. The aim of this study was to assess the utility of highly sensitive mRNA quantitation for the subtyping of advanced NSCLC using small formalin fixing and paraffin embedding (FFPE) biopsy samples. Cytokeratin (CK) 6, CK7, CK14, CK18, and thyroid transcription factor (TTF)-1 mRNA expression levels were measured using semi-nested real-time quantitative (snq) reverse-transcribed polymerase chain reaction (RT-PCR) in microdissected tumor cells collected from 52 lung biopsies. Our results using the present snqRT-PCR method showed an improvement in mRNA quantitation from small FFPE samples, and the mRNA expression level using snqRT-PCR was correlated with the immunohistochemical protein expression level. CK7, CK18, and TTF-1 mRNA were expressed at significantly higher levels (P<0.05) in adenocarcinoma (AD) than in squamous cell carcinoma (SQ), while CK6 and CK14 mRNA expression was significantly higher (P<0.05) in SQ than in AD. Each histology-specific CK, particularly CK18 in AD and CK6 in SQ, were shown to be correlated with a poor prognosis (P=0.02, 0.02, respectively). Our results demonstrated that a quantitative CK subtype mRNA analysis from lung biopsy samples can be useful for predicting the histology subtype and prognosis of advanced NSCLC

  9. DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR.

    Directory of Open Access Journals (Sweden)

    Andrea J Adams

    Full Text Available Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM to Macherey-Nagel DNA FFPE (MN, test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90% when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections, current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from

  10. DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR.

    Science.gov (United States)

    Adams, Andrea J; LaBonte, John P; Ball, Morgan L; Richards-Hrdlicka, Kathryn L; Toothman, Mary H; Briggs, Cheryl J

    2015-01-01

    Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum

  11. Immunohistochemical detection of VHS virus in paraffin-embedded specimens of rainbow trout (Oncorhynchus mykiss); The influence of primary antibody, fixative, and antigen unmasking on method sensitivity

    DEFF Research Database (Denmark)

    Evensen, O.; Olesen, Niels Jørgen

    1997-01-01

    The influence of the primary antibody, the fixative, and the antigen unmasking technique on the method sensitivity of immunohistochemistry as a method for the identification of viral hemorrhagic septicemia (VHS) virus in paraffin-embedded specimens of naturally infected rainbow trout (Oncorhynchus...... performed on parallel specimens, and the virus titer (TCID50/ml) was determined. Purified nucleocapsid protein (N-protein) of the virus was incorporated in an artificial antigen substrate (polymerized bovine serum albumin), fixed as described above, and embedded in paraffin wax. Microwave unmasking...

  12. PCR-RFLP studies on chromosome 3p in formaldehyde-fixed, paraffin-embedded cervical cancer tissues.

    Science.gov (United States)

    Karlsen, F; Rabbitts, P H; Sundresan, V; Hagmar, B

    1994-09-15

    Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p21-22. We examined 31 paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p21. Allele loss was found in 19 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p21-22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehyde-fixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment length polymorphism (RFLP) studies on fixed tissues.

  13. Efficient treatment of paraffin-embedded cervical tissue for HPV DNA testing by HC-II and PCR assays.

    Science.gov (United States)

    Cricca, M; Bonvicini, F; Venturoli, S; Ambretti, S; Gallinella, G; Gentilomi, G; Musiani, M; Zerbini, M

    2004-02-01

    High-risk human papillomaviruses (HR-HPVs) are the primary cause of cervical cancer. In order to meet with clinical requirements, a direct capture test with signal amplification (HC-II), able to detect the 13 prevalent HR-HPVs, has been developed and validated for cytological specimens. the use of HC-II assay with formaldehyde-fixed paraffin-embedded cervical biopsies, for retrospective studies or to support histological findings, was investigated by analysing three different sample treatments. The efficacy of this test was compared with a reference PCR-ELISA, using MY09/11 and GP5+/6+ consensus primers and the use of a single extraction method for both HC-II and PCR-ELISA assays was validated. protease treatment of dewaxed biopsy sections allowed an optimal performance of HC-II and has also been validated for PCR-ELISA. Overall, on the analysis of 50 cervical samples HC-II and PCR-ELISA assays showed a high concordance (K=0.80). Compared with PCR-ELISA, the HC-II had a sensitivity of 86.4% and a specificity of 92.9%. The results showed that short amplimers are necessary for a sensitive PCR-ELISA from formaldehyde-fixed paraffin-embedded biopsies, while HC-II showed a relatively low sensitivity for HPV18 within the HR probe pool. HC-II can be a valid tool for the diagnosis of HPV infection in biopsy material. The possibility to use the same specimen preparation material for both HC-II and PCR-ELISA allows HC-II positive specimens to be further processed by PCR-ELISA if specific genotyping is needed.

  14. Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas

    NARCIS (Netherlands)

    Baay, M. F.; Quint, W. G.; Koudstaal, J.; Hollema, H.; Duk, J. M.; Burger, M. P.; Stolz, E.; Herbrink, P.

    1996-01-01

    We have compared the efficacies of three general primer pairs for the detection of human papillomavirus (HPV) DNA in formaldehyde-fixed paraffin-embedded carcinomas. The use of these primer pairs leads to underestimates of the HPV prevalence (GP5/6, 61.1%; CPI/IIG, 57.4%; MY09/11, 46.9%; combined,

  15. Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas

    NARCIS (Netherlands)

    M.F.D. Baay (Marc); W.G.V. Quint (Wim); J. Koudstaal; H. Hollema; J.M. Duk; M.P.M. Burger; E. Stolz (Ernst); P. Herbrink (Paul)

    1996-01-01

    textabstractWe have compared the efficacies of three general primer pairs for the detection of human papillomavirus (HPV) DNA in formaldehyde-fixed paraffin-embedded carcinomas. The use of these primer pairs leads to underestimates of the HPV prevalence (GP5/6, 61.1%;

  16. Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas

    NARCIS (Netherlands)

    Baay, MFD; Quint, WGV; Koudstaal, J; Hollema, H; Duk, JM; Burger, MPM; Stolz, E; Herbrink, P

    We have compared the efficacies of three general primer pairs for the detection of human papillomavirus (HPV) DNA in formaldehyde-fixed paraffin-embedded carcinomas. The use of these primer pairs leads to underestimates of the HPV prevalence (BP5/6, 61.1%; CPI/IIG, 57.4%; MY09/11, 46.9%; combined,

  17. Avaliação de dois métodos de extração de DNA de material parafinado para amplificação em PCR Evaluation of two methods of DNA extraction from paraffin-embedded material for PCR amplification

    Directory of Open Access Journals (Sweden)

    Luciana Estevam Simonato

    2007-04-01

    Full Text Available INTRODUÇÃO: Diversos métodos para extração de DNA a partir de tecidos biológicos inclusos em parafina encontram-se descritos na literatura, sendo o sucesso desse procedimento de grande importância para a realização de métodos moleculares de diagnóstico empregando a reação em cadeia da polimerase (PCR. OBJETIVO: Este estudo avaliou dois métodos de extração de DNA de material parafinado, visando à amplificação do DNA genômico pela técnica da PCR. MATERIAL E MÉTODO: Foram utilizadas 35 amostras de casos de carcinoma epidermóide de assoalho bucal diagnosticados e tratados no Centro de Oncologia Bucal da Universidade Estadual Paulista (Unesp. Os métodos de extração de DNA avaliados incluíram: 1. digestão com proteinase K seguida por purificação com Chelex 100® (BioRad; e 2. sistema QIAamp DNA minikit® (Qiagen. O DNA obtido foi quantificado por espectrofotometria e amplificado pela técnica da PCR, utilizando-se oligonucleotídeos iniciadores para betaglobina. RESULTADOS: A concentração de DNA obtido do material extraído com o primeiro método apresentou média de 120,62 ng/µl com razão entre as leituras das absorbâncias 260/280 variando de 0,8 a 1,41. Para as amostras extraídas com o segundo procedimento, o rendimento médio foi de 67,38 ng/µl, no entanto a razão 260/280 variou entre 1,11 e 2,53. O material foi submetido à PCR e, das 35 amostras extraídas com cada método, respectivamente, 29 e 30 apresentaram sinal positivo. CONCLUSÃO: Os dois métodos utilizados para obtenção de DNA de material parafinado apresentaram desempenho semelhante, revelando que ambos têm potencial para auxiliar na prática da biologia molecular diagnóstica, assim como no estudo diagnóstico retrospectivo em material parafinizado.BACKGROUND: There are several methods for DNA extraction from formalin-fixed paraffin-embedded tissues reported in the literature. High success rate on this procedure is important for the use of

  18. Characterization of foreign materials in paraffin-embedded pathological specimens using in situ multi-elemental imaging with laser spectroscopy.

    Science.gov (United States)

    Busser, Benoit; Moncayo, Samuel; Trichard, Florian; Bonneterre, Vincent; Pinel, Nicole; Pelascini, Frédéric; Dugourd, Philippe; Coll, Jean-Luc; D'Incan, Michel; Charles, Julie; Motto-Ros, Vincent; Sancey, Lucie

    2018-03-01

    Pathologists typically encounter many disparate exogenous materials in clinical specimens during their routine histopathological examinations, especially within the skin, lymph nodes, and lungs. These foreign substances may be free extracellular deposits or induce several clinical abnormalities or histopathological patterns. However, pathologists almost never investigate or report the chemical nature of exogenous metals in clinical specimens due to a lack of convenient and available technologies. In this paper, a novel strategy based on laser-induced breakdown spectroscopy (LIBS) technology is evaluated for in situ multi-elemental tissue imaging. The improved procedures allow visualization of the presence of chemical elements contained within paraffin-embedded specimens of medical interest with elemental images that are stackable with conventional histology images. We selected relevant medical situations for which the associated pathology reports were limited to the presence of lymphohistiocytic and inflammatory cells containing granules (a granuloma and a pseudolymphoma) or to lymph nodes or skin tissues containing pigments or foreign substances. Exogenous elements such as aluminum, titanium, copper, and tungsten were identified and localized within the tissues. The all-optical LIBS elemental imaging instrument that we developed is fully compatible with conventional optical microscopy used for pathology analysis. When combined with routine histopathological analysis, LIBS is a versatile technology that might help pathologists establish or confirm diagnoses for a wide range of medical applications, particularly when the nature of external agents present in tissues needs to be investigated.

  19. Gelatin in situ zymography on fixed, paraffin-embedded tissue: zinc and ethanol fixation preserve enzyme activity.

    Science.gov (United States)

    Hadler-Olsen, Elin; Kanapathippillai, Premasany; Berg, Eli; Svineng, Gunbjørg; Winberg, Jan-Olof; Uhlin-Hansen, Lars

    2010-01-01

    In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue.

  20. A simple osmium post-fixation paraffin-embedment technique to identify lipid accumulation in fish liver using medaka (Oryziaslatipes) eggs and eleutheroembryos as lipid rich models

    International Nuclear Information System (INIS)

    Mondon, J.A.; Howitt, J.; Tosiano, M.; Kwok, K.W.H.; Hinton, D.E.

    2011-01-01

    Highlights: → Hepatic lipidosis in fish liver is often misdiagnosed or overlooked. → Specific histological fat stains and cryostat sections are not commonly used. → Standard paraffin processing removes lipid leaving vacuoles of unknown origin. → Osmium post-fixed paraffin-embedment is a cost effective alternative. → Medaka trials show suitability for lipid visualization in tissues from egg to adult. - Abstract: Hepatic lipidosis is a non-specific biomarker of effect from pollution exposure in fish. Fatty liver is often misdiagnosed or overlooked in histological assessments due to the decreasing application of specific fat procedures and stains. For example, ethanol dehydration in standard paraffin processing removes lipids, leaving vacuoles of which the precise nature is unknown. Lipids can be identified using osmium post-fixation in semi-thin resin sections or transmission electron microscopy. However, both are expensive and technically demanding procedures, often not available for routine environmental risk assessment and monitoring programs. The current emphasis to reduce and refine animal toxicity testing, requires refinement of the suite of histopathological techniques currently available to maximize information gained from using fish for toxicity testing and as bio-indicators of environmental quality. This investigation has successfully modified an osmium post-fixation technique to conserve lipids in paraffin-embedded tissues using medaka (Oryzias latipes) eleutheroembryos and eggs (embryos) as lipid rich models.

  1. Immunohistochemical detection of SWC3, CD2, CD3, CD4 and CD8 antigens in paraformaldehyde fixed and paraffin embedded porcine lymphoid tissue

    DEFF Research Database (Denmark)

    Tingstedt, Jens Erik; Tornehave, Ditte; Lind, Peter

    2003-01-01

    Identification of the different cell types of the immune system is important for in situ studies on the pathogenesis of infectious diseases in various animals, including the pig. Unfortunately, many monoclonal anti-leukocyte antibodies are only useful for staining frozen tissue sections with inhe......Identification of the different cell types of the immune system is important for in situ studies on the pathogenesis of infectious diseases in various animals, including the pig. Unfortunately, many monoclonal anti-leukocyte antibodies are only useful for staining frozen tissue sections...... with inherent poor tissue morphology, and are not readily adapted to formaldehyde fixed and paraffin embedded tissue with well preserved morphology. Seven well characterised monoclonal antibodies against porcine leukocyte antigens were tested on neutral buffered paraformaldehyde fixed and paraffin embedded...

  2. Comparison between single PCR and nested PCR in detection of human papilloma viruses in paraffin-embedded OSCC and fresh oral mucosa.

    Science.gov (United States)

    Jalouli, Miranda; Jalouli, Jamshid; Ibrahim, Salah O; Hirsch, Jan-Michaél; Sand, Lars

    2015-01-01

    Infection with human papilloma virus (HPV) has been implicated as one of the risk factors for the development of oropharyngeal cancer. Many different HPV tests exist, and information regarding their specific technical, analytical, and clinical properties is increasing. This study aimed to compare the level of detection of HPV using two reliable polymerase chain reaction (PCR) methods, nested PCR (NPCR) and single PCR (SPCR), in archival paraffin-embedded oral squamous cell carcinoma (OSCC) samples and fresh oral mucosa specimens. The presence of HPV genome in two groups of tissue samples was analyzed: (i) 57 paraffin-embedded OSCC samples from Sudan and (ii) eight healthy fresh oral mucosal samples from Swedish volunteers. The specimens were tested by SPCR with primer pair MY9/MY11 and NPCR using GP5+/GP6+ primer sets. Eighteen (32%) out of the 57 paraffin-embedded OSCC samples, and five (62%) out of the eight fresh clinically healthy samples were found to be HPV-positive with NPCR. With SPCR, four (7%) out of the paraffin-embedded OSCC samples were HPV-positive. A statistically significant difference between HPV-positive and -negative samples was found when comparing NPCR and SPCR in OSCC and fresh oral mucosa (p<0.0001). The comparative test between SPCR and NPCR showed 100% sensitivity and 69% specificity for OSCC. The use of the GP5+/GP6+ nested PCR increased the positivity rate, efficiency rate and sensitivity of HPV detection in oral samples significantly and should be considered as the method of choice. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  3. Confirmation of immunoglobulin heavy chain rearrangement by polymerase chain reaction using surgically obtained, paraffin-embedded samples to diagnose primary palate mucosa-associated lymphoid tissue lymphoma: A case study

    Directory of Open Access Journals (Sweden)

    Shigehiro Abe

    2015-01-01

    Conclusion: We suggest that, if histological examination is ambiguous or fresh material is insufficient, PCR can be performed using paraffin-embedded materials to definitively diagnose low-grade lymphomas, such as MALT lymphoma.

  4. Optimization of methods to assess mitochondrial DNA in archival paraffin-embedded tissues from mammary canine tumors Otimização dos métodos para avaliar o DNA mitocondrial obtido a partir de tumores mamários caninos incluídos em parafina

    Directory of Open Access Journals (Sweden)

    Angélica C. Bertagnolli

    2008-08-01

    Full Text Available In this study we describe the alterations used to extract and amplify mitochondrial desoxyribonucleic acid (DNA from formalin-fixed paraffin-embedded samples of canine mammary tumors. The epithelial and mesenchymal components (chondromyxoid and chondroid of each tumor, as well as the normal mammary gland tissues, were manually microdissected from 19 mixed canine mammary tumors (10 benign mixed tumors and nine carcinomas arising in mixed tumors. DNA was extracted by Invisorb® Spin Tissue Mini Kit, with protocol changes proposed by the manufacturer. A 273-bp fragment was amplified by polymerase chain reaction (PCR and submitted to automatic sequence analysis. The fragment was successfully analyzed in 100% of the samples. However, an additional lysis step, the reduction of volume in buffer solutions and PCR, a higher annealing temperature and an increase in the number of PCR cycles were required. The initial PCR products were diluted and re-amplified in six samples so that they could be successfully analyzed.A presente comunicação descreve as modificações usadas para extrair e amplificar o DNA mitocondrial obtido de amostras de tumores mamários caninos fixados em formol tamponado a 10% e incluídos em parafina. Os componentes epiteliais e mesenquimais (condromixóide e condróide, bem como a mama normal adjacente, foram microdissectados manualmente de 19 tumores mamários (10 tumores mistos benignos e nove carcinomas em tumores mistos. O DNA foi extraído utilizando-se o Invisorb® Spin Tissue Mini Kit com modificações do protocolo proposto pelo fabricante. Um fragmento de 273-pb foi amplificado por reação em cadeia da polimerase (PCR e seqüenciado em seqüenciador automático. O fragmento foi analisado em 100% das amostras, entretanto modificações como lise adicional, redução do volume das soluções de extração e PCR, aumento da temperatura de anelamento e do número de ciclos de amplificação foram necessárias. Em seis

  5. Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

    Science.gov (United States)

    Hykin, Sarah M; Bi, Ke; McGuire, Jimmy A

    2015-01-01

    For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for

  6. Temperature-dependent thermal properties of a paraffin phase change material embedded with herringbone style graphite nanofibers

    International Nuclear Information System (INIS)

    Warzoha, Ronald J.; Weigand, Rebecca M.; Fleischer, Amy S.

    2015-01-01

    Highlights: • The thermal properties of a PCM with nanofibers are determined. • The solid-phase thermal conductivity scales exponentially with volume fraction. • The liquid-phase thermal conductivity is only enhanced beyond a critical percolation threshold. • The nanoscale interface resistance depends on the nanoparticle’s dimensionality. • The thermal diffusivity and volumetric heat capacity of the nanoenhanced PCMs are found. - Abstract: In many studies, carbon nanoparticles with high values of thermal conductivity (10–3000 W/m K) have been embedded into phase change thermal energy storage materials (PCMs) in order to enhance their bulk thermal properties. While a great deal of work to date has focused on determining the effect of these nanoparticles on a PCM’s solid phase thermal properties, little is known about their effect on its liquid phase thermal properties. Thus, in this study, the effect of implanting randomly oriented herringbone style graphite nanofibers (HGNF, average diameter = 100 nm, average length = 20 μm) on the bulk thermal properties of an organic paraffin PCM (IGI 1230A, T melt = 329.15 K) in both the solid and liquid phase is quantified. The bulk thermal conductivity, volumetric heat capacity and thermal diffusivity of HGNF/PCM nanocomposites are obtained as a function of temperature and HGNF volume loading level. It is found that the property enhancement varies significantly depending on the material phase. In order to explain the difference between solid and liquid phase thermal properties, heat flow at the nanoparticle–PCM and nanoparticle–nanoparticle interfaces is examined as a function of HGNF loading level and temperature. To do this, the solid and liquid phase thermal boundary resistances (TBRs) between the nanoparticles and the surrounding PCM and/or between contacting nanoparticles are found. Results suggest that the TBR at the HGNF–PCM interface is nearly double the TBR across the HGNF–HGNF interface in

  7. Use of proteinase K for RT-PCR of cytokine mRNA in formalin fixed tissue

    DEFF Research Database (Denmark)

    Davies, G N; Bevan, I S; Banner, Jytte

    1996-01-01

    Fresh tissue from cases of sudden infant death syndrome is becoming increasingly scarce and therefore researchers interesting in studying the aetiology of this syndrome have had to resort to archival tissue, usually in the form of paraffin wax sections. A simple method for isolating mRNA from for....... Transcripts of a housekeeping gene were detected in all tissues. This method should be useful for examining gene expression in archival material....

  8. Comparação de três protocolos de extração de DNA a partir de tecido fixado em formol e incluído em parafina Comparison of three DNA extraction protocols from formaldehyde-fixed and paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    José Veríssimo Fernandes

    2004-06-01

    Full Text Available OBJETIVO: Padronizar um método alternativo para extração de DNA a partir de tecido fixado em formol e conservado em arquivos de blocos de parafina, visando à realização de estudos retrospectivos. MÉTODOS: Comparou-se a eficiência de protocolos de extração de DNA a partir de tecido parafinado, para análise por reação em cadeia de polimerase (PCR, tomando-se como parâmetro um protocolo baseado em um kit comercial. Foram feitas extrações do DNA de 60 espécimes por três métodos: o protocolo A, baseado no kit GlassMAX; o B, utilizando-se o kit GFX TM; e o C, tendo como base o método de Banerjee et al.(2. A integridade e a suficiência do DNA presente na amostra foram avaliadas pela amplificação por PCR de um segmento de 110pb do gene da beta-globina humana, com visualização por meio de eletroforese em gel de poliacrilamida, corado pela prata. Resultados: Das 60 amostras analisadas, 45 apresentaram resultado positivo na PCR quando o DNA foi extraído por qualquer um dos três protocolos. Em seis amostras, a amplificação foi positiva apenas para o DNA extraído pelos protocolos A e C. Em três amostras, o resultado foi positivo apenas para o DNA extraído pelo protocolo A, e em duas, apenas para o DNA extraído pelo protocolo C. CONCLUSÕES: O protocolo C apresentou desempenho semelhante ao do protocolo A, com as vantagens de apresentar menor custo, dispensar o uso de kit comercial, além de não utilizar solventes orgânicos, revelando-se uma alternativa viável para a obtenção de DNA a partir de tecido fixado em formol e incluído em parafina.OBJECTIVE: To set up a method for DNA extraction from paraffin embedded cervical cancer specimens, previously formalin-fixed, aiming to accomplish retrospective analysis. METHODS: Sixty specimens were submitted to DNA extraction by three different methods. All of them involved digestion of the tissues by proteinase K, followed by DNA purification, based in three different approaches

  9. Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas.

    Science.gov (United States)

    Baay, M F; Quint, W G; Koudstaal, J; Hollema, H; Duk, J M; Burger, M P; Stolz, E; Herbrink, P

    1996-03-01

    We have compared the efficacies of three general primer pairs for the detection of human papillomavirus (HPV) DNA in formaldehyde-fixed paraffin-embedded carcinomas. The use of these primer pairs leads to underestimates of the HPV prevalence (GP5/6, 61.1%; CPI/IIG, 57.4%; MY09/11, 46.9%; combined, 72.8%). The efficacy of each primer pair seemed to be inversely correlated to the length of the amplimer produced. By using newly developed type-specific primer pairs (amplimer length, approximately 100 bp), an increase in HPV DNA detection (87.6%) was found.

  10. Cases of cryptosporidiosis co-infections in AIDS patients: a correlation between clinical presentation and GP60 subgenotype lineages from aged formalin-fixed stool samples.

    Science.gov (United States)

    Del Chierico, F; Onori, M; Di Bella, S; Bordi, E; Petrosillo, N; Menichella, D; Cacciò, S M; Callea, F; Putignani, L

    2011-07-01

    Nine cases of cryptosporidiosis co-infections in AIDS patients were clinically categorised into severe (patients 1, 3, 8 and 9), moderate (patients 4 and 5) and mild (patients 2, 6 and 7). Formalin-fixed faecal specimens from these patients were treated to obtain high quality DNA competent for amplification and sequencing of the 60-kDa glycoprotein (GP60) gene. Sequence analysis revealed that one patient was infected with Cryptosporidium hominis whereas the remaining eight patients were infected with C. parvum. Interestingly, the patients showing severe cryptosporidiosis harboured two subtypes within the C. parvum allelic family IIc (IIcA5G3 and IIcA5G3R2), whereas patients with moderate or mild infections showed various subtypes of the C. parvum allelic family IIa (IIaA14G2R1, IIaA15G2R1, IIaA17G3R1 and IIaA18G3R1). DNA extraction and genotyping of Cryptosporidium spp. is a challenging task on formalin-fixed stool samples, whose diagnostic outcome is age-dependent. The method herein reported represents a step forward routine diagnosis and improves epidemiology of HIV-related clinical cases. Due to the need to elucidate genetic richness of Cryptosporidium human isolates, this approach represents a useful tool to correlate individual differences in symptoms to subgenotyping lineages.

  11. Treponema pallidum ssp. pallidum identification by real-time PCR targetting the polA gene in paraffin-embedded samples positive by immunohistochemistry.

    Science.gov (United States)

    Gama, A; Carrillo-Casas, E M; Hernández-Castro, R; Vázquez-Aceituno, V A; Toussaint-Caire, S; Xicohtencatl-Cortes, J; Fernández-Martínez, R; Moreno-Coutiño, G

    2017-11-01

    Syphilis is a systemic and sexually transmitted infection caused by Treponema pallidum ssp. pallidum. This spirochete causes different clinical and subclinical stages depending on the duration of infection and immune status of the host. Several tests have been developed for diagnosis, and are classified into direct and indirect methods. The first one includes dark field microscopy, direct fluorescent antibody test in fluids or tissue, and molecular biology techniques. In the indirect method (serologic), the routine tests are used, and are divided in two categories: non-treponemal and treponemal ones. The objective of this work was to identify T. pallidum ssp. pallidum in paraffin-embedded skin biopsies positive by immunohistochemistry, using conventional polymerase chain reaction (PCR) and quantitative real time PCR (qPCR). We included a sample of 17 paraffin-embedded biopsies. DNA was extracted and processed by conventional PCR and real-time PCR with a TaqMan® probe to identify the polA gene. Using PCR, 11 tested positive (64.7%) and 6 (35.3%) were negative. With qPCR and TaqMan® probe, 100% of samples tested positive. The minimum number of spirochetes detected in each sample was 2. With this work, we can conclude that qPCR is a fast and very accurate method for diagnosis of syphilis in tissue specimens.

  12. Immunohistochemical detection of 3 viral infections in paraffin-embedded tissue from mink (Mustela vison): a tissue-microarray-based study

    Science.gov (United States)

    Hammer, Anne Sofie; Dietz, Hans Henrik; Hamilton-Dutoit, Stephen

    2007-01-01

    Immunohistochemical (IHC) assays were developed and tested for the detection of 3 viral infections in archived paraffin-embedded mink tissue. Specimens had been obtained from mink with diagnoses of acute Aleutian disease (AD), mink parvoviral enteritis (MVE), or canine distemper (CD) made by means of routine diagnostic procedures. To improve the efficiency and reduce the costs of IHC analyses, tissue microarray (TMA) technology was used. Representative cores 2 mm in diameter from each tissue specimen and from positive- and negative-control specimens were collected in a TMA block. Immunohistochemical reactions to viral antigens were assessed and graded. Positive reactions were found in 91% of the 32 specimens from mink with AD, 53% to 80% of the 60 specimens from mink with MVE, and all 66 of the specimens from mink with CD. To validate the use of TMAs, the IHC methods were applied to whole-mount paraffin-embedded sections of 10 of the positive specimens for each disease, together with whole-mount sections of small intestine and lung tissue from 2 healthy mink. The IHC grading of the TMA cores and the whole-mount sections from the same animal corresponded completely. These results suggest that IHC demonstration of viral antigen allows rapid and reliable diagnosis of the 3 viral infections in mink and is a potential supplement to histologic diagnostic procedures. The TMA technique proved useful for screening large numbers of samples for expression of specific viral antigens, while reducing overall costs. PMID:17193876

  13. Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins

    Science.gov (United States)

    Dinhopl, Nora; Mostegl, Meike M.; Richter, Barbara; Nedorost, Nora; Maderner, Anton; Fragner, Karin; Weissenböck, Herbert

    2011-01-01

    In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples. PMID:21711191

  14. Eradication of breast cancer with bone metastasis by autologous formalin-fixed tumor vaccine (AFTV) combined with palliative radiation therapy and adjuvant chemotherapy: a case report.

    Science.gov (United States)

    Kuranishi, Fumito; Ohno, Tadao

    2013-06-04

    Skeletal metastasis of breast carcinoma is refractory to intensive chemo-radiation therapy and therefore is assumed impossible to cure. Here, we report an advanced case of breast cancer with vertebra-Th7 metastasis that showed complete response to combined treatments with formalin-fixed autologous tumor vaccine (AFTV), palliative radiation therapy with 36 Gy, and adjuvant chemotherapy with standardized CEF (cyclophosphamide, epirubicin, and 5FU), zoledronic acid, and aromatase inhibitors following mastectomy for the breast tumor. The patient has been disease-free for more than 4 years after the mammary surgery and remains well with no evidence of metastasis or local recurrence. Thus, a combination of AFTV, palliative radiation therapy, and adjuvant chemotherapy may be an effective treatment for this devastating disease.

  15. Zoonotic onchocerciasis in Hiroshima, Japan, and molecular analysis of a paraffin section of the agent for a reliable identification

    Directory of Open Access Journals (Sweden)

    Fukuda M.

    2011-05-01

    Full Text Available Japan is a country of high specific diversity of Onchocerca with eight species, the adults of two not yet known. Onchocerca dewittei japonica, a common filarial parasite of wild boar, had been proved to be the agent of five zoonotic onchocerciasis in Kyushu island with morphological and molecular studies. The sixth case, at Hiroshima in the main island, was identified to the same Onchocerca species, based on adult characters observed on histological sections. To consolidate the identification, mitochondrial cytochrome c oxidase subunit 1 (CO1 gene analysis was attempted with the formalin-fixed, paraffin-embedded parasite specimen. The sequence (196 bp of a CO1 gene fragment of the parasite successfully PCR-amplified agreed well with those of O. dewittei japonica registered in GenBank, confirming the morphological identification. Moreover a comparison with the CO1 gene sequences of six other Onchocerca species in GenBank excluded the possibility that Onchocerca sp. from wild boar and Onchocerca sp. type A from cattle in Japan, were the causative agents in this case. Mitochondrial DNA analysis proved to be a valuable tool to support the morphological method for the discrimination of zoonotic Onchocerca species in a histological specimen.

  16. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Steven

    2010-01-01

    ABSTRACT: Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18, is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin...... embedded (FFPE) sections of cervical cancer.Tissue blocks from 35 cases of in situ or invasive cervical squamous cell carcinoma and surrogate FFPE sections containing the cell lines HeLa and SiHa were tested for HPV 16 and HPV18 by conventional PCR using type specific primers, and for the housekeeping gene...... beta-actin. Using HPV 16 E7 primers, PCR products with the expected length were detected in 18 of 35 of FFPE sections (51%). HPV 18 E7 specific sequences were detected in 3 of 35 FFPE sections (9%).In our experience, the PCR technique is a robust, simple and sensitive way of type specific detection...

  17. Detection of Hepatitis B Virus Antigens in Paraffin-embedded Liver Specimens from the Amazon Region, Brazil

    Directory of Open Access Journals (Sweden)

    Simonetti SRR

    2002-01-01

    Full Text Available Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in different other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg and hepatitis B core antigen (HBcAg by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4% histological samples were HBsAg reactive and 5 (6.3% were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens.

  18. Quantitative description of the morphology and ossification center in the axial skeleton of 20-week gestation formalin-fixed human fetuses using magnetic resonance images.

    Science.gov (United States)

    Chabert, Steren; Villalobos, Manuel; Ulloa, Patricia; Salas, Rodrigo; Tejos, Cristian; San Martin, Sebastian; Pereda, Jaime

    2012-03-01

    Human tissues are usually studied using a series of two-dimensional visualizations of in vivo or cutout specimens. However, there is no precise anatomical description of some of the processes of human fetal development. The purpose of our study is to develop a quantitative description of the normal axial skeleton by means of high-resolution three-dimensional magnetic resonance (MR) images, collected from six normal 20-week-old human fetuses fixed in formaldehyde. Fetuses were collected after spontaneous abortion and subsequently fixed with formalin. They were imaged using a 1.5 T MR scanner with an isotropic spatial resolution of 200 µm. The correct tissue discrimination between ossified and cartilaginous bones was confirmed by comparing the images achieved by MR scans and computerized axial tomographies. The vertebral column was segmented out from each image using a specially developed semi-automatic algorithm. Vertebral body dimensions and inter-vertebral distances were larger in the lumbar region, in agreement with the beginning of the ossification process from the thoracolumbar region toward the sacral and cephalic ends. In this article, we demonstrate the feasibility of using MR images to study the ossification process in formalin-fixed fetal tissues. A quantitative description of the ossification centers of vertebral bodies and arches is presented. © 2012 John Wiley & Sons, Ltd.

  19. Detection of Lawsonia intracellularis in formalin-fixed porcine intestinal tissue samples: comparison of immunofluorescence and in-situ hybridization, and evaluation of the effects of controlled autolysis.

    Science.gov (United States)

    Jensen, T K; Boesen, H T; Vigre, H; Boye, M

    2010-01-01

    Two methods, an immunofluorescence assay (IFA; with a Lawsonia intracellularis-specific monoclonal antibody) and fluorescent in-situ hybridization (FISH; with a specific oligonucleotide probe targeting 16S ribosomal RNA of the bacterium), were compared for their ability to detect L. intracellularis (the cause of porcine proliferative enteritis [PE]) in formalin-fixed samples of intestinal tissue. Of 69 intestinal samples with gross lesions of PE, 63 were positive by both FISH and IFA, but six were positive only by IFA. This indicated that the sensitivity of FISH was 91% that of IFA. However, both methods had a specificity of 100%. Fifty normal porcine intestines were negative by both tests. IFA was much less susceptible than FISH to the effects of autolysis. Thus, three of nine samples from pigs with PE were FISH-negative after being kept at 20 degrees C for 4 days, and seven were FISH negative after 2 weeks; after 4 weeks at this temperature, however, six of the nine samples were still IFA positive. After being kept at 4 degrees C for 12 weeks, the majority of samples (> or = 66%) were positive by both methods.

  20. Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections

    Energy Technology Data Exchange (ETDEWEB)

    Park, J.S.; Kurman, R.J.; Kessis, T.D.; Shah, K.V. (Johns Hopkins Medical Institutions, Baltimore, MD (USA))

    1991-01-01

    A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and {sup 35}S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.

  1. PCR-based clonality analysis of B-cell lymphomas in paraffin-embedded tissues: diagnostic value of immunoglobulin kappa and lambda light chain gene rearrangement investigation.

    Science.gov (United States)

    Amara, Khaled; Trimeche, Mounir; Ziadi, Sonia; Sriha, Badreddine; Mokni, Moncef; Korbi, Sadok

    2006-01-01

    Polymerase chain reaction (PCR)-based analysis, employed for detecting immunoglobulin heavy chain (IgH) gene rearrangements, has become a diagnostic tool widely used in the investigation of B-cell lymphomas, but the overall sensitivity of these methods does not exceed 80%, notably in germinal center (GC) and post-GC B-cell origin lymphomas. Many PCR strategies devised for detecting immunoglobulin light chain (IgL) gene rearrangements have been developed to enhance the clonality detection rates. However, the feasibility of these methods in routine clinical diagnosis using paraffin-embedded tissues has not yet been investigated sufficiently. We studied a large series of 108 cases of B-cell lymphomas, as well as 20 reactive lymphoid tissues using degenerate primers to amplify immunoglobulin kappa (Igkappa) and lambda (Iglambda) light chain genes. B-cell clonality was further investigated using semi-nested PCR for IgH gene rearrangements. B-cell clonality was detected in 74%, 56.5%, and 43.5% of cases using IgH, Igkappa, and Iglambda PCR, respectively. By combining these methods, the clonality detection rate increased to 93.5%. Only polyclonal patterns were noted in reactive lymphoid samples. We concluded that in addition to the established methods for IgH analysis, a PCR-based approach for IgL gene rearrangements analysis improves the clonality detection rate in over 90% of B-cell lymphoma cases using routine histological specimens with poor preservation of the genomic DNA.

  2. Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

    Science.gov (United States)

    Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-Ichi; Ono, Ken-Ichiro; Kishi, Yoshiro; Mekada, Eisuke

    2016-04-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

  3. Gene Expression Analysis of Immunostained Endothelial Cells Isolated from Formaldehyde-fixated Paraffin Embedded Tumors Using Laser Capture Microdissection – a Technical Report

    Science.gov (United States)

    Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E.

    2009-01-01

    Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by RT-PCR and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4°C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. GAPDH and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. PMID:19425073

  4. Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection--a technical report.

    Science.gov (United States)

    Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E

    2009-12-01

    Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by reverse transcription-PCR (RT-PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4 degrees C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. Glyceraldehyde-3-phosphate dehydrogenase and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM.

  5. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma.

    Science.gov (United States)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Steven; Koch, Jørn

    2010-01-22

    Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18, is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin embedded (FFPE) sections of cervical cancer.Tissue blocks from 35 cases of in situ or invasive cervical squamous cell carcinoma and surrogate FFPE sections containing the cell lines HeLa and SiHa were tested for HPV 16 and HPV18 by conventional PCR using type specific primers, and for the housekeeping gene beta-actin. Using HPV 16 E7 primers, PCR products with the expected length were detected in 18 of 35 of FFPE sections (51%). HPV 18 E7 specific sequences were detected in 3 of 35 FFPE sections (9%).In our experience, the PCR technique is a robust, simple and sensitive way of type specific detection of HPV16 and HPV18 genes in FFPE tissue. That makes this technique applicable to routine practices of HPV detection.

  6. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma

    Directory of Open Access Journals (Sweden)

    Hamilton-Dutoit Steven

    2010-01-01

    Full Text Available Abstract Human papillomavirus (HPV infection, and in particularly infection with HPVs 16 and 18, is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin embedded (FFPE sections of cervical cancer. Tissue blocks from 35 cases of in situ or invasive cervical squamous cell carcinoma and surrogate FFPE sections containing the cell lines HeLa and SiHa were tested for HPV 16 and HPV18 by conventional PCR using type specific primers, and for the housekeeping gene β-actin. Using HPV 16 E7 primers, PCR products with the expected length were detected in 18 of 35 of FFPE sections (51%. HPV 18 E7 specific sequences were detected in 3 of 35 FFPE sections (9%. In our experience, the PCR technique is a robust, simple and sensitive way of type specific detection of HPV16 and HPV18 genes in FFPE tissue. That makes this technique applicable to routine practices of HPV detection.

  7. Evaluation of ALK rearrangement in Chinese non-small cell lung cancer using FISH, immunohistochemistry, and real-time quantitative RT- PCR on paraffin-embedded tissues.

    Directory of Open Access Journals (Sweden)

    Yun-Gang Zhang

    Full Text Available Patients with ALK gene rearrangements often manifest dramatic responses to crizotinib, an ALK inhibitor. Accurate identification of patients with ALK-positive non-small cell lung cancer (NSCLC is essential for the clinical application of ALK-targeted therapy. However, assessing EML4-ALK rearrangement in NSCLC remains challenging in routine pathology practice. The aim of this study was to compare the diagnostic accuracy of FISH, immunohistochemistry (IHC, and real-time quantitative RT-PCR (QPCR methodologies for detection of EML4-ALK rearrangement in NSCLC and to appraise immunohistochemistry as a pre-screening tool. In this study, a total of 473 paraffin-embedded NSCLC samples from surgical resections and biopsies were analyzed by IHC with ALK antibody. ALK rearrangement was further confirmed by FISH and QPCR. ALK protein expression was detected in twenty patients (20/473, 4.2%. Of the 20 ALK-positive cases by IHC, 15 cases were further confirmed as ALK rearrangement by FISH, and 5 cases were not interpretable. Also, we evaluated 13 out of the 20 IHC-positive tissues by QPCR in additional to FISH, and found that 9 cases were positive and 2 cases were equivocal, whereas 2 cases were negative although they were positive by both IHC and FISH. The ALK status was concordant in 5 out of 8 cases that were interpretable by three methods. Additionally, none of the 110 IHC-negative cases with adenocarcinoma histology showed ALK rearrangements by FISH. Histologically, almost all the ALK-rearranged cases were adenocarcinoma, except that one case was sarcomatoid carcinoma. A solid signet-ring cell pattern or mucinous cribriform pattern was presented at least focally in all ALK-positive tumors. In conclusion, our findings suggested that ALK rearrangement was associated with ALK protein expression. The conventional IHC assay is a valuable tool for the pre-screening of patients with ALK rearrangement in clinical practice and a combination of FISH and QPCR is

  8. Changing patterns of keratin expression could be associated with functional maturation of the developing human bladder

    NARCIS (Netherlands)

    de la Rosette, Jean; Smedts, Frank; Schoots, Coen; Hoek, Hans; Laguna, Pilar

    2002-01-01

    PURPOSE: We investigate the keratin phenotype of human transitional epithelium at various gestational ages and whether keratin composition of transitional epithelium is related to bladder function and morphology. MATERIALS AND METHODS: Consecutive sections from formalin fixed paraffin embedded

  9. Changing patterns of keratin expression could be associated with functional maturation of the developing human bladder.

    NARCIS (Netherlands)

    Rosette, J.J.M.H.C. de la; Smedts, F.; Schoots, C.; Hoek, H.; Laguna, P.M.

    2002-01-01

    PURPOSE: We investigate the keratin phenotype of human transitional epithelium at various gestational ages and whether keratin composition of transitional epithelium is related to bladder function and morphology. MATERIALS AND METHODS: Consecutive sections from formalin fixed paraffin embedded

  10. Cadmium, Zinc, and Selenium Levels in Carcinoma of the Human Prostate

    National Research Council Canada - National Science Library

    Sarafanov, Andrey; Centeno, Jose A

    2008-01-01

    .... The objectives are: 1) to establish reliability of using formalin-fixed paraffin-embedded (FFPE) prostate tissue for analysis of Zn, Se and Cd tissue by comparing their levels in the fresh specimen...

  11. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Stephen Jacques

    2010-01-01

    ABSTRACT: Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18 is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin...

  12. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Steven

    2010-01-01

    ABSTRACT: Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18, is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin...

  13. Embedding

    DEFF Research Database (Denmark)

    Høyrup, Jens

    2016-01-01

    “Embedding” as a technical concept comes from linguistics, more precisely from grammar. The present paper investigates whether it can be applied fruitfully to certain questions that have been investigated by historians (and sometimes philosophers) of mathematics: 1. The construction of numeral...... systems, in particular place-value and quasi place-value systems. 2. The development of algebraic symbolisms. 3. The discussion whether “scientific revolutions” ever take place in mathematics, or new conceptualizations always include what preceded them. A final section investigates the relation between...... spatial and linguistic embedding and concludes that the spatio-linguistic notion of embedding can be meaningfully applied to the former two discussions, whereas the apparent embedding of older within new theories is rather an ideological mirage....

  14. Embedding

    DEFF Research Database (Denmark)

    Høyrup, Jens

    2015-01-01

    to become the starting point not only for theoretical algebra, but for the whole transformation of mathematics from his time onward: the possibility of embedding, that is, of making a symbol or an element of a calculation stand not only for a single number, determined or undetermined, but for a whole...

  15. Paraffin ingestion - the problem

    African Journals Online (AJOL)

    mistake it for liquid refreshment. Rom et al.5 found that 33% of children ingested paraffin from intermediate containers. These are containers used to dispense paraffin into appliances. Overcrowding and limited storage space often result in paraffin being left within easy reach of children. The incidence of paraffin ingestion is ...

  16. Long-lasting complete response status of advanced stage IV gall bladder cancer and colon cancer after combined treatment including autologous formalin-fixed tumor vaccine: two case reports.

    Science.gov (United States)

    Imaoka, Yuki; Kuranishi, Fumito; Miyazaki, Tsubasa; Yasuda, Hiroko; Ohno, Tadao

    2017-09-11

    The prognosis of advanced (stage IV) cancer of the digestive organs is very poor. We have previously reported a case of advanced breast cancer with bone metastasis that was successfully treated with combined treatments including autologous formalin-fixed tumor vaccine (AFTV). Herein, we report the success of this approach in advanced stage IV (heavily metastasized) cases of gall bladder cancer and colon cancer. Case 1: A 61-year-old woman with stage IV gall bladder cancer (liver metastasis and lymph node metastasis) underwent surgery in May 2011, including partial resection of the liver. She was treated with AFTV as the first-line adjuvant therapy, followed by conventional chemotherapy. This patient is still alive without any recurrence, as confirmed with computed tomography, for more than 5 years. Case 2: A 64-year-old man with stage IV colon cancer (multiple para-aortic lymph node metastases and direct abdominal wall invasion) underwent non-curative surgery in May 2006. Following conventional chemotherapy, two courses of AFTV and radiation therapy were administered sequentially. This patient has had no recurrence for more than 5 years. We report the success of combination therapy including AFTV in cases of liver-metastasized gall bladder cancer and abdominal wall-metastasized colon cancer. Both patients experienced long-lasting, complete remission. Therefore, combination therapies including AFTV should be considered in patients with advanced cancer of the digestive organs.

  17. First detection and genotyping of Giardia intestinalis in stool samples collected from children in Ghazni Province, eastern Afghanistan and evaluation of the PCR assay in formalin-fixed specimens.

    Science.gov (United States)

    Lass, Anna; Karanis, Panagiotis; Korzeniewski, Krzysztof

    2017-08-01

    It is estimated that faecal-orally transmitted diseases are common in Afghanistan, as a consequence of poor hygienic standards of life and widespread contamination of water and food with both human and animal faeces. However, there is little information in the literature concerning infections caused by intestinal parasites in the Afghan population. In this study, we report the occurrence of Giardia intestinalis assemblages (A and B) in formalin-fixed stool samples collected from 245 Afghan schoolchildren living in Ghazni Province in eastern Afghanistan. Detection of the parasite's DNA and genotyping was performed using real-time PCR, specific to the β-giardin gene of G. intestinalis. Positive results were recorded in 52 (21.2%) samples. Genotyping was successful in 39 faecal samples and showed the predominance of assemblage B of G. intestinalis in this population (15 assemblage A and 24 assemblage B). Co-infection with both genotypes A and B was detected in four samples. Additionally, we evaluated the effect of 10% buffered formalin fixative on the detection of G. intestinalis DNA using real-time PCR and nested PCR characterised by different lengths of PCR products (74 and 479 bp, respectively). The human faeces containing the Giardia cysts were tested for 16 weeks. Amplification of G. intestinalis DNA with real-time PCR was possible up to 6 weeks of preservation of stool sample in formalin, compared to only 2 weeks with nested PCR. This suggests that real-time PCR is a more suitable tool in cases where stool samples have to be kept in formalin for longer periods of time.

  18. Modified paraffin wax for improvement of histological analysis efficiency.

    Science.gov (United States)

    Lim, Jin Ik; Lim, Kook-Jin; Choi, Jin-Young; Lee, Yong-Keun

    2010-08-01

    Paraffin wax is usually used as an embedding medium for histological analysis of natural tissue. However, it is not easy to obtain enough numbers of satisfactory sectioned slices because of the difference in mechanical properties between the paraffin and embedded tissue. We describe a modified paraffin wax that can improve the histological analysis efficiency of natural tissue, composed of paraffin and ethylene vinyl acetate (EVA) resin (0, 3, 5, and 10 wt %). Softening temperature of the paraffin/EVA media was similar to that of paraffin (50-60 degrees C). The paraffin/EVA media dissolved completely in xylene after 30 min at 50 degrees C. Physical properties such as the amount of load under the same compressive displacement, elastic recovery, and crystal intensity increased with increased EVA content. EVA medium (5 wt %) was regarded as an optimal composition, based on the sectioning efficiency measured by the numbers of unimpaired sectioned slices, amount of load under the same compressive displacement, and elastic recovery test. Based on the staining test of sectioned slices embedded in a 5 wt % EVA medium by hematoxylin and eosin (H&E), Masson trichrome (MT), and other staining tests, it was concluded that the modified paraffin wax can improve the histological analysis efficiency with various natural tissues. (c) 2010 Wiley-Liss, Inc.

  19. Design and Testing of Digitally Manufactured Paraffin Acrylonitrile-Butadiene-Styrene Hybrid Rocket Motors

    OpenAIRE

    McCulley, Jonathan M.

    2013-01-01

    This research investigates the application of additive manufacturing techniques for fabricating hybrid rocket fuel grains composed of porous Acrylonitrile-butadiene-styrene impregnated with paraffin wax. The digitally manufactured ABS substrate provides mechanical support for the paraffin fuel material and serves as an additional fuel component. The embedded paraffin provides an enhanced fuel regression rate while having no detrimental effect on the thermodynamic burn properties of the fuel g...

  20. Preventing Paraffin-Related Injury

    OpenAIRE

    C. Schwebel, David; Swart, Dehran

    2009-01-01

    Paraffin (called kerosene in North America and other parts of the world) is the most commonly used fuel in ‎non-electrified dwellings worldwide. It is especially popular in Africa and South Asia. Although paraffin ‎offers many advantages – especially its comparatively low cost to produce – it poses two major risks of ‎injury. First, paraffin poisoning is common, either through ingestion or through inhalation of smoke and ‎fumes. Second, paraffin is highly flammable, and poses fire risk t...

  1. The prognostic value of oncogenic antigen 519 (OA-519) expression and proliferative activity detected by antibody MIB-1 in node-negative breast cancer

    DEFF Research Database (Denmark)

    Jensen, V; Ladekarl, M; Holm-Nielsen, P

    1995-01-01

    of invasion of skin or deep fascia (= T1N0M0 and T2N0M0). The median follow-up time was 104 months (range 5-143 months). Immunohistochemical analysis of OA-519 expression was performed on formalin-fixed, paraffin-embedded tissue. The proliferative activity was estimated using a Ki-67 equivalent monoclonal...

  2. An ER activity profile including ER, PR, Bcl-2 and IGF-IR may have potential as selection criterion for letrozole or tamoxifen treatment of patients with advanced breast cancer

    DEFF Research Database (Denmark)

    Henriksen, Katrine L; Rasmussen, Birgitte B; Lykkesfeldt, Anne E

    2009-01-01

    microarrays from formalin fixed paraffin embedded primary tumor material from a subgroup of patients (9.4%), who have participated in the international, randomized, phase III clinical trial PO25 comparing letrozole with tamoxifen in 907 patients with advanced breast cancer. The expression levels of ER...

  3. Overexpression of p53 in Nigerian breast cancers and its ...

    African Journals Online (AJOL)

    This study sought to determine the expression of p53 protein as well as the relationship with oestrogen receptor (ER) and progesterone receptor (PR) proteins. Methodology: Formalin-fixed, paraffin-embedded tissue samples of diagnosed invasive breast cancer were obtained from the Department of Anatomic and ...

  4. Temporal lobe pathology in amyotrophic lateral sclerosis. Do amyotrophic lateral sclerosis and Alzheimer's disease share a common etiological factor?

    NARCIS (Netherlands)

    Smitt, P. A.; Troost, D.; Louwerse, E. S.; de Jong, J. M.; van Kessel, D. T.; de Leeuw, M. A.

    1993-01-01

    An autopsy study was performed on temporal lobe samples from 20 non-demented patients with amyotrophic lateral sclerosis (ALS), 17 age-matched non-demented controls and 4 Alzheimer's disease (AD) patients. Formalin fixed, paraffin embedded sections from the hippocampus with adjacent parahippocampal

  5. Assessment of the topoisomerase I gene copy number as a predictive biomarker of objective response to irinotecan in metastatic colorectal cancer

    DEFF Research Database (Denmark)

    Nygård, Sune Boris; Christensen, Ib Jarle; Nielsen, Signe Lykke

    2014-01-01

    (TOP1) copy number and objective response following irinotecan treatment in patients with metastatic colorectal cancer. Materials and methods. Formalin-fixed, paraffin-embedded tumor samples from 78 patients, who received irinotecan monotherapy in second line, were included. TOP1 was assessed...

  6. A generally applicable sequential alkaline phosphatase immunohistochemical double staining

    NARCIS (Netherlands)

    van der Loos, Chris M.; Teeling, Peter

    2008-01-01

    A universal type of sequential double alkaline phosphatase immunohistochemical staining is described that can be used for formalin-fixed, paraffin-embedded and cryostat tissue sections from human and mouse origin. It consists of two alkaline phosphatase detection systems including enzymatic

  7. Overexpression of cyclin D1 indicates a poor prognosis in squamous cell carcinoma of the head and neck

    NARCIS (Netherlands)

    Michalides, R. J.; van Veelen, N. M.; Kristel, P. M.; Hart, A. A.; Loftus, B. M.; Hilgers, F. J.; Balm, A. J.

    1997-01-01

    To evaluate the overexpression of cyclin D1 and p53 as a prognostic marker of squamous cell carcinoma of the head and neck and to investigate whether deregulation of these genes is associated with an unfavorable course of disease. Retrospective study. Formalin-fixed, paraffin-embedded tumor

  8. The effects of mode of delivery and sex of newborn on placental morphology in control and diabetic pregnancies

    DEFF Research Database (Denmark)

    Mayhew, T M; Sørensen, Flemming Brandt; Klebe, J G

    1993-01-01

    of glucose and glycated haemoglobin levels. Neonatal and placental weights were recorded and placentae sampled in a systematic random fashion. Fields of view on formalin-fixed, paraffin-embedded sections were analysed to obtain estimates of volumes, surface areas, lengths and diffusion (harmonic mean...

  9. Original paper

    African Journals Online (AJOL)

    Hawkins RA. Receptors in the management of breast cancer Br J Hos Med 1985; PP 160. - 164. 10. Katoh AK, Stemmler N et al. Immunoperoxidase staining for oestrogen and progesterone receptors in archival formalin fixed, paraffin embedded breast carcinomas after microwave antigen retrieval. Biotech- histochem 1997 ...

  10. A prognostic profile of hypoxia-induced genes for localised high-grade soft tissue sarcoma

    DEFF Research Database (Denmark)

    Aggerholm-Pedersen, Ninna; Sørensen, Brita Singers; Overgaard, Jens

    2016-01-01

    sarcoma (STS). METHODS: The hypoxia-induced gene quantification was performed by real-time quantitative PCR (RT-qPCR) of formalin-fixed, paraffin-embedded tissue samples. The gene expression cut-points were determined in a test cohort of 55 STS patients and used to allocate each patient into a more...

  11. Resistance to first line platinum paclitaxel chemotherapy in serous epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Smoter, Marta; Waldstrøm, Marianne

    2014-01-01

    of sensitivity to platinum/paclitaxel treatment. The primary aim of the study was to investigate whether ERCC1 and Tau protein expression correlates with patient outcome in newly diagnosed epithelial ovarian cancer (EOC) patients. Formalin-fixed, paraffin-embedded tissue sections from 227 newly diagnosed EOC...

  12. The relationship of platinum resistance and ERCC1 protein expression in epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Waldstrøm, Marianne; Jakobsen, Anders

    2009-01-01

    : Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian cancer were used for immunohistochemical staining for the ERCC1 protein. All patients received carboplatin-paclitaxel combination chemotherapy. RESULTS: Excision repair cross-complementation group 1 enzyme...

  13. Association of ERCC1 protein expression to platinum resistance in epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Dahl Steffensen, Karina; Waldstrøm, Marianne; Jakobsen, Anders

    was to investigate if immunohistochemical expression of ERCC1 protein was associated with resistance to standard combination carboplatin and paclitaxel chemotherapy in newly diagnosed ovarian cancer patients. Methods: Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian...

  14. Nigerian Veterinary Journal - Vol 38, No 3 (2017)

    African Journals Online (AJOL)

    Formalin-fixed and paraffin-embedded tissues of chickens are useful for retrospective studies on pathology of H5N1 Highly Pathogenic Avian Influenza Virus (HPAI) outbreaks in Nigeria · EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. B.O. Akanbi, S Fereidouni, V.O. ...

  15. Long noncoding RNA metastasis-associated lung adenocarcinoma ...

    African Journals Online (AJOL)

    Subjects and methods: The relative expression of MALAT1 was determined in 37 human glioblastoma formalin-fixed paraffin embedded (FFPE) tissue samples and 10 FFPE non-neoplastic brain tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR) technology. Results: The current results ...

  16. The use of special stains in liver biopsy interpretation: Implications ...

    African Journals Online (AJOL)

    Materials and Methods: The formalin fixed paraffin embedded blocks of liver biopsies reported in two histopathology laboratories between 2008 and 2013 were retrieved. These were stained with H and E and the following standard special stains for liver tissue histology – Perl's Prussian blue, reticulin, Sirius red, Shikata ...

  17. Immunohistochemical expression of latent membrane protein 1 ...

    African Journals Online (AJOL)

    Methods: Archival formalin-fixed, paraffin-embedded NPC biopsies were evaluated in 23 Moroccan patients for the presence of LMP1 and p53 using immunohistochemistry (IHC). Results: No LMP1 expression was observed whereas 8 of 23 cases (34. 7%) had detectable p53 protein in the nuclei of tumor cells.

  18. Immunophenotyping of Gastrointestinal Mesenchymal Tumours in ...

    African Journals Online (AJOL)

    METHODS: Materials were formalin fixed paraffin embedded blocks of GMT diagnosed in Lagos Nigeria between January 1995 and February 2007. Sections were stained with CD117, CD34, SMA, S100 and Desmin antibodies at the research Laboratory of The Leeds General Infirmary, United Kingdom following standard ...

  19. Mucin expression patterns in histological grades of colonic cancers ...

    African Journals Online (AJOL)

    Pathological expression of mucins has been noted in cancer development and progression. This study sought to identify and quantify the types of mucins produced during various histological grades of colon cancer and to assess the diagnostic significance. Methods: Formalin fixed, paraffin-embedded tissue blocks, ...

  20. Survivorship patterns of histopathological variants and molecular ...

    African Journals Online (AJOL)

    Objective: To study the relationship of histopathological characteristics, molecular subtypes of breast cancer and survival in a low resource setting. Design: Tumours from prospectively ascertained patients newly diagnosed with breast cancer were analyzed. Formalin-fixed and paraffin-embedded sections were constructed ...

  1. Antikeratin antibodies in routine diagnostic pathology. A comparison of 10 different commercial antikeratins

    DEFF Research Database (Denmark)

    Mygind, H; Nielsen, B; Moe, D

    1988-01-01

    Ten commercially available antikeratin antisera were tested immunohistochemically on fresh frozen and formalin-fixed paraffin-embedded tissue. Eight of the antisera were in addition tested on protein-immunoblottings. For six of the antisera a good correspondence was found between our immunoblots ...

  2. Megakaryocytic features useful for the diagnosis of myeloproliferative disorders can be obtained by a novel unsupervised software analysis

    OpenAIRE

    Tripodo, C.; Valenti, C.; Ballarò, B.; Rudzki, Z.; Tegolo, D.; Di Gesù, V.; Florena, A.M.; Franco, V.

    2006-01-01

    An unsupervised method for megakaryocyte detection and analysis is proposed, in order to validate supplementary tools which can be of help in supporting the pathologist in the classification of Philadelphia negative chronic myeloproliferative disorders with thrombocytosis. The experiment was conducted on high power magnification photomicrographs taken from hematoxylin-and-eosin 3 µm thick sections of formalin fixed, paraffin embedded bone marrow biopsies from ...

  3. Concordance of genotype for polymorphisms in DNA isolated from peripheral blood and colorectal cancer tumor samples

    NARCIS (Netherlands)

    van Huis-Tanja, Lieke; Kweekel, Dinemarie; Gelderblom, Hans; Koopman, Miriam; Punt, Kees; Guchelaar, Henk-Jan; van der Straaten, Tahar

    2013-01-01

    Background & aim: Results from different pharmacogenetic association studies in colorectal cancer are often conflicting. Both peripheral blood and formalin-fixed, paraffin-embedded (FFPE) tissue are routinely used as DNA source. This could cause bias due to somatic alterations in tumor tissue, such

  4. Influence of Liquid Paraffin, White Soft Paraffin and Initial Hydration ...

    African Journals Online (AJOL)

    hydrated white soft paraffin on the viscosity of a cream formulated with a corticosteroid. Methods: The formulations were prepared via homogenization with variable velocity in the range 3300 - 4000 rpm. Individual series of preparations contained the ...

  5. TULSA UNIVERSITY PARAFFIN DEPOSITION PROJECTS

    Energy Technology Data Exchange (ETDEWEB)

    Michael Volk; Cem Sarica

    2003-10-01

    As oil and gas production moves to deeper and colder water, subsea multiphase production systems become critical for economic feasibility. It will also become increasingly imperative to adequately identify the conditions for paraffin precipitation and predict paraffin deposition rates to optimize the design and operation of these multiphase production systems. Although several oil companies have paraffin deposition predictive capabilities for single-phase oil flow, these predictive capabilities are not suitable for the multiphase flow conditions encountered in most flowlines and wellbores. For deepwater applications in the Gulf of Mexico, it is likely that multiphase production streams consisting of crude oil, produced water and gas will be transported in a single multiphase pipeline to minimize capital cost and complexity at the mudline. Existing single-phase (crude oil) paraffin deposition predictive tools are clearly inadequate to accurately design these pipelines because they do not account for the second and third phases, namely, produced water and gas. The objective of this program is to utilize the current test facilities at The University of Tulsa, as well as member company expertise, to accomplish the following: enhance our understanding of paraffin deposition in single and two-phase (gas-oil) flows; conduct focused experiments to better understand various aspects of deposition physics; and, utilize knowledge gained from experimental modeling studies to enhance the computer programs developed in the previous JIP for predicting paraffin deposition in single and two-phase flow environments. These refined computer models will then be tested against field data from member company pipelines. The following deliverables are scheduled during the first three projects of the program: (1) Single-Phase Studies, with three different black oils, which will yield an enhanced computer code for predicting paraffin deposition in deepwater and surface pipelines. (2) Two

  6. Nitrous Paraffin Hybrid, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — The Nitrous Oxide Paraffin Hybrid engine (N2OP) is a proposed technology designed to provide small launch vehicles with high specific impulse, indefinitely storable...

  7. Analysis of hepcidin expression: in situ hybridization and quantitative polymerase chain reaction from paraffin sections.

    Science.gov (United States)

    Sakuraoka, Yuhki; Sawada, Tokihiko; Shiraki, Takayuki; Park, Kyunghwa; Sakurai, Yuhichiro; Tomosugi, Naohisa; Kubota, Keiichi

    2012-07-28

    To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.

  8. Specific detection of Pasteurella multocida in chickens with fowl cholera and in pig lung tissues using fluorescent rRNA in situ hybridization

    DEFF Research Database (Denmark)

    Mbuthia, P.G.; Christensen, H.; Boye, Mette

    2001-01-01

    in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues P. multocida cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood...... and fast method for specific detection of P. multocida in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to P. multocida....

  9. Fibrinogen Demonstration in Oral Lichen Planus: An Immunofluorescence Study on Archival Tissues.

    Science.gov (United States)

    Shirol, Pallavi D; Naik, Veena; Kale, Alka

    2015-10-01

    Lichen planus is a premalignant condition with minimal diagnostic aids. This study is an attempt to use paraffin embedded sections of lichen planus with immunofluorescein stain and to evaluate the immunofluorescent sections to establish pattern of fibrinogen deposition. Thirty-five paraffin embedded sections of old and new cases of oral lichen planus (study group) and five normal oral mucosa (control group) were chosen. Two sections of each (H & E) case were taken, one was stained with hematoxylin and eosin and another with fluorescein isothiocynate conjugate (FITC) polyclonal rabbit antibody against fibrinogen. Fluorescent findings were examined with a fluorescent microscope. A high statistical significant correlation was found in respect to fluorescence positivity, intensity of fluorescence and distribution of fluorescence each with p direct immunofluorescence staining in routine set up where only formalin fixed tissues are received. Paraffin embedded sections can be successfully used in direct immunofluorescence staining when only formalin fixed tissues are received.

  10. Detection of Chlamydia in postmortal formalin-fixed tissue

    DEFF Research Database (Denmark)

    Lundemose, AG; Lundemose, JB; Birkelund, Svend

    1989-01-01

    examined and the effect of autolysis and tetracycline treatment was evaluated. Furthermore, lung tissue from two patients who died of ornithosis was examined. Inclusions detected in lung sections showed a bright apple-green fluorescence, and had a characteristic and easily recognizable morphology...

  11. TULSA UNIVERSITY PARAFFIN DEPOSITION PROJECTS

    Energy Technology Data Exchange (ETDEWEB)

    Cem Sarica; Michael Volk

    2004-06-01

    As oil and gas production moves to deeper and colder water, subsea multiphase production systems become critical for economic feasibility. It will also become increasingly imperative to adequately identify the conditions for paraffin precipitation and predict paraffin deposition rates to optimize the design and operation of these multi-phase production systems. Although several oil companies have paraffin deposition predictive capabilities for single-phase oil flow, these predictive capabilities are not suitable for the multiphase flow conditions encountered in most flowlines and wellbores. For deepwater applications in the Gulf of Mexico, it is likely that multiphase production streams consisting of crude oil, produced water and gas will be transported in a single multiphase pipeline to minimize capital cost and complexity at the mudline. Existing single-phase (crude oil) paraffin deposition predictive tools are clearly inadequate to accurately design these pipelines, because they do not account for the second and third phases, namely, produced water and gas. The objective of this program is to utilize the current test facilities at The University of Tulsa, as well as member company expertise, to accomplish the following: enhance our understanding of paraffin deposition in single and two-phase (gas-oil) flows; conduct focused experiments to better understand various aspects of deposition physics; and, utilize knowledge gained from experimental modeling studies to enhance the computer programs developed in the previous JIP for predicting paraffin deposition in single and two-phase flow environments. These refined computer models will then be tested against field data from member company pipelines.

  12. A rapid method of reprocessing for electronic microscopy of cut histological in paraffin

    International Nuclear Information System (INIS)

    Hernandez Chavarri, F.; Vargas Montero, M.; Rivera, P.; Carranza, A.

    2000-01-01

    A simple and rapid method is described for re-processing of light microscopy paraffin sections to observe they under transmission electron microscopy (TEM) and scanning electron microscopy (SEM) The paraffin-embedded tissue is sectioned and deparaffinized in toluene; then exposed to osmium vapor under microwave irradiation using a domestic microwave oven. The tissues were embedded in epoxy resin, polymerized and ultrathin sectioned. The method requires a relatively short time (about 30 minutes for TEM and 15 for SEM), and produces a reasonable quality of the ultrastructure for diagnostic purposes. (Author) [es

  13. 21 CFR 890.5110 - Paraffin bath.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Paraffin bath. 890.5110 Section 890.5110 Food and... PHYSICAL MEDICINE DEVICES Physical Medicine Therapeutic Devices § 890.5110 Paraffin bath. (a) Identification. A paraffin bath is a device intended for medical purposes that consists of a tub to be filled...

  14. Otimização da imunoistoquímica para detecção de herpesvírus bovino tipo 5 (BHV-5 em tecidos do sistema nervoso central fixados com formaldeído Optimization of immunohistochemistry for Bovine Herpesvirus 5 (BHV-5 detection on formalin-fixed tissues of the central nervous system

    Directory of Open Access Journals (Sweden)

    S.O. Hübner

    2005-02-01

    Full Text Available Com o objetivo de otimizar a técnica de imunoistoquímica para detecção de herpesvírus bovino tipo 5 (BHV-5 em tecidos do sistema nervoso central fixado em formaldeído, foram avaliados diferentes métodos de digestão enzimática, diferentes anticorpos e reagentes para bloqueio de reações inespecíficas. As reações apresentaram a máxima intensidade de coloração específica e a quantidade mínima de coloração de fundo quando foram usadas protease de Streptomyces griseus (0,1% ou proteinase K de Tritirachium album limber (0,05%, mediante incubação durante 15 minutos a 37°C. Entre os anticorpos monoclonais analisados, dois foram capazes de detectar BHV-5. As reações inespecíficas foram bloqueadas mais efetivamente pela incubação do tecido com caseína (0,5%, durante cinco minutos, ou leite em pó (2,5%, durante 60 minutos, ou soro eqüino (2,5%, durante 60 minutos. Com a técnica otimizada foi possível a detecção de BHV-5 em material de arquivo.In order to optimize immunohistochemical technique (IHC for detection of Bovine herpesvirus type 5 (BHV-5 on formalin-fixed sections of central nervous system, different methods of enzymatic digestion, use of different antibodies and products for blocking of nonspecific reactivity were evaluated. The reactions showed the highest intensity of specific coloration and the minimum amounts of background when protease from Streptomyces griseus (0.1% or proteinase K from Tritirachium album limber (0.05% were used, incubating for 15 minutes at 37°C. Only two of the tested monoclonal antibodies specifically labelled BHV-5 antigen. The nonspecific reactions were blocked through incubation of tissues with casein (0.5% for five minutes or powdered milk (2.5% for 60 minutes or equine serum (2.5% for 60 minutes. The optimized immunohistochemical method allowed the detection of BHV-5 antigen in histopathological archives.

  15. Quantification of microRNA-21 and microRNA-125b in melanoma tissue

    DEFF Research Database (Denmark)

    Wandler, Anne; Riber-Hansen, Rikke; Hager, Henrik

    2017-01-01

    the important involvement of different miRNAs in melanoma biology and may serve as solid basics for further miRNA investigations in melanoma formalin-fixed and paraffin-embedded tissue. In particular, there is increased expression of miR-21 in melanomas compared with benign nevi.......Although microRNAs (miRNAs) have emerged as potent mediators of melanoma development and progression, a precise understanding of their oncogenic role remains unclear. In this study, we analysed formalin-fixed and paraffin-embedded tissues from two separate melanoma cohorts and from a series...... with array analysis could not be confirmed using qPCR. ISH with digital quantification showed expression of miR-21 and miR-125b in the melanocytic lesions. miR-21 ISH was increased in melanomas, whereas quantification of miR-125b showed uniform ISH expression across nevi and melanomas. Our results support...

  16. The prognostic value of oncogenic antigen 519 (OA-519) expression and proliferative activity detected by antibody MIB-1 in node-negative breast cancer

    DEFF Research Database (Denmark)

    Jensen, V; Ladekarl, M; Holm-Nielsen, P

    1995-01-01

    of invasion of skin or deep fascia (= T1N0M0 and T2N0M0). The median follow-up time was 104 months (range 5-143 months). Immunohistochemical analysis of OA-519 expression was performed on formalin-fixed, paraffin-embedded tissue. The proliferative activity was estimated using a Ki-67 equivalent monoclonal...... antibody (MIB-1), which is applicable on formalin-fixed, paraffin-embedded tissue after microwave pretreatment. OA-519 was expressed in about one-third of the tumours and the percentage of proliferating cells (the MIB-1 index) ranged between 1 and 72 per cent (median 17 per cent). Using multivariate Cox...... analysis, both the MIB-1 index and OA-519 expression were of independent prognostic value (2p

  17. Embedded Leverage

    DEFF Research Database (Denmark)

    Frazzini, Andrea; Heje Pedersen, Lasse

    find that asset classes with embedded leverage offer low risk-adjusted returns and, in the cross-section, higher embedded leverage is associated with lower returns. A portfolio which is long low-embedded-leverage securities and short high-embedded-leverage securities earns large abnormal returns...

  18. Nitrous Oxide/Paraffin Hybrid Rocket Engines

    Science.gov (United States)

    Zubrin, Robert; Snyder, Gary

    2010-01-01

    Nitrous oxide/paraffin (N2OP) hybrid rocket engines have been invented as alternatives to other rocket engines especially those that burn granular, rubbery solid fuels consisting largely of hydroxyl- terminated polybutadiene (HTPB). Originally intended for use in launching spacecraft, these engines would also be suitable for terrestrial use in rocket-assisted takeoff of small airplanes. The main novel features of these engines are (1) the use of reinforced paraffin as the fuel and (2) the use of nitrous oxide as the oxidizer. Hybrid (solid-fuel/fluid-oxidizer) rocket engines offer advantages of safety and simplicity over fluid-bipropellant (fluid-fuel/fluid-oxidizer) rocket en - gines, but the thrusts of HTPB-based hybrid rocket engines are limited by the low regression rates of the fuel grains. Paraffin used as a solid fuel has a regression rate about 4 times that of HTPB, but pure paraffin fuel grains soften when heated; hence, paraffin fuel grains can, potentially, slump during firing. In a hybrid engine of the present type, the paraffin is molded into a 3-volume-percent graphite sponge or similar carbon matrix, which supports the paraffin against slumping during firing. In addition, because the carbon matrix material burns along with the paraffin, engine performance is not appreciably degraded by use of the matrix.

  19. 7 CFR 58.427 - Paraffin tanks.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Paraffin tanks. 58.427 Section 58.427 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards....427 Paraffin tanks. The metal tank should be adequate in size, have wood rather than metal racks to...

  20. TruSeq Stranded mRNA and Total RNA Sample Preparation Kits

    Science.gov (United States)

    Total RNA-Seq enabled by ribosomal RNA (rRNA) reduction is compatible with formalin-fixed paraffin embedded (FFPE) samples, which contain potentially critical biological information. The family of TruSeq Stranded Total RNA sample preparation kits provides a unique combination of unmatched data quality for both mRNA and whole-transcriptome analyses, robust interrogation of both standard and low-quality samples and workflows compatible with a wide range of study designs.

  1. Levels of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (APE1, APEX, Ref-1) are associated with the intrinsic radiosensitivity of cervical cancers.

    OpenAIRE

    Herring, C. J.; West, C. M.; Wilks, D. P.; Davidson, S. E.; Hunter, R. D.; Berry, P.; Forster, G.; MacKinnon, J.; Rafferty, J. A.; Elder, R. H.; Hendry, J. H.; Margison, G. P.

    1998-01-01

    A study was made of the relationship between the intrinsic radiosensitivity of human cervical tumours and the expression of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (HAP1). The radiosensitivity of clonogenic cells in tumour biopsies was measured as surviving fraction at 2 Gy (SF2) using a soft agar assay. HAP1 expression levels were determined after staining of formalin-fixed paraffin-embedded tumour sections with a rabbit antiserum raised against recombinant HAP1. Both ...

  2. Becker Muscular Dystrophy-Like Myopathy Regarded as So-Called “Fatty Muscular Dystrophy” in a Pig: A Case Report and Its Diagnostic Method

    OpenAIRE

    HORIUCHI, Noriyuki; AIHARA, Naoyuki; MIZUTANI, Hiroshi; KOUSAKA, Shinichi; NAGAFUCHI, Tsuneyuki; OCHIAI, Mariko; OCHIAI, Kazuhiko; KOBAYASHI, Yoshiyasu; FURUOKA, Hidefumi; ASAI, Tetsuo; OISHI, Koji

    2013-01-01

    ABSTRACT We describe a case of human Becker muscular dystrophy (BMD)-like myopathy that was characterized by the declined stainability of dystrophin at sarcolemma in a pig and the immunostaining for dystrophin on the formalin-fixed, paraffin-embedded (FFPE) tissue. The present case was found in a meat inspection center. The pig looked appeared healthy at the ante-mortem inspection. Muscular abnormalities were detected after carcass dressing as pale, discolored skeletal muscles with prominent ...

  3. PREVALENCE OF HUMAN PAPILLOMAVIRUS GENOTYPES IN LOW AND HIGH GRADE SQUAMOUS INTRAEPITHELIAL LESIONS AT CERVICAL TISSUE

    OpenAIRE

    Prasetyo, Rizki Eko; Mastutik, Gondo; Mustokoweni, Sjahjenny

    2017-01-01

    HPV infection is known to cause cervical cancer. This study aimed to identify the variant of HPV genotypes of cervical precancerous lesions from low grade squamous intraepithelial lesion  (LSIL) and high grade squamous intraepithelial lesion (HSIL). This was an explorative study using formalin fix paraffin embedded (FFPE) from cervical precancerous lesions at Dr. Soetomo Hospital, Surabaya. DNA was extracted from FFPE and hybridized for HPV genotyping using Ampliquality HPV Type Express kit (...

  4. KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

    OpenAIRE

    Piton, Nicolas; Borrini, Francesco; Bolognese, Antonio; Lamy, Aude; Sabourin, Jean-Christophe

    2015-01-01

    KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR) monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE) samples, ...

  5. Chromogenic in situ hybridization (CISH): a novel alternative in screening archival breast cancer tissue samples for HER-2/neu status

    OpenAIRE

    Madrid, Manuelito A; Lo, Raymundo W

    2004-01-01

    Background Chromogenic in situ hybridization (CISH) is emerging as a practical, cost-effective, and valid alternative to fluorescent in situ hybridization in testing for gene alteration, especially in centers primarily working with immunohistochemistry (IHC). Methods We assessed Her-2/neu alteration using CISH on formalin-fixed paraffin-embedded primary invasive ductal carcinoma tumors in which IHC (CB11 antibody) had previously been performed, and we compared the results with IHC. The 160 se...

  6. Tracing overlapping biological signals in mid-infrared using colonic tissues as a model system

    OpenAIRE

    Sahu, Ranjit Kumar; Salman, Ahmad; Mordechai, Shaul

    2017-01-01

    AIM To understand the interference of carbohydrates absorbance in nucleic acids signals during diagnosis of malignancy using Fourier transform infrared (FTIR) spectroscopy. METHODS We used formalin fixed paraffin embedded colonic tissues to obtain infrared (IR) spectra in the mid IR region using a bruker II IR microscope with a facility for varying the measurement area by varying the aperture available. Following this procedure we could measure different regions of the crypt circles containin...

  7. On alterations in the refractive index and scattering properties of biological tissue caused by histological processing

    Science.gov (United States)

    Aung, Htet; DeAngelo, Bianca; Soldano, John; Kostyk, Piotr; Rodriguez, Braulio; Xu, M.

    2013-02-01

    Clinical tissue processing such as formalin fixing, paraffin-embedding and histological staining alters significantly the optical properties of the tissue. We document the alterations in the optical properties of prostate cancer tissue specimens in the 500nm to 700nm spectral range caused by histological processing with quantitative differential interference contrast (qDIC) microscopy. A simple model to explain these alterations is presented at the end.

  8. Rubidium dimers in paraffin-coated cells

    International Nuclear Information System (INIS)

    Acosta, V M; Windes, D; Corsini, E; Ledbetter, M P; Karaulanov, T; Budker, D; Jarmola, A; Auzinsh, M; Rangwala, S A; Jackson Kimball, D F

    2010-01-01

    Measurements were made to determine the density of rubidium dimer vapor in paraffin-coated cells. The number density of dimers and atoms in similar paraffin-coated and uncoated cells was measured by optical spectroscopy. Due to the relatively low melting point of paraffin, a limited temperature range of 43-80 0 C was explored, with the lower end corresponding to a dimer density of less than 10 7 cm -3 . With 1 min integration time, a sensitivity to dimer number density of better than 10 6 cm -3 was achieved. No significant difference in dimer density between the cells was observed.

  9. The human placenta from heavy smokers: evaluation of vasoactive peptides by immunohistochemistry

    DEFF Research Database (Denmark)

    Clausen, H V; Larsen, L Grupe; Jørgensen, A

    2007-01-01

    The study aimed to demonstrate the expression of nitric oxide converting enzyme, nitric oxide synthase (e-NOS), and endothelin-1 (Et-1) in formalin-fixed paraffin-embedded placental tissue, and to demonstrate a difference in staining intensity between heavy smokers and non-smokers. Term placentas...... from pregnancies from otherwise healthy women smoking 15 or more cigarettes per day (heavy smokers) and term placentas from a matching group of non-smokers were included. The antibodies for Et-1 and e-NOS are recommended for cryostat sections. We evaluated the antibodies on paraffin-embedded tissue...

  10. MYC RNAi-Pt Combination Nanotherapy for Metastatic Prostate Cancer Treatment

    Science.gov (United States)

    2017-10-01

    Figures 5a and 5b show that the administration of the MYC siRNA loaded NPs lead to ~55% knockdown in MYC protein expression compared to the control...fixed in formalin and embedded into paraffin blocks (formalin fixed and paraffin embedded or FFPE), as well as by RNAseq. Figure 9 shows that MYC protein ...by IHC was detected at high levels whereas Pten was absent in both cell lines, consistent with the Figure 6. Serum levels of IL-6 (a), IL-12 (b

  11. Solubilization of paraffinic deposits for microemulsions

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Erika A.S.; Soares, Ranieri G.F.; Nascimento, Roseane E.S.; Dantas Neto, Afonso A.; Barros Neto, Eduardo L. [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil)

    2008-07-01

    The oil company has been intensifying its efforts to find more efficient solutions for the problems related to the paraffin in wells and transport lines. When applied in the flow lines, the solvents dissolve the paraffin and they must be used hot, since the temperature increases the solubility of the wax and, consequently, its removal rate. The microemulsions appear as an alternative capable of acting in the solubilization and in the inhibition of the formation of deposits due to its great interfacial area, low superficial tension and high capacity of solubilization. They present some advantages in relation to the methods of use of chemical products due to its flexibility of composition in which they can be used, presenting low toxicity and inflammability, without any loss of its capacity of solubilization. The use of oil-in-water microemulsion aims to solubilized paraffin in the disperse phase, where one can find the apolar part of the molecule of the surfactant and the also apolar chain of paraffin, occurring, therefore the 'encapsulation' of the crystal, prohibiting the growth of the chain due to the affinity of paraffin and oil. In this in case, it is possible to transport the inserted paraffin in direct micelles, reducing the precipitation and optimizing the transport. (author)

  12. In-house Manual Construction of High-Density and High-Quality Tissue Microarrays by Using Homemade Recipient Agarose-Paraffin Blocks.

    Science.gov (United States)

    Kim, Kyu Ho; Choi, Suk Jin; Choi, Yeon Il; Kim, Lucia; Park, In Suh; Han, Jee Young; Kim, Joon Mee; Chu, Young Chae

    2013-06-01

    Self-made tissue punches can be effectively used to punch holes in blank recipient paraffin blocks and extract tissue cores from the donor paraffin blocks for the low-cost construction of tissue microarrays (TMAs). However, variable degrees of section distortion and loss of the tissue cores can occurs during cutting of the TMAs, posing technical problems for in-house manual construction of high-density TMAs. We aimed to update the method for in-house manual TMA construction to improve the quality of high-density TMAs. Blocks of agarose gel were subjected to the standard tissue processing and embedding procedure to prepare recipient agarose-paraffin blocks. The self-made tissue punches and recipient agarose-paraffin blocks were used to construct TMAs, which were completely melted and re-embedded in paraffin to make finished TMA blocks. The donor tissue cores were completely integrated into the surrounding paraffin of the recipient blocks. This method enabled us to construct high-density TMAs with significantly less section distortion or loss of tissue cores during microtomy. Simple and inexpensive construction of high-density and high-quality TMAs can be warranted by using paraffinized agarose gels as recipient blocks.

  13. Formulation and Testing of Paraffin-Based Solid Fuels Containing Energetic Additives for Hybrid Rockets

    Science.gov (United States)

    Larson, Daniel B.; Boyer, Eric; Wachs,Trevor; Kuo, Kenneth K.; Story, George

    2012-01-01

    with both paraffin and RDX, the mixture will be combined with the melted paraffin. With the melting point of the paraffin far below the decomposition temperature of the RDX, the solvent will be boiled off, leaving the crystallized RDX embedded in the paraffin. At low percentages of RDX additive and with crystallized RDX surrounded by paraffin, the fuel grains will remain inert, maintaining a key benefit of hybrids in the safety of the solid fuel.

  14. Embedded Systems

    Indian Academy of Sciences (India)

    sumer electronic systems, they are cost sensitive. Thus their cost must be low. Robustness: Embedded systems should be robust since they operate in a harsh environment. They should endure vibrations, power supply fluctuations and excessive heat. Due to limited power supply in an embedded system, the power ...

  15. Biodegradation of paraffin wax by crude Aspergillus enzyme preparations for potential use in removing paraffin deposits.

    Science.gov (United States)

    Zhang, Junhui; Xue, Quanhong; Gao, Hui; Wang, Ping

    2015-11-01

    Paraffin deposition problems have plagued the oil industry. Whist mechanical and chemical methods are problematic, microbiological method of paraffin removal is considered an alternative. However, studies have mainly investigated the use of bacteria, with little attention to the potential of fungi. The performance of six Aspergillus isolates to degrade paraffin wax was evaluated under laboratory conditions using solid enzyme preparations. The results showed that all the six enzyme preparations efficiently improved the solubility of paraffin wax in n-hexane and degraded n-alkanes in paraffin wax. The degradation process was accompanied by dynamic production of gases (CO2 and H2 ) and organic acids (oxalate and propionate). The shape of wax crystals markedly changed after enzymatic degradation, with a rough surface and a loose structure. This study indicates that extracellular enzymes from Aspergillus spp. can efficiently degrade paraffin wax. These enzyme preparations have the potential for use in oil wells with paraffin deposition problems. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Recrystallization phenomena of solution grown paraffin dendrites

    NARCIS (Netherlands)

    Hollander, F.F.A.; Hollander, F.; Stasse, O.; van Suchtelen, J.; van Enckevort, W.J.P.

    2001-01-01

    Paraffin crystals were grown from decane solutions using a micro-Bridgman set up for in-situ observation of the morphology at the growth front. It is shown that for large imposed velocities, dendrites are obtained. After dendritic growth, aging or recrystallization processes set in rather quickly,

  17. Topical analgesic added to paraffin enhances paraffin bath treatment of individuals with hand osteoarthritis.

    Science.gov (United States)

    Myrer, Joseph William; Johnson, Aaron Wayne; Mitchell, Ulrike H; Measom, Gary J; Fellingham, Gilbert W

    2011-01-01

    To compare treating patients with symptomatic hand osteoarthritis (OA) with paraffin baths only (PO) (100% wax) or paraffin baths 80% wax with 20% topical analgesic (PTA). Subjects met criteria of the American College of Rheumatology for classifying symptomatic hand OA and had a Dreiser's index score >5 points. Current and average pain at rest and with movement was assessed with visual analogue scales. Hand function was assessed by the functional index for hand OA (FIHOA). Both groups had a significant reduction in their 'current' pain 15 min after the first and twelfth treatments compared to pre-treatment but there was no difference between groups (t = 0.10, p > 0.05). The PTA group had greater improvement over the 12 treatment sessions for their pain at rest (t = 2.92, p paraffin produced significantly greater pain relief at rest and during movement than paraffin baths alone after 12 treatments. Additionally, the PTA group experienced greater improved hand function.

  18. 21 CFR 172.275 - Synthetic paraffin and succinic derivatives.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Synthetic paraffin and succinic derivatives. 172... FOOD FOR HUMAN CONSUMPTION Coatings, Films and Related Substances § 172.275 Synthetic paraffin and succinic derivatives. Synthetic paraffin and succinic derivatives identified in this section may be safely...

  19. Paraffin (kerosene) poisoning in childhood is prevention affordable ...

    African Journals Online (AJOL)

    Of the 436 children with paraffin poisoning, 288 (66%) were black and 99 (23%) coloured. Only 4 patients (1 %) in the series were white. Paraffin poisoning occurred more frequently (62,5%) in boys. Age distribution. From Fig. 1, it is clear that paraffin poisoning in the Cape. Peninsula occurred mainly in children under 3 ...

  20. Magnetization of Paraffin-Based Magnetic Nanocolloids

    Science.gov (United States)

    Dikanskii, Yu. I.; Ispiryan, A. G.; Kunikin, S. A.; Radionov, A. V.

    2018-01-01

    Using paraffin-based magnetic nanocolloids as an example, the reasons for maxima in the temperature dependence of the magnetic susceptibility of magnetic colloids have been discussed. The behavior of these dependences in a wide temperature interval has been analyzed for colloids in solid and liquid states. It has been concluded that the maximum observed at the melting point of paraffin can be attributed to freezing Brownian degrees of freedom in magnetite coarse particles, the magnetic moment of which is intimately related to the solid matrix. The second main maximum, which arises in the solid state, is explained by the superparamagnetic-magnetically hard transition of most fine particles at lower temperatures. It has been noted that the flatness of this maximum results from the polydispersity of the magnetic nanoparticle ensemble.

  1. Interaction of oil and paraffin with sodium

    International Nuclear Information System (INIS)

    Kozlov, F.A.; Zagorulko, Yu.I.; Kovalev, Yu.P.; Lamykin, E.V.; Alekseev, V.V.; Sergeev, G.P.

    1980-01-01

    This report presents the investigation results on the kinetics of a gaseous oil pyrolysis product composition change in the presence of sodium under static conditions at 350 deg. C, 550 deg. C and 700 deg. C as well as the behaviour of oil and paraffin within the sodium circulating loops. The calculated estimations of the critical oil physical state parameters in sodium flows are given. (author)

  2. Embedded Systems

    Indian Academy of Sciences (India)

    system programmers should take into consideration all possi- bilities and write programs that do not fail. Responsiveness: Embedded systems should respond to events as soon as possible. For example, a patient monitoring system should process the patient'S heart signals quickly and immedi- ately notify if any abnormality ...

  3. Embedded defects

    International Nuclear Information System (INIS)

    Barriola, M.; Vachaspati, T.; Bucher, M.

    1994-01-01

    We give a prescription for embedding classical solutions and, in particular, topological defects in field theories which are invariant under symmetry groups that are not necessarily simple. After providing examples of embedded defects in field theories based on simple groups, we consider the electroweak model and show that it contains the Z string and a one-parameter family of strings called the W(α) string. It is argued that although the members of this family are gauge equivalent when considered in isolation, each member becomes physically distinct when multistring configurations are considered. We then turn to the issue of stability of embedded defects and demonstrate the instability of a large class of such solutions in the absence of bound states or condensates. The Z string is shown to be unstable for all values of the Higgs boson mass when θ W =π/4. W strings are also shown to be unstable for a large range of parameters. Embedded monopoles suffer from the Brandt-Neri-Coleman instability. Finally, we connect the electroweak string solutions to the sphaleron

  4. Cerebral phaeohyphomycosis in a green iguana (Iguana iguana).

    Science.gov (United States)

    Olias, P; Hammer, M; Klopfleisch, R

    2010-07-01

    Cerebral phaeohyphomycosis was diagnosed in a 4-year-old green iguana (Iguana iguana) with paroxysmal spastic paralysis of all limbs and circling motion. Formalin-fixed tissues were collected at necropsy examination and submitted for evaluation. The left cerebrum and the left ventricle were replaced by a solid brown coloured mass. Microscopical examination revealed the presence of necrotizing and granulomatous encephalitis affecting the cerebrum, cerebellum and brainstem, with severe vasculitis and intralesional dematiaceous fungal hyphae. No other lesions or fungi were found in other organs. Fungi were identified as Oidiodendron spp. by sequence analysis of the internal transcribed spacer (ITS) region 1 extracted from formalin-fixed and paraffin wax-embedded brain tissue. This case represents the first report of phaeohyphomycosis with tropism for the central nervous system in a reptile. In the absence of fresh tissue, the diagnosis in such cases may be assisted by molecular analysis of fixed tissue specimens. (c) 2009 Elsevier Ltd. All rights reserved.

  5. Immunohistochemical detection of PGL-1, LAM, 30 kD and 65 kD antigens in leprosy infected paraffin preserved skin and nerve sections

    NARCIS (Netherlands)

    van den Bos, I. C.; Khanolkar-Young, S.; Das, P. K.; Lockwood, D. N.

    1999-01-01

    A panel of lipid, carbohydrate and protein antibodies were optimized for use in detecting M. leprae antigens in paraffin embedded material. Skin and nerve biopsies from 13 patients across the leprosy spectrum were studied. All antibodies detected antigen in tissues with a BI > 1. Phenolic-glycolipid

  6. Bio-paraffins: alternative products to petroleum paraffins; Bioparafinas: produtos alternativos as parafinas de petroleo

    Energy Technology Data Exchange (ETDEWEB)

    Lima, Anie Daniela Medeiros [PETROBRAS, Rio de Janeiro, RJ (Brazil). Centro de Pesquisas (CENPES). Gerencia de Hidrorrefino e Processos Especiais; Oliveira, Claudia Cristina Cardoso Calvano de; Carvalho, Ivone de Freitas; Silva, Danilo do Carmo Santos; Cruz, Valeria Senra da Silva [PETROBRAS, Rio de Janeiro, RJ (Brazil). Centro de Pesquisas (CENPES). Gerencia de Lubrificantes e Produtos Especiais]. E-mails: anie.lima, claudiacristina, ivone, danilosilva, vsenra@petrobras.com.br

    2007-04-15

    Market trends and social and environmental issues encouraged the vegetal wax presence in the world-wide paraffin market. This work presents the most commercialized vegetal waxes, soy and pal, comparing their physicochemical characteristics and their applicability with the paraffins obtained from petroleum. It also presents a characterization of the carnauba wax, produced exclusively in Brazil and a comparison with paraffins from petroleum. The carnauba wax is an alternative product, with good applicability as a substitute for waxes from petroleum or a petroleum/vegetal mixture. The characteristics of palm and soy waxes show the possible application in candles, cosmetics, foods and others industries. Brazil, having a great agricultural potential, represents a source of vegetal wax that could be use to meet the market demands. (author)

  7. Development of paraffin and paraffin/bitumen composites with additions of B2O3 for thermal neutron shielding applications

    International Nuclear Information System (INIS)

    Toyen, Donruedee; Saenboonruang, Kiadtisak

    2017-01-01

    In this work, paraffin and paraffin/bitumen composites with additions of boron oxide (B 2 O 3 ) were prepared to evaluate the viscosity, flexural, and thermal neutron shielding properties for uses as thermal neutron shielding materials. The results showed that the addition of 3 wt% or 9 wt% bitumen to paraffin increased the overall flexural properties with the content of 9 wt% bitumen having the highest values. The improvement in flexural properties made the composites less brittle, stiffer, and longer-lasting. Furthermore, different contents of B 2 O 3 (0, 7, 14, 21, 28, and 35 wt%) were added to paraffin and paraffin/bitumen composites to investigate the effects of the B 2 O 3 contents. The results indicated that an increase in B 2 O 3 contents improved the shielding properties but slightly reduced the flexural properties. Specifically for 5-mm paraffin and 5-mm paraffin/bitumen samples with 35 wt% of B 2 O 3 , both samples could reduce neutron flux by more than 70%. The overall results suggested that the paraffin and paraffin/bitumen composites with additions of B 2 O 3 showed improved properties for utilization as effective thermal neutron shielding materials. (author)

  8. Compression embedding

    Science.gov (United States)

    Sandford, M.T. II; Handel, T.G.; Bradley, J.N.

    1998-07-07

    A method and apparatus for embedding auxiliary information into the digital representation of host data created by a lossy compression technique and a method and apparatus for constructing auxiliary data from the correspondence between values in a digital key-pair table with integer index values existing in a representation of host data created by a lossy compression technique are disclosed. The methods apply to data compressed with algorithms based on series expansion, quantization to a finite number of symbols, and entropy coding. Lossy compression methods represent the original data as ordered sequences of blocks containing integer indices having redundancy and uncertainty of value by one unit, allowing indices which are adjacent in value to be manipulated to encode auxiliary data. Also included is a method to improve the efficiency of lossy compression algorithms by embedding white noise into the integer indices. Lossy compression methods use loss-less compression to reduce to the final size the intermediate representation as indices. The efficiency of the loss-less compression, known also as entropy coding compression, is increased by manipulating the indices at the intermediate stage. Manipulation of the intermediate representation improves lossy compression performance by 1 to 10%. 21 figs.

  9. Radiometric determination of uniformity of putting paraffin on textile threads

    International Nuclear Information System (INIS)

    Ridel', Z.; Kherrmann, Eh.; Shefer, I.; Tseiner, A.

    1979-01-01

    To improve processing of the natural and synthetic fiber threads on stocking-frames, they are treated by paraffin. Paraffin is applied in the amounts nearly equal to 0.1 -1.0 g for 10 4 m of the thread's length. To determine amount of paraffin on thread and to determine uniformity if its application, a radiometric method has been developed. As a radioactive label, didocilephosphate of rare earths was used. This compound has good solubility in hydrocarbons and does not change physical properties of paraffin in the investigated field of its application as well as its concentration. It is possible to add to paraffin this radioactive label or a non-active label with subsequent its activation. Amount of paraffin, applied on a thread is determined by means of measurement of activity of thread samples of different length. Information about uniformity of paraffin application on thread have been obtained by means of autoradiography. It has been found that paraffin in mainly applied on the thread's bulges [ru

  10. Paraffin poisoning in children: what can we do differently? | Malangu ...

    African Journals Online (AJOL)

    Background: The purpose of this study was to describe the occurrence, health cost and management of paraffin poisoning in a rural South African hospital. Methods: A retrospective study was undertaken of 145 children admitted with a diagnosis of paraffin poisoning at Philadelphia Hospital, Mpumalanga from January 2000 ...

  11. Paraffin ingestion - the problem | Ellis | South African Medical Journal

    African Journals Online (AJOL)

    Paraffin ingestion is the commonest cause of accidental childhood poisoning in South Africa. Children from the lower socio-economic group are affected most. They drink paraffin in the summer months from bottles or intermediate containers, mistaking it for water or colddrink. The children are predominantly male with a ...

  12. Modulated structure formation in demixing paraffin blends

    CERN Document Server

    Gilbert, E P

    2002-01-01

    Small angle scattering (SANS and SAXS) and differential scanning calorimetry have been measured from C sub 2 sub 8 :C sub 3 sub 6 normal paraffin mixtures of varying composition quenched from the melt. Satellite peaks are observed in the SAXS whose offset in Q, relative to Bragg diffraction peaks associated with the average structure, are composition dependent. The offset is close to the position of the most intense peak observed in SANS. Scattering from the quenched structures is consistent with a correlated displacement and substitutional disorder model yielding modulations that are incommensurate with the average lattice. DSC shows an additional endotherm in the mixtures that is not present in the pure components and is associated with this superstructure formation. (orig.)

  13. Elemental analysis of histological specimens: a method to unmask nano asbestos fibers.

    Science.gov (United States)

    Scimeca, M; Pietroiusti, A; Milano, F; Anemona, L; Orlandi, A; Marsella, L T; Bonanno, E

    2016-02-01

    There is recent mounting evidence that nanoparticles may have enhanced toxicological potential in comparison to the same material in the bulk form. The aim of this study was to develop a new method for unmask asbestos nanofibers from Formalin-Fixed, Paraffin-Embedded tissue. There is an increasing amount of evidence that nanoparticles may enhance toxicological potential in comparison to the same material in the bulk form. The aim of this study was to develop a new method to unmask asbestos nanofibers from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. For the first time, in this study we applied Energy Dispersive X-ray (EDX) microanalysis through transmission electron microscopy to demonstrate the presence of asbestos nanofibers in histological specimens of patients with possible occupational exposure to asbestos. The diagnostic protocol was applied to 10 randomly selected lung cancer patients with no history of previous asbestos exposure. We detected asbestos nanofibers in close contact with lung cancer cells in two lung cancer patients with previous possible occupational exposure to asbestos. We were also able to identify the specific asbestos iso-type, which in one of the cases was the same rare variety used in the workplace of the affected patient. By contrast, asbestos nanofibers were not detected in lung cancer patients with no history of occupational asbestos exposure. The proposed technique can represent a potential useful tool for linking the disease to previous workplace exposure in uncertain cases. Furthermore, Formalin-Fixed Paraffin-Embedded (FFPE) tissues stored in the pathology departments might be re-evaluated for possible etiological attribution to asbestos in the case of plausible exposure. Since diseases acquired through occupational exposure to asbestos are generally covered by workers' insurance in most countries, the application of the protocol used in this study may have also relevant social and economic implications.

  14. Bacillary haemoglobinuria diagnosis by the peroxidase-antiperoxidase (PAP) technique.

    Science.gov (United States)

    Uzal, F A; Belak, K; Rivera, E; Robles, C A; Feinstein, R E

    1992-10-01

    A peroxidase-antiperoxidase (PAP) technique was used to diagnose bacillary haemoglobinuria in formalin-fixed, paraffin-embedded liver tissues of cattle. The PAP method revealed Clostridium haemolyticum in the zone of liver necrosis characteristic of the disease and also in culture smears of this microorganism, but C. novyi type B, C. chauvoei, C. septicum and C. perfringens types B and C remained unstained by the PAP reaction. The PAP technique performed provides a specific, simple and rapid method to diagnose bacillary haemoglobinuria.

  15. BRAF mutation testing with a rapid, fully integrated molecular diagnostics system

    OpenAIRE

    Janku, Filip; Claes, Bart; Huang, Helen J.; Falchook, Gerald S.; Devogelaere, Benoit; Kockx, Mark; Bempt, Isabelle Vanden; Reijans, Martin; Naing, Aung; Fu, Siqing; Piha-Paul, Sarina A.; Hong, David S.; Holley, Veronica R.; Tsimberidou, Apostolia M.; Stepanek, Vanda M.

    2015-01-01

    Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel IdyllaTM BRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those ob...

  16. Optimisation and validation of methods to assess single nucleotide polymorphisms (SNPs) in archival histological material

    DEFF Research Database (Denmark)

    Andreassen, C N; Sørensen, Flemming Brandt; Overgaard, J

    2004-01-01

    only archival specimens are available. This study was conducted to validate protocols optimised for assessment of SNPs based on paraffin embedded, formalin fixed tissue samples. PATIENTS AND METHODS: In 137 breast cancer patients, three TGFB1 SNPs were assessed based on archival histological specimens...... precipitation). RESULTS: Assessment of SNPs based on archival histological material is encumbered by a number of obstacles and pitfalls. However, these can be widely overcome by careful optimisation of the methods used for sample selection, DNA extraction and PCR. Within 130 samples that fulfil the criteria...

  17. Association between in vivo iododeoxyuridine labeling, MIB-1 expression, malignancy grade and clinical stage in human prostate cancer

    DEFF Research Database (Denmark)

    Borre, M; Høyer, M; Sørensen, Flemming Brandt

    1998-01-01

    Large variability in the biological behavior of prostate cancer makes prognostic markers important. The extent of tumor cell proliferation has been suggested as an important predictor of clinical outcome. Fifty-five patients suspected of having or with previously diagnosed prostate cancer were...... proliferation rates measured by in vivo IdUrd labeling and MIB-1 expression in formalin-fixed paraffin-embedded tumors. Good correlations were also found between S-phase fraction, MIB-1 expression, clinical stage and malignancy grade. These results make larger retrospective studies on archival tissue meaningful....

  18. The Role of Interleukin-6/GP130 Signaling in Prostate Cancer Progression and its Contribution to Bone Metastasis Morbidity

    Science.gov (United States)

    2008-11-01

    Those that can initiate signaling via gp130 include IL-6, IL-11, LIF, CT-1, CNTF and OSM (Heinrich et al., 1998). Notably, BM18 tumors of all...gp130 and STAT3, e.g. IL-11, LIF, CT-1, CNTF or OSM. 2c) Measure apoptosis and cellular proliferation in formalin fixed paraffin embedded BM18 xenograft...Pellegrini, S., et al. (1994). Association and activation of Jak-Tyk kinases by CNTF -LIF-OSM-IL-6 beta receptor components. Science (New York, NY 263

  19. The Thomsen-Friedenreich (T) simple mucin-type carbohydrate antigen in salivary gland carcinomas

    DEFF Research Database (Denmark)

    Therkildsen, M H; Mandel, U; Christensen, M

    1995-01-01

    The simple mucin-type T (Thomsen-Friedenreich) antigen is a marker of carcinomas, and has been related to aggressiveness of malignant tumours. We studied the expression of T, sialosyl-T, A and H blood group antigens in salivary gland carcinomas. The aim was to study whether the tumours, based...... on the expression of these structures, could be divided into new diagnostic groups that may later show prognostic significance. Formalin fixed paraffin-embedded tissue sections from 77 salivary gland carcinomas of different histological types were studied using immunohistology and monoclonal antibodies (MAbs...

  20. The effects of mode of delivery and sex of newborn on placental morphology in control and diabetic pregnancies

    DEFF Research Database (Denmark)

    Mayhew, T M; Sørensen, Flemming Brandt; Klebe, J G

    1993-01-01

    Placentae from control and diabetic subjects were analysed using stereological techniques in order to assess the effects of mode of delivery (vaginal versus caesarean) and sex of neonate on parenchymal morphology. Effects were assessed using indices of peripheral villous and fetal capillary growth...... of glucose and glycated haemoglobin levels. Neonatal and placental weights were recorded and placentae sampled in a systematic random fashion. Fields of view on formalin-fixed, paraffin-embedded sections were analysed to obtain estimates of volumes, surface areas, lengths and diffusion (harmonic mean...

  1. Romanowsky-Giemsa as a counterstain for immunohistochemistry: optimizing a traditional reagent.

    Science.gov (United States)

    Stefanović, D; Stefanović, M; Nikin, Z

    2013-08-01

    We describe a detailed protocol for using Romanowsky-Giemsa (RG) counterstaining on formalin fixed, paraffin embedded tissue sections that are stained immunohistochemically (IHC) after antigen retrieval using hot acidic citrate buffer. RG staining is easy to perform and provides consistent results that are similar to hematoxylin and eosin (HE) staining. The counterstaining was applied after a variety of antibodies that used the DAB chromogen and the intensity of IHC stained structures was preserved. Moreover, RG counterstaining provided finer cell detail than HE, methyl green or nuclear fast red. A detailed troubleshooting guide is provided for the RG staining protocol.

  2. Is TIMP-1 immunoreactivity alone or in combination with other markers a predictor of benefit from anthracyclines in the BR9601 adjuvant breast cancer chemotherapy trial?

    DEFF Research Database (Denmark)

    Munro, Alison F.; Bartels, Annette; Balslev, Eva

    2013-01-01

    copy number can be used to predict benefit from epirubicin (E) containing chemotherapy compared with cyclophosphamide, methotrexate and fluorouracil (CMF) treatment. METHODS: For the purpose of this study, formalin fixed paraffin embedded tumor tissue from women recruited into the BR9601 clinical trial...... immunoreactive and HER2 negative) and anthracycline responsive (all other cases). RESULTS: In total, 288 tumors were available for TIMP-1 analysis with (183/274) 66.8%, and (181/274) 66.0% being classed as 2T and HT responsive, respectively. TIMP-1 was neither associated with patient prognosis (relapse free...

  3. Phylogeny and distribution of an unknown Treponema sp. associated with porcine colitis by using in situ hybridization and laser capture microdissection (LCM)

    DEFF Research Database (Denmark)

    Mølbak, Lars; Schou, Kirstine Klitgaard; Jensen, Tim Kåre

    Helical-shaped bacteria resembling Spirochaetes commonly are present in the gastrointestinal tract of animals and humans. Culturing of Spirochaetes is in general fastidious and not always successful. Here, a new DNA isolation approach for prokaryotic cells in formalin-fixed paraffin-embedded tissue...... species was recognized in pigs by using fluorescent in situ hybridization, followed by laser capture microdissection (LCM) of the targeted cells from the colonic mucosa. Direct 16S rRNA gene PCR was performed from the dissected micro-colonies. A phylogenetic analysis clustered the 16S rRNA gene...

  4. Prognostic impact of chromosomal aberrations and GNAQ, GNA11 and BAP1 mutations in uveal melanoma

    DEFF Research Database (Denmark)

    Staby, Kjersti M; Gravdal, Karsten; Mørk, Sverre J

    2018-01-01

    PURPOSE: To evaluate clinico-pathological and molecular prognostic factors in a well-defined series of posterior uveal melanoma (UM) with focus on chromosomal aberrations and mutations in the GNAQ, GNA11 and BRCA1-associated protein 1 (BAP1) genes. METHODS: Formalin-fixed paraffin-embedded (FFPE......) tissue samples were obtained from 50 consecutive eyes enucleated for UM between 1993 and 2005. The material was tested for loss of chromosome 3 and gain of chromosome 8q gene signatures by selective molecular gene markers using multiplex ligation-dependent probe amplification (MLPA), and for DNA...

  5. High prevalence of TP53 mutations is associated with poor survival and an EMT signature in gliosarcoma patients

    OpenAIRE

    Cho, Sung-Yup; Park, Changho; Na, Deukchae; Han, Jee Yun; Lee, Jieun; Park, Ok-Kyoung; Zhang, Chengsheng; Sung, Chang Ohk; Moon, Hyo Eun; Kim, Yona; Kim, Jeong Hoon; Kim, Jong Jae; Khang, Shin Kwang; Nam, Do-Hyun; Choi, Jung Won

    2017-01-01

    Gliosarcoma (GS) is a rare variant (2%) of glioblastoma (GBM) that poses clinical genomic challenges because of its poor prognosis and limited genomic information. To gain a comprehensive view of the genomic alterations in GS and to understand the molecular etiology of GS, we applied whole-exome sequencing analyses for 28 GS cases (6 blood-matched fresh-frozen tissues for the discovery set, 22 formalin-fixed paraffin-embedded tissues for the validation set) and copy-number variation microarra...

  6. ERG Protein Expression in Diagnostic Specimens Is Associated with Increased Risk of Progression During Active Surveillance for Prostate Cancer

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Vainer, Ben; Thomsen, Frederik Birkebæk

    2014-01-01

    : This study included 265 patients followed on AS with prostate-specific antigen (PSA) measurements, clinical examinations, and 10-12 core rebiopsies from 2002 to 2012 in a prospectively maintained database. ERG immunohistochemical staining was performed on diagnostic paraffin-embedded formalin-fixed sections...... examination and ultrasound, and/or histopathologic progression: upgrade of Gleason score, more than three positive cores or bilateral positive cores, and/or PSA progression: PSA doubling time ..., the ERG-positive group showed significantly higher incidences of overall AS progression (pPSA progression (p

  7. Embedded Processor Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — The Embedded Processor Laboratory provides the means to design, develop, fabricate, and test embedded computers for missile guidance electronics systems in support...

  8. Photothermally Driven Refreshable Microactuators Based on Graphene Oxide Doped Paraffin.

    Science.gov (United States)

    Hou, Sichao; Wang, Miao; Guo, Shouwu; Su, Ming

    2017-08-09

    Actuators based on phase change materials (paraffin) can simultaneously produce large stroke length and large force due to thermal expansion, but the low thermal conductivity of paraffin requires high power input and long actuation time. The graphene oxide (GO) doped paraffin dynamic actuator addresses the key challenges in the design of thermal phase change actuators: Thermal conductivity and light absorbing are increased, and the response time is reduced compared to the standard phase change actuator designed with metal heating resistors. The thermal properties of GO-paraffin composites with varied loading amount are characterized to confirm the optimal loading amount of 1.0%. A multicell phase change actuator was integrated into a digital micromirror controlled optical system. A series of photothermally driven refreshable patterns were generated and confirmed with infrared imaging.

  9. Observations of radiation damage effects in paraffin and polyethylene crystals

    International Nuclear Information System (INIS)

    Petermann, J.; Gleiter, H.; Bochum Univ.

    1973-01-01

    A report is given on electron microscopic observations on n-paraffin and polyethylene monocrystals after irradiating with electrons. The observations show that the cross-links in n-paraffin monocrystals form agglomerates which preferably occur in the neighbourhood of lattice defects. In polyethylene monocrystals, the cross-links line up in long rows parallel to the [100] or [010] direction. (orig./LH) [de

  10. Numerical Analysis of a Paraffin/Metal Foam Composite for Thermal Storage

    Science.gov (United States)

    Di Giorgio, P.; Iasiello, M.; Viglione, A.; Mameli, M.; Filippeschi, S.; Di Marco, P.; Andreozzi, A.; Bianco, N.

    2017-01-01

    In the last decade, the use of Phase Change Materials (PCMs) as passive thermal energy storage has been widely studied both analytically and experimentally. Among the PCMs, paraffins show many advantages, such as having a high latent heat, a low vapour pressure, being chemically inert, stable and non-toxic. But, their thermal conductivity is very low with a high volume change during the melting process. An efficient way to increase their poor thermal conductivity is to couple them with open cells metallic foams. This paper deals with a theoretical analysis of paraffin melting process inside an aluminum foam. A mathematical model is developed by using the volume-averaged governing equations for the porous domain, made up by the PCM embedded into the metal foam. Non-Darcian and buoyancy effects are considered in the momentum equation, while the energy equations are modelled with the Local Thermal Non-Equilibrium (LTNE) approach. The PCM liquefaction is treated with the apparent heat capacity method and the governing equations are solved with a finite-element scheme by COMSOL Multiphysics®. A new method to calculate the coupling coefficients needed for the thermal model has been developed and the results obtained have been validated comparing them to experimental data in literature.

  11. Polymorphic Embedding of DSLs

    DEFF Research Database (Denmark)

    Hofer, Christian; Ostermann, Klaus; Rendel, Tillmann

    2008-01-01

    propose polymorphic embedding of DSLs, where many different interpretations of a DSL can be provided as reusable components, and show how polymorphic embedding can be realized in the programming language Scala. With polymorphic embedding, the static type-safety, modularity, composability and rapid...... prototyping of pure embedding are reconciled with the flexibility attainable by external toolchains....

  12. Synthesis and application of polyaminoamide as new paraffin inhibitor from vegetable oil

    OpenAIRE

    Chen, Gang; Tang, Ying; Zhang, Jie

    2011-01-01

    Abstract In this work, a series of novel paraffin inhibitor, polyaminoamide (PAA), was designed and prepared by aminolysis and poly-condensation using soybean oil and canola oil as the raw material. The property of the PAAs as paraffin inhibitor was investigated, the results show several PAA samples are potent in paraffin inhibition, and PPC-2 is the most effective one. Besides, the paraffin crystal morphology analysis was carried out to provide the mechanism of paraffin inhibition.

  13. Non-hazardous organic solvents in the paraffin-embedding technique

    DEFF Research Database (Denmark)

    Holm, Inger Lindebo; Lyon, Hans; Prentø, Poul

    1995-01-01

    The aim of this study was to substitute hazardous compounds, used in tissue processing and dewaxing, with compounds having lowest possible toxicity and inflammability without impairing the morphology, staining characteristics, or diagnostic value of the tissue sections. All aromatic compounds...... to that obtained with xylene. For dewaxing, it is used at 30-35 degrees C. Butyldecanoate is not suitable as a pre-mounting agent. In practice, this is no problem as modern mounting agents permit mounting of coverslips directly from ethanol without impairing the appearance of the section in the microscope....... Butyldecanoate has only a slight odour, insignificant vapour pressure (degrees C), and does not present a fire hazard (flash point 134 degrees C). The introduction of this compound in the laboratory poses no health hazard, and the substance is biodegradable....

  14. Liquid Paraffin vs Hyaluronic Acid in Preventing Intraperitoneal Adhesions.

    Science.gov (United States)

    Kataria, Hanish; Singh, Vinod Prem

    2017-12-01

    Adhesion formation after abdominal and pelvic operations remains a challenging problem. Role of adjuvant barriers have been studied but there is no comparative study between liquid paraffin and hyaluronic acid as a barrier method. Hence, we planned to compare the effectiveness of 0.4 % hyaluronic acid and liquid paraffin in the prevention of postoperative intraperitoneal adhesions in rats. This prospective, randomized and controlled study was conducted in 60 adult Wistar albino rats. Surgical trauma by caecal abrasion and 1 g talcum powder was used in the rat model to induce adhesion formation. After trauma, 3 ml normal saline was instilled in the peritoneal cavity in control group ( n  = 20), 3 ml liquid paraffin was instilled in experimental group A ( n  = 20) and 3 ml 0.4 % hyaluronic acid was instilled in experimental group B ( n  = 20). Two weeks after laparotomy, repeat laparotomy was performed and the adhesions were scored according to Zuhlke classification. Liquid paraffin and hyaluronic acid both reduce the extent and grade of adhesions both macroscopically ( p  = 0.018, p  = 0.017) and microscopically ( p  = 0.019, p  = 0.019) respectively. Although there was significant reduction in adhesions by hyaluronic acid at certain specific sites as compared with liquid paraffin, its overall effectiveness in preventing postoperative intraperitoneal adhesions is not significantly different from liquid paraffin ( p  = 0.092, p  = 0.193) respectively. The presence of liquid paraffin and hyaluronic acid in the peritoneal cavity reduce postoperative intraperitoneal adhesions significantly in rats. However, there is no overall significant difference in the effectiveness of two groups. Dosage and safety of these chemicals in human beings remains to be established.

  15. Effect of radiation on the crystals of polyethylene and paraffins

    International Nuclear Information System (INIS)

    Ungar, G.; Grubb, D.T.; Keller, A.

    1980-01-01

    Paraffins were irradiated with electrons in the electron microscope. The electron microscopic image and the electron diffraction patterns were followed as a function of dose. The objectives were: (a) to establish a connection between the 'polyethylene-type' and 'paraffin-like' behaviour and (b) to identify the phase segregation, by visual means. Increasing chain length, increasing dose rate and decreasing temperature individually and in combination, were found to favour the 'polyethylene-type' behaviour (crystal destruction through increasing lattice defects) while the reverse trend of the above three variables favoured the 'paraffin-like' behaviour (phase-segregated damaged and undamaged species). Segregated phases could in some circumstances be identified as non-diffracting 'droplets' within a crystalline matrix, with the lattice hardly affected, in the electron microscopic image. These droplets remain constant in number but increase in size as the irradiation progresses, the number of droplets depending on the chain length of the paraffin, on the irradiation temperature and on the dose rate. This behaviour, together with some further observations, reveals that the radiation-induced active species do not form crosslinks in situ but migrate over distances which can amount to μm. In contrast to the above, in the case of the lowest paraffin investigated, (C 23 H 48 ), the lattice became uniformly distorted as judged from the diffraction pattern, but the damage was observed to 'heal-out' with time. The results are discussed. (author)

  16. The effects of normal paraffins mobilizers on irradiated polypropylene

    International Nuclear Information System (INIS)

    Chen Wenxiu; Gao Ling

    1995-01-01

    The n-paraffins blended with polypropylene (PP) as mobilizer had been investigated. The effectiveness of mobilizer (n-paraffins) on irradiated polypropylene is dependent on the molecular weight of mobilizer and its content on polypropylene. The n-docosame (n-C 22 ) possesses the best effectiveness of radiation tolerance on PP among the mobilizer paraffins: n-decane (n-C 10 ), n-hexadecane (n-C 16 ), n-docosane (n-C 22 ) and n-hexatriacontane (n-C 36 ). The 2% (w/w) content of a given mobilizer is the most effective at reducing the embrittlement of irradiated PP as evidenced by the elongation at break. The physical properties of polypropylene with mobilizers such as density, Young's modulus, the Fraction of free volume and the weight swelling ratio in p-xylene at room temperature were measured. Above phenomena are related with the constructive of blended PP and demonstrated by its physical properties

  17. Reduction in spasticity in stroke patient with paraffin therapy.

    Science.gov (United States)

    Wang, Jing; Yu, Peng; Zeng, Ming; Gu, Xudong; Liu, Yan; Xiao, Mingyue

    2017-01-01

    The aim of the study was to confirm whether paraffin therapy offer clinical value in the treatment of spasticity due to stroke. Fifty-two patients with spasticity in the upper limb were included. The patients were randomized into the experimental group with paraffin therapy (n = 27) and the control group with placebo therapy (n = 25). The outcome measures besides temperature examination were undertaken at time points of 0 (T0), 2 (T1) and 4 weeks (T2) following therapy treatment. The extent of spasticity was measured using Modified Ashworth Score (MAS) during passive movement at the shoulder, elbow, wrist and finger joints. Visual analogue scale (VAS) was used to evaluate the hemiplegic upper limb pain and functional activity of the upper limb motor function was evaluated by Brunnstrom recovery stage. All adverse events were recorded. MAS decreased significantly in Exp group compared with Con group, at the time points of T1 and T2, both before and immediately after paraffin therapy. Paraffin treatment failed to show remarkable improvement in pain compared with placebo-treated patient at movement at any time point. But VAS in Exp exhibited a tendency to decrease over time in shoulder, elbow, wrist and hand. With regard to the Brunnstrom score, patients in Exp showed significant improvement at the end of trial compared to the beginning. The values of temperature showed significant increment immediately after paraffin therapy at each time point in Exp group. Paraffin therapy may be a kind of noninvasive, promising method to reduce spasticity of stroke patients.

  18. Toxicoepidemiology in Zimbabwe: admissions resulting from exposure to paraffin (kerosene).

    Science.gov (United States)

    Tagwireyi, D; Ball, D E; Nhachi, C F B

    2006-01-01

    Paraffin (kerosene) ingestion is the most common form of childhood poisoning in most developing countries. Despite this, there is a paucity of toxicoepidemiological data which could potentially be used in measures to reduce preventable exposures. This article reports on the patterns of hospital admissions resulting from paraffin exposure in Zimbabwe. All cases of paraffin ingestion admitted to eight major referral hospitals in Zimbabwe from January 1998 to December 1999 (inclusive), were identified using ICD-9 codes and ward registers and relevant information recorded on a standard data collection sheet. There were a total of 327 admissions due to oral exposure to paraffin. This represented 11.8% of all the poisoning admissions to the eight study hospitals. Most exposures (300; 91.7%) occurred accidentally, with only 6.7% resulting from deliberate ingestion of the chemical. The median age on admission was 2 years (interquartile range [IQR] 1-2 yrs) with over 85% of cases in the 0-5 year age range and less than 10% above the age of 12 years. The median age on admission was much higher for deliberate self poisoning (23 yrs; IQR 19-26 yrs) compared to that for accidental poisoning (1.5 yrs; IQR 1-2 yrs). Accidental poisoning from paraffin occurred throughout the year. Over three-quarters of patients received an antibiotic either alone, or in combination with another antibiotic or drug. Paracetamol (24.3%) was the next most commonly encountered treatment. The case fatality rate (CFR) was therefore 0.3 deaths per 100 admissions (95% Confidence Interval 0.0-1.7). Paraffin ingestion remains an important cause of poisoning morbidity in Zimbabwe throughout the year, particularly in children. Clinical management appears adequate with a low mortality, although there may be overuse of prophylactic antibiotics. Further study specific to this area is warranted to prevent unnecessary antibiotic use and wastage of resources.

  19. Magnetic orientation of paraffin in a magnetic levitation furnace

    Science.gov (United States)

    Takahashi, K.; Umeki, C.; Mogi, I.; Koyama, K.; Awaji, S.; Motokawa, M.; Watanabe, K.

    2004-04-01

    Containerless melting of paraffin under a magnetic levitation condition has been performed by using a cryogen-free hybrid magnet and two kinds of laser furnaces. One is local irradiation of CO 2 laser light at the top of the sample. The other is homogeneous irradiation of YAG laser light with a concave ring mirror. In the latter case, reduction of the Marangoni convection on the surface of the sample and the magnetic orientation of paraffin molecules were observed. The magnetic anisotropy of the spherical sample was confirmed by the measurement of magnetization and X-ray diffraction.

  20. Magnetic orientation of paraffin in a magnetic levitation furnace

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, K.; Umeki, C.; Mogi, I.; Koyama, K.; Awaji, S.; Motokawa, M.; Watanabe, K

    2004-04-30

    Containerless melting of paraffin under a magnetic levitation condition has been performed by using a cryogen-free hybrid magnet and two kinds of laser furnaces. One is local irradiation of CO{sub 2} laser light at the top of the sample. The other is homogeneous irradiation of YAG laser light with a concave ring mirror. In the latter case, reduction of the Marangoni convection on the surface of the sample and the magnetic orientation of paraffin molecules were observed. The magnetic anisotropy of the spherical sample was confirmed by the measurement of magnetization and X-ray diffraction.

  1. Magnetic orientation of paraffin in a magnetic levitation furnace

    International Nuclear Information System (INIS)

    Takahashi, K.; Umeki, C.; Mogi, I.; Koyama, K.; Awaji, S.; Motokawa, M.; Watanabe, K.

    2004-01-01

    Containerless melting of paraffin under a magnetic levitation condition has been performed by using a cryogen-free hybrid magnet and two kinds of laser furnaces. One is local irradiation of CO 2 laser light at the top of the sample. The other is homogeneous irradiation of YAG laser light with a concave ring mirror. In the latter case, reduction of the Marangoni convection on the surface of the sample and the magnetic orientation of paraffin molecules were observed. The magnetic anisotropy of the spherical sample was confirmed by the measurement of magnetization and X-ray diffraction

  2. Spectral distribution measurements of neutrons in paraffin borax mixtures

    International Nuclear Information System (INIS)

    El-Khatib, A.M.; Gaber, M.; Abou El-Khier, M.A.

    1987-01-01

    Neutron fluxes from a compact D-T neutron source has been measured in paraffin-borax mixtures by using activation foil detectors with successive threshold energies. The absorbed doses, backscattering coefficients and build-up factors were determined as well. The contribution of thermal and intermediate neutron dose is much lower, compared to that of fast neutrons. Among the used mediums, paraffin loaded with 4% borax concentration was found to be the best absorbing medium against neutrons at near depths within the blocks, while at a depth around 12 cm the neutron absorption (or scattering) is independent on the type of the used medium. (author)

  3. Extracorporeal membrane oxygenation after intravenous injection of paraffin oil

    DEFF Research Database (Denmark)

    Pasgaard, Thomas; Huynh, Anh-Nhi Thi; Gjedsted, Jakob

    2016-01-01

    We present a rare cause of acute respiratory distress syndrome (ARDS) due to an accidental intravascular injection of paraffin oil. While there is no specific therapy, we decided to support the patient with veno-venous extracorporeal membrane oxygenation (V-V ECMO) to allow the ARDS to resolve....... A previously healthy 30-year-old man was admitted to the Emergency Department with acute onset respiratory distress following an injection with paraffin oil for cosmetic purposes. In 36 hours, the patient developed severe ARDS and respiratory support with V-V ECMO was initiated. The patient was successfully...

  4. From an equilibrium based MOF adsorbent to a kinetic selective carbon molecular sieve for paraffin/iso-paraffin separation

    KAUST Repository

    Li, Baiyan

    2016-11-04

    We unveil a unique kinetic driven separation material for selectively removing linear paraffins from iso-paraffins via a molecular sieving mechanism. Subsequent carbonization and thermal treatment of CD-MOF-2, the cyclodextrin metal-organic framework, afforded a carbon molecular sieve with a uniform and reduced pore size of ca. 5.0 Å, and it exhibited highly selective kinetic separation of n-butane and n-pentane from iso-butane and iso-pentane, respectively. © The Royal Society of Chemistry.

  5. Conceptualizing Embedded Configuration

    DEFF Research Database (Denmark)

    Oddsson, Gudmundur Valur; Hvam, Lars; Lysgaard, Ole

    2006-01-01

    Installing and servicing complex electromechanical systems is more tedious than is necessary. By putting the product knowledge into the product itself, which then would allow automation in constructing the product from modules, could solve that. It would support personnel in aftersales installation...... and services. The general idea can be named embedded configuration. In this article we intend to conceptualize embedded configuration, what it is and is not. The difference between embedded configuration, sales configuration and embedded software is explained. We will look at what is needed to make embedded...... configuration systems. That will include requirements to product modelling techniques. An example with consumer electronics will illuminate the elements of embedded configuration in settings that most can relate to. The question of where embedded configuration would be relevant is discussed, and the current...

  6. Expression of autocrine motility factor mRNA is a poor prognostic factor in high-grade astrocytoma.

    Science.gov (United States)

    Tanizaki, Yoshinori; Sato, Yuichi; Oka, Hidehiro; Utsuki, Satoshi; Kondo, Koji; Miyajima, Yoshiteru; Nagashio, Ryo; Fujii, Kiyotaka

    2006-09-01

    It has been reported that tumor infiltration is correlated with the expression of autocrine motility factor (AMF) and its receptor 78 kDa glycoprotein (gp78). The purpose of the present study was to detect AMF and gp78 mRNA expression levels and their localization in high-grade astrocytomas (glioblastoma and anaplastic astrocytoma) and to determine whether AMF and gp78 are important prognostic factors. A total of 32 formalin-fixed and paraffin-embedded glioblastomas and 23 formalin-fixed and paraffin-embedded anaplastic astrocytomas was used. The expressions of AMF and gp78 mRNA were detected using the highly sensitive in situ hybridization method. The expression of AMF mRNA was detected in 27 of 32 glioblastomas (84.4%) and 11 of 23 anaplastic astrocytomas (47.8%). The positivity of AMF mRNA was significantly higher in glioblastomas than in anaplastic astrocytomas (P = 0.0094), but gp78 mRNA was detected in most cases and no statistical significance was observed. The overall survival of patients with AMF expression was significantly shorter than patients without AMF expression (P = 0.0175). In anaplastic astrocytomas, the overall survival of patients with AMF expression was also significantly shorter than in patients without AMF expression (P = 0.0058). This study demonstrated that AMF is a poor prognostic factor in high-grade astrocytomas.

  7. Analysis of Fusion Genes by NanoString System: A Role in Lung Cytology?

    Science.gov (United States)

    Alì, Greta; Bruno, Rossella; Savino, Mauro; Giannini, Riccardo; Pelliccioni, Serena; Menghi, Maura; Boldrini, Laura; Proietti, Agnese; Chella, Antonio; Ribechini, Alessandro; Fontanini, Gabriella

    2018-01-26

    - Patients with non-small cell lung cancer harboring ALK receptor tyrosine kinase ( ALK), ROS proto-oncogene 1 ( ROS1), and ret proto-oncogene ( RET) gene rearrangements can benefit from specific kinase inhibitors. Detection of fusion genes is critical for determining the best treatment. Assessing rearrangements in non-small cell lung cancer remains challenging, particularly for lung cytology. - To examine the possible application of the multiplex, transcript-based NanoString system (NanoString Technologies, Seattle, Washington) in the evaluation of fusion genes in lung adenocarcinoma samples. - This study is a narrative literature review. Studies about NanoString, gene fusions, and lung adenocarcinoma were collected from PubMed (National Center for Biotechnology Information, Bethesda, Maryland). We found 7 articles about the application of the NanoString system to detect fusion genes on formalin-fixed, paraffin-embedded tumor tissues and one article evaluating the adequacy of lung cytologic specimens for NanoString gene expression analysis. - To maximize the yield of molecular tests on small lung biopsies, the NanoString nCounter system has been suggested to detect fusion genes. NanoString fusion gene assays have been successfully applied on formalin-fixed, paraffin-embedded tissues. Although there are only few studies available, the application of NanoString assays may also be feasible in lung cytology. According to available data, the NanoString system could strengthen the routine molecular characterization of lung adenocarcinoma.

  8. A high-throughput method to detect RNA profiling by integration of RT-MLPA with next generation sequencing technology.

    Science.gov (United States)

    Wang, Jing; Yang, Xue; Chen, Haofeng; Wang, Xuewei; Wang, Xiangyu; Fang, Yi; Jia, Zhenyu; Gao, Jidong

    2017-07-11

    RNA in formalin-fixed and paraffin-embedded (FFPE) tissues provides large amount of information indicating disease stages, histological tumor types and grades, as well as clinical outcomes. However, Detection of RNA expression levels in formalin-fixed and paraffin-embedded samples is extremely difficult due to poor RNA quality. Here we developed a high-throughput method, Reverse Transcription-Multiple Ligation-dependent Probe Sequencing (RT-MLPSeq), to determine expression levels of multiple transcripts in FFPE samples. By combining Reverse Transcription-Multiple Ligation-dependent Amplification method and next generation sequencing technology, RT-MLPSeq overcomes the limit of probe length in multiplex ligation-dependent probe amplification assay and thus could detect expression levels of transcripts without quantitative limitations. We proved that different RT-MLPSeq probes targeting on the same transcripts have highly consistent results and the starting RNA/cDNA input could be as little as 1 ng. RT-MLPSeq also presented consistent relative RNA levels of selected 13 genes with reverse transcription quantitative PCR. Finally, we demonstrated the application of the new RT-MLPSeq method by measuring the mRNA expression levels of 21 genes which can be used for accurate calculation of the breast cancer recurrence score - an index that has been widely used for managing breast cancer patients.

  9. Head and Neck Squamous Cell Carcinomas Do Not Express EGFRvIII

    Energy Technology Data Exchange (ETDEWEB)

    Melchers, Lieuwe J., E-mail: l.j.melchers@umcg.nl [Department of Oral and Maxillofacial Surgery, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Clausen, Martijn J.A.M. [Department of Oral and Maxillofacial Surgery, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Mastik, Mirjam F. [Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Slagter-Menkema, Lorian [Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Department of Otorhinolaryngology/Head and Neck Surgery, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Langendijk, Johannes A. [Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Laan, Bernard F.A.M. van der [Department of Otorhinolaryngology/Head and Neck Surgery, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Wal, Jacqueline E. van der; Vegt, Bert van der [Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Roodenburg, Jan L.N. [Department of Oral and Maxillofacial Surgery, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Schuuring, Ed [Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands)

    2014-10-01

    Purpose: To assess the prevalence of EGFRvIII, a specific variant of EGFR (epidermal growth factor receptor), in 3 well-defined cohorts of head and neck squamous cell carcinoma (HNSCC). Methods and Materials: Immunohistochemistry for the specific detection of EGFRvIII using the L8A4 antibody was optimized on formalin-fixed, paraffin-embedded tissue using glioblastoma tissue. It was compared with EGFR and EGFRvIII RNA expression using a specific reverse transcription–polymerase chain reaction also optimized for formalin-fixed, paraffin-embedded tissue. Tissue microarrays including 531 HNSCCs of various stages with complete clinicopathologic and follow-up data were tested for the presence of EGFRvIII. Results: None of the 531 cases showed EGFRvIII protein expression. Using an immunohistochemistry protocol reported by others revealed cytoplasmic staining in 8% of cases. Reverse transcription–polymerase chain reaction for the EGFRvIII transcript of the 28 highest cytoplasmic staining cases, as well as 69 negative cases, did not show expression in any of the tested cases, suggesting aspecific staining by a nonoptimal protocol. Conclusions: The EGFRvIII mutation is not present in HNSCC. Therefore, EGFRvIII does not influence treatment response in HNSCC and is not a usable clinical prognostic marker.

  10. Head and Neck Squamous Cell Carcinomas Do Not Express EGFRvIII

    International Nuclear Information System (INIS)

    Melchers, Lieuwe J.; Clausen, Martijn J.A.M.; Mastik, Mirjam F.; Slagter-Menkema, Lorian; Langendijk, Johannes A.; Laan, Bernard F.A.M. van der; Wal, Jacqueline E. van der; Vegt, Bert van der; Roodenburg, Jan L.N.; Schuuring, Ed

    2014-01-01

    Purpose: To assess the prevalence of EGFRvIII, a specific variant of EGFR (epidermal growth factor receptor), in 3 well-defined cohorts of head and neck squamous cell carcinoma (HNSCC). Methods and Materials: Immunohistochemistry for the specific detection of EGFRvIII using the L8A4 antibody was optimized on formalin-fixed, paraffin-embedded tissue using glioblastoma tissue. It was compared with EGFR and EGFRvIII RNA expression using a specific reverse transcription–polymerase chain reaction also optimized for formalin-fixed, paraffin-embedded tissue. Tissue microarrays including 531 HNSCCs of various stages with complete clinicopathologic and follow-up data were tested for the presence of EGFRvIII. Results: None of the 531 cases showed EGFRvIII protein expression. Using an immunohistochemistry protocol reported by others revealed cytoplasmic staining in 8% of cases. Reverse transcription–polymerase chain reaction for the EGFRvIII transcript of the 28 highest cytoplasmic staining cases, as well as 69 negative cases, did not show expression in any of the tested cases, suggesting aspecific staining by a nonoptimal protocol. Conclusions: The EGFRvIII mutation is not present in HNSCC. Therefore, EGFRvIII does not influence treatment response in HNSCC and is not a usable clinical prognostic marker

  11. Real-time PCR improves Helicobacter pylori detection in patients with peptic ulcer bleeding.

    Science.gov (United States)

    Ramírez-Lázaro, María José; Lario, Sergio; Casalots, Alex; Sanfeliu, Esther; Boix, Loreto; García-Iglesias, Pilar; Sánchez-Delgado, Jordi; Montserrat, Antònia; Bella-Cueto, Maria Rosa; Gallach, Marta; Sanfeliu, Isabel; Segura, Ferran; Calvet, Xavier

    2011-01-01

    Histological and rapid urease tests to detect H. pylori in biopsy specimens obtained during peptic ulcer bleeding episodes (PUB) often produce false-negative results. We aimed to examine whether immunohistochemistry and real-time PCR can improve the sensitivity of these biopsies. We selected 52 histology-negative formalin-fixed paraffin-embedded biopsy specimens obtained during PUB episodes. Additional tests showed 10 were true negatives and 42 were false negatives. We also selected 17 histology-positive biopsy specimens obtained during PUB to use as controls. We performed immunohistochemistry staining and real-time PCR for 16S rRNA, ureA, and 23S rRNA for H. pylori genes on all specimens. All controls were positive for H. pylori on all PCR assays and immunohistochemical staining. Regarding the 52 initially negative biopsies, all PCR tests were significantly more sensitive than immunohistochemical staining (pdetection of H. pylori infection in histology-negative formalin-fixed paraffin-embedded biopsy samples obtained during PUB episodes. The low reported prevalence of H. pylori in PUB may be due to the failure of conventional tests to detect infection.

  12. Automated RNA In Situ Hybridization for 18 High Risk Human Papilloma Viruses in Squamous Cell Carcinoma of the Head and Neck: Comparison With p16 Immunohistochemistry.

    Science.gov (United States)

    Drumheller, Bradley; Cohen, Cynthia; Lawson, Diane; Siddiqui, Momin T

    2017-08-02

    Detection of human papilloma virus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is important, as HPV-associated HNSCCs respond better to therapy. The RNAscope HPV-test is a novel RNA in situ hybridization (ISH) technique which strongly stains transcripts of E6 and E7 mRNA in formalin-fixed, paraffin-embedded tissue, with the potential to replace the indirect immunohistochemical (IHC) marker for p16 protein. A direct clinical comparison between p16 IHC and an automated RNA ISH using 18 probes has not been established. Samples from 27 formalin-fixed, paraffin-embedded HNSCC cases from the Emory University Hospital archives were stained using 18 individual RNA ISH probes for high-risk HPV (RNAscope 2.5 LS Probe ) on a Leica autostainer (Buffalo Grove, IL) and were compared with p16 IHC. Two pathologists reviewed and reached a consensus on all interpretations. The RNAscope technique was positive in 89% (24/27) and the p16 IHC was positive in 78% (21/27). The RNAscope was negative in 11.1% of samples (3/27) and the p16 IHC-negative in 22.2% (6/27). The RNA ISH detected 100% of the p16-positive IHC-stained slides and had a concordance of 88.9% (24/27). This easy to interpret automated staining method for 18 high-risk HPV genotypes is a feasible replacement for the indirect p16 IHC method.

  13. Implementation of immunohistochemistry on frozen ear notch tissue samples in diagnosis of bovine viral diarrhea virus in persistently infected cattle

    Directory of Open Access Journals (Sweden)

    Bedeković Tomislav

    2011-12-01

    Full Text Available Abstract Background Bovine viral diarrhea is a contagious disease of domestic and wild ruminants and one of the most economically important diseases in cattle. Bovine viral diarrhea virus belongs to the genus Pestivirus, within the family Flaviviridae. The identification and elimination of the persistently infected animals from herds is the initial step in the control and eradication programs. It is therefore necessary to have reliable methods for diagnosis of bovine viral diarrhea virus. One of those methods is immunohistochemistry. Immunohistochemistry on formalin fixed, paraffin embedded tissue is a routine technique in diagnosis of persistently infected cattle from ear notch tissue samples. However, such technique is inappropriate due to complicated tissue fixation process and it requires more days for preparation. On the contrary, immunohistochemistry on frozen tissue was usually applied on organs from dead animals. In this paper, for the first time, the imunohistochemistry on frozen ear notch tissue samples was described. Findings Seventeen ear notch tissue samples were obtained during the period 2008-2009 from persistently infected cattle. Samples were fixed in liquid nitrogen and stored on -20°C until testing. Ear notch tissue samples from all persistently infected cattle showed positive results with good section quality and possibility to determinate type of infected cells. Conclusions Although the number of samples was limited, this study indicated that immunohistochemistry on formalin fixed paraffin embedded tissue can be successfully replaced with immunohistochemistry on frozen ear notch tissue samples in diagnosis of persistently infected cattle.

  14. The measurements of thermal neutron flux distribution in a paraffin ...

    Indian Academy of Sciences (India)

    neutron flux density, the foil activation method was used. Thermal neutron flux determination in paraffin phantom by counting the emitted rays of indium foils with two different detectors (Geiger–. Muller counter and NaI(Tl)) was the aim of this project. The relative differences of the outcome of the experiments were between ...

  15. The measurements of thermal neutron flux distribution in a paraffin

    Indian Academy of Sciences (India)

    The term `thermal flux' implies a Maxwellian distribution of velocity and energy corresponding to the most probable velocity of 2200 ms-1 at 293.4 K. In order to measure the thermal neutron flux density, the foil activation method was used. Thermal neutron flux determination in paraffin phantom by counting the emitted rays of ...

  16. Organic Rankine Cycle with Solar Heat Storage in Paraffin Way

    Directory of Open Access Journals (Sweden)

    Constantin LUCA

    2015-06-01

    Full Text Available The paper presents an electricity generation system based on an Organic Rankine Cycle and proposed storing the amount of the heat produced by the solar panels using large volume of paraffin wax. The proposed working fluid is R-134a refrigerant. The cycle operates at very low temperatures. A efficiency of 6,55% was obtained.

  17. [Methods for the rapid preparation of paraffin blocks].

    Science.gov (United States)

    Shmurun, R I

    1992-01-01

    Two accelerated chloroform-paraffin processings of materials with the use of ultrasound (US) and microwave (MW) irradiation in the stove "Electronica" as well as a combined method with US- and MW-irradiation are proposed to shorten drastically the duration of the prehistologic processing.

  18. Bismuth toxicity in patients treated with bismuth iodoform paraffin packs.

    Science.gov (United States)

    Atwal, A; Cousin, G C S

    2016-01-01

    Bismuth is a heavy metal used in bismuth iodoform paraffin paste (BIPP) antiseptic dressings and in a number of other medical preparations. It can be absorbed systemically and cause toxicity. We report 2 cases of such neurotoxicity after it was used in operations on the jaws. Copyright © 2015. Published by Elsevier Ltd.

  19. resistant containers on the incidence of paraffin (kerosene)

    African Journals Online (AJOL)

    The commonest cause of accidental poisoning in the. South African black paediatric population is paraffin ingestion. In this intervention study a specificall¥ designed child-resistant container (CRG) was introduced·to evaluate whether its use would decrease the incidence ofparaffin ingestion. CRCs were distributed to 20 ...

  20. The measurements of thermal neutron flux distribution in a paraffin ...

    Indian Academy of Sciences (India)

    at 293.4 K. In order to measure the thermal neutron flux density, the foil activation method was used. Thermal neutron flux determination in paraffin phantom by counting the emitted rays of indium foils with two different detectors (Geiger–. Muller counter and NaI(Tl)) was the aim of this project. The relative differences of the ...

  1. The measurements of thermal neutron flux distribution in a paraffin ...

    Indian Academy of Sciences (India)

    The term `thermal flux' implies a Maxwellian distribution of velocity and energy corresponding to the most probable velocity of 2200 ms-1 at 293.4 K. In order to measure the thermal neutron flux density, the foil activation method was used. Thermal neutron flux determination in paraffin phantom by counting the emitted rays of ...

  2. Contamination of grape seed oil with mineral oil paraffins.

    Science.gov (United States)

    Fiorini, Dennis; Fiselier, Katell; Biedermann, Maurus; Ballini, Roberto; Coni, Ettore; Grob, Konrad

    2008-12-10

    The contamination of 11 commercial grape seed oils with paraffins of mineral oil origin was analyzed by online-coupled HPLC-HPLC-GC-FID and ranged from 43 to 247 mg kg(-1). The analysis of the marc and seeds indicated that the contamination is primarily from the peels. Since superficial extraction of the seeds with hexane removed most of the mineral paraffins, the contamination of the seeds is largely on the surface, perhaps transferred from the peels during storage of the marc. Mechanical purification of the seeds combined with washing with hexane reduced the contamination of the oil by a factor of about 10. The refining process removed 30% of the mineral paraffins, primarily the more volatile components. Oil obtained from the seeds of fresh grapes, including grapes not having undergone any phytochemical treatment, contained clearly less mineral paraffins (up to 14 mg kg(-1)), and the peels were less contaminated, suggesting an environmental background contamination. To this an additional contamination might be added by a treatment of the grapes used for wine making.

  3. Study on Thermal Degradation Characteristics and Regression Rate Measurement of Paraffin-Based Fuel

    Directory of Open Access Journals (Sweden)

    Songqi Hu

    2015-09-01

    Full Text Available Paraffin fuel has been found to have a regression rate that is higher than conventional HTPB (hydroxyl-terminated polybutadiene fuel and, thus, presents itself as an ideal energy source for a hybrid rocket engine. The energy characteristics of paraffin-based fuel and HTPB fuel have been calculated by the method of minimum free energy. The thermal degradation characteristics were measured for paraffin, pretreated paraffin, HTPB and paraffin-based fuel in different working conditions by the using differential scanning calorimetry (DSC and a thermogravimetric analyzer (TGA. The regression rates of paraffin-based fuel and HTPB fuel were tested by a rectangular solid-gas hybrid engine. The research findings showed that: the specific impulse of paraffin-based fuel is almost the same as that of HTPB fuel; the decomposition temperature of pretreated paraffin is higher than that of the unprocessed paraffin, but lower than that of HTPB; with the increase of paraffin, the initial reaction exothermic peak of paraffin-based fuel is reached in advance, and the initial reaction heat release also increases; the regression rate of paraffin-based fuel is higher than the common HTPB fuel under the same conditions; with the increase of oxidizer mass flow rate, the regression rate of solid fuel increases accordingly for the same fuel formulation.

  4. Rheology of petrolatum-paraffin oil mixtures : Applications to analogue modelling of geological processes

    NARCIS (Netherlands)

    Duarte, João C.; Schellart, Wouter P.; Cruden, Alexander R.

    2014-01-01

    Paraffins have been widely used in analogue modelling of geological processes. Petrolatum and paraffin oil are commonly used to lubricate model boundaries and to simulate weak layers. In this paper, we present rheological tests of petrolatum, paraffin oil and several homogeneous mixtures of the two.

  5. Embedded data representations

    DEFF Research Database (Denmark)

    Willett, Wesley; Jansen, Yvonne; Dragicevic, Pierre

    2017-01-01

    We introduce embedded data representations, the use of visual and physical representations of data that are deeply integrated with the physical spaces, objects, and entities to which the data refers. Technologies like lightweight wireless displays, mixed reality hardware, and autonomous vehicles ......-situated, situated, and embedded data displays, including both visualizations and physicalizations. Based on our observations, we identify a variety of design challenges for embedded data representation, and suggest opportunities for future research and applications....

  6. Characterization of Fast-Scan Cyclic Voltammetric Electrodes Using Paraffin as an Effective Sealant with In Vitro and In Vivo Applications.

    Science.gov (United States)

    Ramsson, Eric S; Cholger, Daniel; Dionise, Albert; Poirier, Nicholas; Andrus, Avery; Curtiss, Randi

    2015-01-01

    Fast-scan cyclic voltammetry (FSCV) is a powerful technique for measuring sub-second changes in neurotransmitter levels. A great time-limiting factor in the use of FSCV is the production of high-quality recording electrodes; common recording electrodes consist of cylindrical carbon fiber encased in borosilicate glass. When the borosilicate is heated and pulled, the molten glass ideally forms a tight seal around the carbon fiber cylinder. It is often difficult, however, to guarantee a perfect seal between the glass and carbon. Indeed, much of the time spent creating electrodes is in an effort to find a good seal. Even though epoxy resins can be useful in this regard, they are irreversible (seals are permanent), wasteful (epoxy cannot be reused once hardener is added), hazardous (hardeners are often caustic), and require curing. Herein we characterize paraffin as an electrode sealant for FSCV microelectrodes. Paraffin boasts the advantages of near-immediate curing times, simplicity in use, long shelf-life and stable waterproof seals capable of withstanding extended cycling. Borosilicate electrode tips were left intact or broken and dipped in paraffin embedding wax. Excess wax was removed from the carbon surface with xyelenes or by repeated cycling at an extended waveform (-0.4 to 1.4V, 400 V/s, 60 Hz). Then, the waveform was switched to a standard waveform (-0.4 to 1.3V, 400 V/s, 10 Hz) and cycled until stable. Wax-sealing does not inhibit electrode sensitivity, as electrodes detected linear changes in dopamine before and after wax (then xylenes) exposure. Paraffin seals are intact after 11 days of implantation in the mouse, and still capable of measuring transient changes in in vivo dopamine. From this it is clear that paraffin wax is an effective sealant for FSCV electrodes that provides a convenient substitute to epoxy sealants.

  7. Characterization of Fast-Scan Cyclic Voltammetric Electrodes Using Paraffin as an Effective Sealant with In Vitro and In Vivo Applications.

    Directory of Open Access Journals (Sweden)

    Eric S Ramsson

    Full Text Available Fast-scan cyclic voltammetry (FSCV is a powerful technique for measuring sub-second changes in neurotransmitter levels. A great time-limiting factor in the use of FSCV is the production of high-quality recording electrodes; common recording electrodes consist of cylindrical carbon fiber encased in borosilicate glass. When the borosilicate is heated and pulled, the molten glass ideally forms a tight seal around the carbon fiber cylinder. It is often difficult, however, to guarantee a perfect seal between the glass and carbon. Indeed, much of the time spent creating electrodes is in an effort to find a good seal. Even though epoxy resins can be useful in this regard, they are irreversible (seals are permanent, wasteful (epoxy cannot be reused once hardener is added, hazardous (hardeners are often caustic, and require curing. Herein we characterize paraffin as an electrode sealant for FSCV microelectrodes. Paraffin boasts the advantages of near-immediate curing times, simplicity in use, long shelf-life and stable waterproof seals capable of withstanding extended cycling. Borosilicate electrode tips were left intact or broken and dipped in paraffin embedding wax. Excess wax was removed from the carbon surface with xyelenes or by repeated cycling at an extended waveform (-0.4 to 1.4V, 400 V/s, 60 Hz. Then, the waveform was switched to a standard waveform (-0.4 to 1.3V, 400 V/s, 10 Hz and cycled until stable. Wax-sealing does not inhibit electrode sensitivity, as electrodes detected linear changes in dopamine before and after wax (then xylenes exposure. Paraffin seals are intact after 11 days of implantation in the mouse, and still capable of measuring transient changes in in vivo dopamine. From this it is clear that paraffin wax is an effective sealant for FSCV electrodes that provides a convenient substitute to epoxy sealants.

  8. Embedding beyond electrostatics

    DEFF Research Database (Denmark)

    Nåbo, Lina J.; Olsen, Jógvan Magnus Haugaard; Holmgaard List, Nanna

    2016-01-01

    We study excited states of cholesterol in solution and show that, in this specific case, solute wave-function confinement is the main effect of the solvent. This is rationalized on the basis of the polarizable density embedding scheme, which in addition to polarizable embedding includes non-electrostatic...... repulsion that effectively confines the solute wave function to its cavity. We illustrate how the inclusion of non-electrostatic repulsion results in a successful identification of the intense π → π∗ transition, which was not possible using an embedding method that only includes electrostatics....... This underlines the importance of non-electrostatic repulsion in quantum-mechanical embedding-based methods....

  9. Embedded systems handbook

    CERN Document Server

    Zurawski, Richard

    2005-01-01

    Embedded systems are nearly ubiquitous, and books on individual topics or components of embedded systems are equally abundant. Unfortunately, for those designers who thirst for knowledge of the big picture of embedded systems there is not a drop to drink. Until now. The Embedded Systems Handbook is an oasis of information, offering a mix of basic and advanced topics, new solutions and technologies arising from the most recent research efforts, and emerging trends to help you stay current in this ever-changing field.With preeminent contributors from leading industrial and academic institutions

  10. Radiotherapic Valuation of Paraffin Wax for Patients with Oral Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Na, Kyung Su; Seo, Seuk Jin; Lee, Je Hee; Yoo, Sook Heun [Dept. of Radiation Oncology, Seoul National University Hosdital, Seoul (Korea, Republic of)

    2011-03-15

    This study is designed to investigate radiotherapic valuation of Paraffin Wax, which is newly formed for this study and generally utilized in dentistry, and Mouth Piece and Putty impression, which are commonly used in radiotherapy, for oral cavity as a compensator. Each compensator was formed by 10 x 10 x 1 cm and measured radiation dose attenuation ratio with reference of water phantom which is made of tissue-equivalent materials. Two patients with oral cancer underwent DRR (Digitally Reconstructed Radiogrph) of Offline Review Program of Aria System and Portal vision for 5 times for each material to evaluate reproducibility by each filling materials. Moreover, MU (monitor unit) changes by dose absorption were considered in the case of inevitable implication of an filling materials in the range for radiotherapy. Radiation dose attenuation ratios were shown -0.7{approx}+3.7% for Mouth Piece, +0.21{approx}+0.39% for Paraffin Wax and -2.71{approx}-1.76% for Putty impression. Error ranges of reproducibility of positions were measured {+-}3 mm for Mouth Piece, {+-}2 mm for Paraffin Wax and {+-}2 mm for Putty impression. Difference of prescription MU from dose absorption with an filling material increased +7.8% (250 MU) in Putty impression and -0.9% (230 MU) in Paraffin Wax as converted into a percentage from the standard phantom, Water 232 MU. Dose reduction of boundary between cavity and tissue was observed for Mouth Piece. Mouth Piece also had low reproducibility of positions as it had no reflection of anatomy of oral cavity even though it was a proper material to separate Maxilla and Mandible during therapy. On the other hand, Putty impression was a suitable material to correctly re-position oral cavity as before. However, it risked normal tissues getting unnecessary over irradiation and it caused radiation dose decrease by -2.5% for 1cm volume in comparison of it of water phantom. Dose reduction in Paraffin Wax, Fat Tissue-Equivalent Material, was smaller than other

  11. Melt Channel Formation in Paraffin With Applications to Lava Channels

    Science.gov (United States)

    Whitehead, J. A.; Mills, C. J.

    2005-12-01

    We present the results from laboratory experiments designed to explore channel formation caused by instabilities in fluid flow, and to provide a simple experimental basis to facilitate the extension of lava channel theory. Kelemen et. al. [1995] reported on a series of experiments that explored channel formation resulting from fluid instabilities caused by the ``reactive infiltration instability'' (RII). However, the material used in portions of their experiments was undesirable because of a poorly suited rheology. Therefore, our experiments were initiated as an extension of those earlier experiments, using instead a material with more robust rheological properties. The material chosen for this series of experiments is a paraffin with a freezing temperature of 4°C, which is transparent when liquid and opaque-white when frozen. The paraffin is pumped at a constant and controlled rate into a narrow gap between two circular plates, both with a diameter of ~51cm. The gap is open along the circumference of the plates, allowing the paraffin to flow out of the gap once it reaches the edge of the plate. The upper plate is constructed of Plexiglas to allow observation of the wax below, and it has a hole in the center through which the paraffin is pumped. The lower plate is constructed of aluminum, painted black, and kept at -5°C by circulating coolant through a reservoir below the it. As the paraffin, which is kept at 20°C before being pumped into the gap, flows over the cold lower plate, it begins to freeze and eventually fills up the gap between the plates. A single channel then forms, surrounded by solid paraffin, and the liquid being pumped in is transported via this channel to the edge of the plate. This is a stable state for all but the lowest flow rates attempted and verifies the results from the earlier work exploring the RII. We conducted experiments at different flows rates from 270mL/min to <30mL/min, and recorded the dimensions of the steady state channel

  12. Radiotherapic Valuation of Paraffin Wax for Patients with Oral Cancer

    International Nuclear Information System (INIS)

    Na, Kyung Su; Seo, Seuk Jin; Lee, Je Hee; Yoo, Sook Heun

    2011-01-01

    This study is designed to investigate radiotherapic valuation of Paraffin Wax, which is newly formed for this study and generally utilized in dentistry, and Mouth Piece and Putty impression, which are commonly used in radiotherapy, for oral cavity as a compensator. Each compensator was formed by 10 x 10 x 1 cm and measured radiation dose attenuation ratio with reference of water phantom which is made of tissue-equivalent materials. Two patients with oral cancer underwent DRR (Digitally Reconstructed Radiogrph) of Offline Review Program of Aria System and Portal vision for 5 times for each material to evaluate reproducibility by each filling materials. Moreover, MU (monitor unit) changes by dose absorption were considered in the case of inevitable implication of an filling materials in the range for radiotherapy. Radiation dose attenuation ratios were shown -0.7∼+3.7% for Mouth Piece, +0.21∼+0.39% for Paraffin Wax and -2.71∼-1.76% for Putty impression. Error ranges of reproducibility of positions were measured ±3 mm for Mouth Piece, ±2 mm for Paraffin Wax and ±2 mm for Putty impression. Difference of prescription MU from dose absorption with an filling material increased +7.8% (250 MU) in Putty impression and -0.9% (230 MU) in Paraffin Wax as converted into a percentage from the standard phantom, Water 232 MU. Dose reduction of boundary between cavity and tissue was observed for Mouth Piece. Mouth Piece also had low reproducibility of positions as it had no reflection of anatomy of oral cavity even though it was a proper material to separate Maxilla and Mandible during therapy. On the other hand, Putty impression was a suitable material to correctly re-position oral cavity as before. However, it risked normal tissues getting unnecessary over irradiation and it caused radiation dose decrease by -2.5% for 1cm volume in comparison of it of water phantom. Dose reduction in Paraffin Wax, Fat Tissue-Equivalent Material, was smaller than other impressions and

  13. Embedded systems handbook networked embedded systems

    CERN Document Server

    Zurawski, Richard

    2009-01-01

    Considered a standard industry resource, the Embedded Systems Handbook provided researchers and technicians with the authoritative information needed to launch a wealth of diverse applications, including those in automotive electronics, industrial automated systems, and building automation and control. Now a new resource is required to report on current developments and provide a technical reference for those looking to move the field forward yet again. Divided into two volumes to accommodate this growth, the Embedded Systems Handbook, Second Edition presents a comprehensive view on this area

  14. Efeito dos fixadores formalina e Bouin na preservação de biópsias do endométrio de éguas após inclusão em resina plástica Effect of formalin and Bouin fixation upon the mare's endometrial biopsies embedded in plastic resin

    Directory of Open Access Journals (Sweden)

    D. Amaral

    2004-02-01

    Full Text Available Biópsias do endométrio de 16 éguas sexualmente maduras, em estro e diestro, foram processadas para microscopia de luz utilizando-se fixação em formalina ou Bouin e inclusão em resina plástica à base de glicol metacrilato. Análises morfológicas de 46 biópsias demonstraram que o epitélio de revestimento do endométrio, o epitélio glandular, as fibras do tecido conjuntivo e os diferentes tipos celulares presentes na lâmina própria, tais como fibroblastos, plasmócitos, mastócitos e macrófagos, apresentaram-se melhor preservados quando os fragmentos de tecidos foram fixados em formalina. O epitélio de revestimento mostrou grau mais acentuado de retração tecidual nas biópsias fixadas em Bouin, independente da fase do ciclo estral. A fixação em formalina aliada à inclusão em resina plástica resultou em melhor resolução das células ao microscópio de luz, permitindo um estudo citológico mais acurado do endométrio eqüino.Endometrial biopsies were performed in 16 mares at estrus and diestrus and tissues were processed for light microscopy using formalin or Bouin fixatives and plastic resin glycol methacrylate for embedding. Results of the tissue processing demonstrated that the luminal and glandular epithelium, connective tissue fibers and many cell types present in the lamina propria such as fibroblasts, plasmocytes, mast cells and macrophages were best preserved in formalin fixed samples. The luminal epithelium showed increased shrinkage in Bouin fixed specimens when compared to formalin fixed ones. Those morphological findings were present throughout the estral cycle. The formalin fixation procedure associated with plastic resin embedding yielded increased tissue resolution as seen by light microscopy, and allowed a more accurate cytological study of the endometrium of the mare.

  15. Preparation and Characterization of Modified Montmorillonite/Paraffin Phase Change Microcapsules for Energy Storage

    Directory of Open Access Journals (Sweden)

    LIN Sen

    2017-03-01

    Full Text Available The phase change microcapsules of modified montmorillonite/paraffin were prepared by Pickering emulsion method. Analytic techniques of optical microscopy, scanning electron microscopy(SEM, infrared spectroscopy(FTIR, differential scanning calorimetry(DSC and thermogravimetry(TG were utilized for characterizing chemical structure, morphology and thermal properties. Results show that modified montmorillonite as a new type wall material has excellent performance for protecting core material of paraffin. FTIR spectra of phase change of modified montmorillonite/paraffin microcapsules shows that their characteristic peaks match with corresponding peaks of pure paraffin and modified montmorillonite. DSC results indicate that modified montmorillonite/paraffin microcapsules have similar solid-liquid phase change temperature with pure paraffin. The phase transition enthalpy values of microcapsules with paraffin contents varying from 55% to 80% are 110.5-147.2J/g, indicating that microcapsules have excellent thermal storage performance and the phase change properties can be adjusted by changing contents of paraffin. TG results confirm that modified montmorillonite/paraffin microcapsules have outstanding thermal stability. The presented study indicates that modified montmorillonite is a suitable wall material for preparing paraffin microcapsule. Modified montmorillonite/paraffin microcapsules have advantages of low cost and high performance with a great application potential in the field of thermal storage.

  16. Preparation and thermal energy storage properties of paraffin/expanded graphite composite phase change material

    International Nuclear Information System (INIS)

    Zhang, Zhengguo; Zhang, Ni; Peng, Jing; Fang, Xiaoming; Gao, Xuenong; Fang, Yutang

    2012-01-01

    Highlights: ► EG was obtained by microwave irradiation to prepare the paraffin/EG composite PCM. ► Composite PCM was characterized by XRD to investigate the chemical compatibility. ► Temperature profiles of the composite PCM were obtained during thermal energy storage. -- Abstract: The paraffin/expanded graphite (EG) composite phase change material (PCM) was prepared by absorbing liquid paraffin into EG, in which paraffin was chosen as the PCM. EG was produced by microwave irradiation performed at room temperature. It was found that the EG prepared at 800 W irradiation power for 10 s exhibited the maximum sorption capacity of 92 wt% for paraffin. Scanning electron microscopy images showed that paraffin was uniformly dispersed in the pores of EG. Differential scanning calorimeter analysis indicated that the melting temperature of the composite PCM was close to that of paraffin, and its latent heat was equivalent to the calculated value based on the mass fraction of paraffin in the composite. X-ray diffraction analysis showed that the composite PCM was just a combination of paraffin with EG, and no new substance was produced. Thermal energy storage performance of the composite PCM was tested in a latent thermal energy storage (LTES) system. Transients of axial and radial temperature profiles were obtained in the LTES for the composite PCM and paraffin. The thermal energy storage charging duration for the composite PCM was reduced obviously compared to paraffin.

  17. The data embedding method

    Energy Technology Data Exchange (ETDEWEB)

    Sandford, M.T. II; Bradley, J.N.; Handel, T.G.

    1996-06-01

    Data embedding is a new steganographic method for combining digital information sets. This paper describes the data embedding method and gives examples of its application using software written in the C-programming language. Sandford and Handel produced a computer program (BMPEMBED, Ver. 1.51 written for IBM PC/AT or compatible, MS/DOS Ver. 3.3 or later) that implements data embedding in an application for digital imagery. Information is embedded into, and extracted from, Truecolor or color-pallet images in Microsoft{reg_sign} bitmap (.BMP) format. Hiding data in the noise component of a host, by means of an algorithm that modifies or replaces the noise bits, is termed {open_quote}steganography.{close_quote} Data embedding differs markedly from conventional steganography, because it uses the noise component of the host to insert information with few or no modifications to the host data values or their statistical properties. Consequently, the entropy of the host data is affected little by using data embedding to add information. The data embedding method applies to host data compressed with transform, or {open_quote}lossy{close_quote} compression algorithms, as for example ones based on discrete cosine transform and wavelet functions. Analysis of the host noise generates a key required for embedding and extracting the auxiliary data from the combined data. The key is stored easily in the combined data. Images without the key cannot be processed to extract the embedded information. To provide security for the embedded data, one can remove the key from the combined data and manage it separately. The image key can be encrypted and stored in the combined data or transmitted separately as a ciphertext much smaller in size than the embedded data. The key size is typically ten to one-hundred bytes, and it is in data an analysis algorithm.

  18. Quantum Embedding Theories.

    Science.gov (United States)

    Sun, Qiming; Chan, Garnet Kin-Lic

    2016-12-20

    In complex systems, it is often the case that the region of interest forms only one part of a much larger system. The idea of joining two different quantum simulations-a high level calculation on the active region of interest, and a low level calculation on its environment-formally defines a quantum embedding. While any combination of techniques constitutes an embedding, several rigorous formalisms have emerged that provide for exact feedback between the embedded system and its environment. These three formulations: density functional embedding, Green's function embedding, and density matrix embedding, respectively, use the single-particle density, single-particle Green's function, and single-particle density matrix as the quantum variables of interest. Many excellent reviews exist covering these methods individually. However, a unified presentation of the different formalisms is so far lacking. Indeed, the various languages commonly used, functional equations for density functional embedding, diagrammatics for Green's function embedding, and entanglement arguments for density matrix embedding, make the three formulations appear vastly different. In this Account, we introduce the basic equations of all three formulations in such a way as to highlight their many common intellectual strands. While we focus primarily on a straightforward theoretical perspective, we also give a brief overview of recent applications and possible future developments. The first section starts with density functional embedding, where we introduce the key embedding potential via the Euler equation. We then discuss recent work concerning the treatment of the nonadditive kinetic potential, before describing mean-field density functional embedding and wave function in density functional embedding. We finish the section with extensions to time-dependence and excited states. The second section is devoted to Green's function embedding. Here, we use the Dyson equation to obtain equations that

  19. Polarizable Density Embedding

    DEFF Research Database (Denmark)

    Reinholdt, Peter; Kongsted, Jacob; Olsen, Jógvan Magnus Haugaard

    2017-01-01

    and diffuse basis sets that are otherwise questionable-due to electron spill-out effects-in standard embedding models. Based on our analysis, we find the PDE model to be robust and much more systematic than less sophisticated focused embedding models, and thus outline the PDE model as a very efficient...

  20. Embeddings of Heyting Algebras

    NARCIS (Netherlands)

    Jongh, D.H.J. de; Visser, A.

    In this paper we study embeddings of Heyting Algebras. It is pointed out that such embeddings are naturally connected with Derived Rules. We compare the Heyting Algebras embeddable in the Heyting Algebra of the Intuitionistic Propositional Calculus (IPC), i.e. the free Heyting Algebra on countably

  1. Paraffin scintillator for radioassay of solid support samples

    International Nuclear Information System (INIS)

    Fujii, Haruo; Takiue, Makoto

    1989-01-01

    A new paraffin scintillator used for solid support sample counting has been proposed, and its composition and various characteristics are described. The solid support sample treated with this scintillator can be easily handled because of rigid sample conditions. This technique provides great advantages such as the elimination of a large volume of scintillator and little radioactive waste material by using an economical polyethylene bag instead of the conventional counting vial. (author)

  2. Embedded engineering education

    CERN Document Server

    Kaštelan, Ivan; Temerinac, Miodrag; Barak, Moshe; Sruk, Vlado

    2016-01-01

    This book focuses on the outcome of the European research project “FP7-ICT-2011-8 / 317882: Embedded Engineering Learning Platform” E2LP. Additionally, some experiences and researches outside this project have been included. This book provides information about the achieved results of the E2LP project as well as some broader views about the embedded engineering education. It captures project results and applications, methodologies, and evaluations. It leads to the history of computer architectures, brings a touch of the future in education tools and provides a valuable resource for anyone interested in embedded engineering education concepts, experiences and material. The book contents 12 original contributions and will open a broader discussion about the necessary knowledge and appropriate learning methods for the new profile of embedded engineers. As a result, the proposed Embedded Computer Engineering Learning Platform will help to educate a sufficient number of future engineers in Europe, capable of d...

  3. Ultrasound assisted manufacturing of paraffin wax nanoemulsions: process optimization.

    Science.gov (United States)

    Jadhav, A J; Holkar, C R; Karekar, S E; Pinjari, D V; Pandit, A B

    2015-03-01

    This work reports on the process optimization of ultrasound-assisted, paraffin wax in water nanoemulsions, stabilized by modified sodium dodecyl sulfate (SDS). This work focuses on the optimization of major emulsification process variables including sonication time, applied power and surfactant concentration. The effects of these variables were investigated on the basis of mean droplet diameter and stability of the prepared emulsion. It was found that the stable emulsion with droplet diameters about 160.9 nm could be formed with the surfactant concentration of 10 mg/ml and treated at 40% of applied power (power density: 0.61 W/ml) for 15 min. Scanning electron microscopy (SEM) was used to study the morphology of the emulsion droplets. The droplets were solid at room temperature, showing bright spots under polarized light and a spherical shape under SEM. The electrophoretic properties of emulsion droplets showed a negative zeta potential due to the adsorption of head sulfate groups of the SDS surfactant. For the sake of comparison, paraffin wax emulsion was prepared via emulsion inversion point method and was checked its intrinsic stability. Visually, it was found that the emulsion get separated/creamed within 30 min. while the emulsion prepared via ultrasonically is stable for more than 3 months. From this study, it was found that the ultrasound-assisted emulsification process could be successfully used for the preparation of stable paraffin wax nanoemulsions.

  4. Automatic Measuring System for Oil Stream Paraffin Deposits Parameters

    Science.gov (United States)

    Kopteva, A. V.; Koptev, V. Yu

    2018-03-01

    This paper describes a new method for monitoring oil pipelines, as well as a highly efficient and automated paraffin deposit monitoring method. When operating oil pipelines, there is an issue of paraffin, resin and salt deposits on the pipeline walls that come with the oil stream. It ultimately results in frequent transportation suspension to clean or even replace pipes and other equipment, thus shortening operation periods between repairs, creating emergency situations and increasing production expenses, badly affecting environment, damaging ecology and spoil underground water, killing animals, birds etc. Oil spills contaminate rivers, lakes, and ground waters. Oil transportation monitoring issues are still subject for further studying. Thus, there is the need to invent a radically new automated process control and management system, together with measurement means intellectualization. The measurement principle is based on the Lambert-Beer law that describes the dependence between the gamma-radiation frequency and the density together with the linear attenuation coefficient for a substance. Using the measuring system with high accuracy (± 0,2%), one can measure the thickness of paraffin deposits with an absolute accuracy of ± 5 mm, which is sufficient to ensure reliable operation of the pipeline system. Safety is a key advantage, when using the proposed control system.

  5. Evaluation of performance loss of paraffin oil loaded filtering facepieces.

    Science.gov (United States)

    Tombolini, Francesca; Listrani, Stefano; Campopiano, Antonella; Plebani, Carmela

    2016-01-01

    Penetration measurements through commercially available filtering facepieces were performed with monodisperse DEHS aerosols ranging from 0.03 μm to 0.40 μm (either singly charged or neutralized), before and after 500 mg of paraffin oil loading. The distinct behavior of Coulomb and polarization capture efficiency is studied: as in the case of non loading also in the case of loading 500 mg of paraffin oil, the electrostatic capture mechanisms are mainly due to the Coulomb contribution up to aerosol particle diameter of about 0.10 μm, just when the polarization contribution becomes substantial. Both Coulomb and polarization capture mechanisms are influenced by the presence of 500 mg of paraffin oil, resulting less effective than the oil unloaded case of about 12% and 11%, respectively. By the occupational hygiene point of view, there is a degradation in the filter performance due to oil loading that the user does not realize because there is no remarkable variation in the breathing resistance.

  6. Effective melanin depigmentation of human and murine ocular tissues: an improved method for paraffin and frozen sections.

    Directory of Open Access Journals (Sweden)

    Caroline Manicam

    Full Text Available PURPOSE: The removal of excessive melanin pigments that obscure ocular tissue morphology is important to address scientific questions and for differential diagnosis of ocular tumours based on histology. Thus, the goal of the present study was to establish an effective and fast melanin bleaching method for paraffin and frozen mouse and human ocular tissues. METHODS: Paraffin-embedded and frozen ocular specimens from mice and human donors were subjected to bleaching employing two methods. The first employed potassium permanganate (KMnO4 with oxalic acid, and the second 10% hydrogen peroxide (H2O2. To determine optimal bleaching conditions, depigmentation was carried out at various incubation times. The effect of diluents used for 10% H2O2 was assessed using phosphate-buffered saline (PBS, and deionized water. Three different slide types and two fixatives, which were ice-cold acetone with 80% methanol, and 4% paraformaldehyde (PFA were used to determine the optimal conditions for better tissue adherence during bleaching. All tissues were stained in hematoxylin and eosin for histological evaluation. RESULTS: Optimal bleaching was achieved using warm 10% H2O2 diluted in PBS at 65°C for 120 minutes. Chromium-gelatin-coated slides prevented tissue detachment. Adherence of cryosections was also improved with post-fixation using 4% PFA and overnight air-drying at RT after cryosectioning. Tissue morphology was preserved under these conditions. Conversely, tissues bleached in KMnO4/oxalic acid demonstrated poor depigmentation with extensive tissue damage. CONCLUSIONS: Warm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues.

  7. Embedded Linux in het onderwijs

    NARCIS (Netherlands)

    Dr Ruud Ermers

    2008-01-01

    Embedded Linux wordt bij steeds meer grote bedrijven ingevoerd als embedded operating system. Binnen de opleiding Technische Informatica van Fontys Hogeschool ICT is Embedded Linux geïntroduceerd in samenwerking met het lectoraat Architectuur van Embedded Systemen. Embedded Linux is als vakgebied

  8. Influence des additifs sur la cristallisation des n-paraffines en milieu hydrocarbure. Influence of Additives on the Crystallization of N-Paraffins in a Hydrocarbon Medium

    Directory of Open Access Journals (Sweden)

    Petinelli J. C.

    2006-11-01

    Full Text Available On montre que les additifs qui ont une longueur de chaîne et une structure physico-chimique voisines de celles des n-paraffines ne retardent pas, ou très peu, la cristallisation des n-paraffines en milieu hydrocarbure, et ne perturbent pas de façon significative le faciès des cristaux.Par contre, les additifs qui présentent soit de très longues chaînes avec plusieurs groupements non paraffiniques, soit un encombrement stérique suffisamment grand avec des radicaux alkyles greffés sur un groupement non paraffinique retardent la cristallisation et modifient le faciès des cristaux de n-paraffines. This article shows that additives which have a chain length and a physicochemical structure similar to those of n-paraffins do not or hardly slow down the crystallization of n-paraffins in a hydrocarbon medium and do not create any significant disturbance in the crystal facies.On the other hand, additives having either very long chains with several non-paraffinic groups or sufficiently great steric bulk with alkyl radicals grafted to a non-paraffinic group delay crystallization and modify the facies of n-paraffin crystals.

  9. Increased renal gene transcription of protein kinase C-beta in human diabetic nephropathy: relationship to long-term glycaemic control

    DEFF Research Database (Denmark)

    Langham, R.G.; Kelly, D.J.; Gow, R.M.

    2008-01-01

    are unknown. We sought to determine whether in addition to activation, diabetes may lead to increased transcription of two PKC isoforms that have been implicated in the pathogenesis of diabetic nephropathy, PKC-alpha and PKC-beta. METHODS: Recent advances in molecular biological techniques now permit...... quantitative analysis of mRNA from archival, formalin-fixed, paraffin-embedded tissue sections. RNA was extracted from scraped 6 microm sections of biopsy tissue, and PRKC-alpha and PRKC-beta (also known as PRKCA and PRKCB) mRNA measured using real-time PCR. Expression of genes encoding PKC isoforms...... was examined in renal biopsies (n=25) with classical histological features of diabetic nephropathy and compared with that in normal control tissue (n=6). Peptide localisation of PKC-alpha, PKC-beta and the activated forms phosphorylated PKC-alpha and -beta was also performed on matched paraffin...

  10. Brauer type embedding problems

    CERN Document Server

    Ledet, Arne

    2005-01-01

    This monograph is concerned with Galois theoretical embedding problems of so-called Brauer type with a focus on 2-groups and on finding explicit criteria for solvability and explicit constructions of the solutions. The advantage of considering Brauer type embedding problems is their comparatively simple condition for solvability in the form of an obstruction in the Brauer group of the ground field. This book presupposes knowledge of classical Galois theory and the attendant algebra. Before considering questions of reducing the embedding problems and reformulating the solvability criteria, the

  11. Electronics for embedded systems

    CERN Document Server

    Bindal, Ahmet

    2017-01-01

    This book provides semester-length coverage of electronics for embedded systems, covering most common analog and digital circuit-related issues encountered while designing embedded system hardware. It is written for students and young professionals who have basic circuit theory background and want to learn more about passive circuits, diode and bipolar transistor circuits, the state-of-the-art CMOS logic family and its interface with older logic families such as TTL, sensors and sensor physics, operational amplifier circuits to condition sensor signals, data converters and various circuits used in electro-mechanical device control in embedded systems. The book also provides numerous hardware design examples by integrating the topics learned in earlier chapters. The last chapter extensively reviews the combinational and sequential logic design principles to be able to design the digital part of embedded system hardware.

  12. Smart Multicore Embedded Systems

    DEFF Research Database (Denmark)

    This book provides a single-source reference to the state-of-the-art of high-level programming models and compilation tool-chains for embedded system platforms. The authors address challenges faced by programmers developing software to implement parallel applications in embedded systems, where very...... specificities of various embedded systems from different industries. Parallel programming tool-chains are described that take as input parameters both the application and the platform model, then determine relevant transformations and mapping decisions on the concrete platform, minimizing user intervention...... and hiding the difficulties related to the correct and efficient use of memory hierarchy and low level code generation. Describes tools and programming models for multicore embedded systems Emphasizes throughout performance per watt scalability Discusses realistic limits of software parallelization Enables...

  13. Embedded Fragments Registry (EFR)

    Data.gov (United States)

    Department of Veterans Affairs — In 2009, the Department of Defense estimated that approximately 40,000 service members who served in OEF/OIF may have embedded fragment wounds as the result of small...

  14. FCJ-130 Embedding response:

    DEFF Research Database (Denmark)

    Ramsgaard Thomsen, Mette; Bech, Karin

    2011-01-01

    Ubiquitous computing positions a world where computation is embedded into our surrounding environment. Rather than retrieving information and communication from distinct devices (PCs) removed from contexts and activities, ubiquitous computing proposes that the mediated can become an integral part...

  15. Effect of duration of fixation on quantitative reverse transcription polymerase chain reaction analyses.

    Science.gov (United States)

    Macabeo-Ong, Maricris; Ginzinger, David G; Dekker, Nusi; McMillan, Alex; Regezi, Joseph A; Wong, David T W; Jordan, Richard C K

    2002-09-01

    Increasingly, there is the need to analyze gene expression in tumor tissues and correlate these findings with clinical outcome. Because there are few tissue banks containing enough frozen material suitable for large-scale genetic analyses, methods to isolate and quantify messenger RNA (mRNA) from formalin-fixed, paraffin-embedded tissue sections are needed. Recovery of RNA from routinely processed biopsies and quantification by the polymerase chain reaction (PCR) has been reported; however, the effects of formalin fixation have not been well studied. We used a proteinase K-salt precipitation RNA isolation protocol followed by TaqMan quantitative PCR to compare the effect of formalin fixation for 24, 48, and 72 hours and for 1 week in normal (2), oral epithelial dysplasia (3), and oral squamous cell carcinoma (4) specimens yielding 9 fresh and 36 formalin-fixed samples. We also compared mRNA and protein expression levels using immunohistochemistry for epidermal growth factor receptor (EGFR), matrix metalloproteinase (MMP)-1, p21, and vascular endothelial growth factor (VEGF) in 15 randomly selected and routinely processed oral carcinomas. We were able to extract RNA suitable for quantitative reverse transcription (RT) from all fresh (9/9) and formalin-fixed (36/36) specimens fixed for differing lengths of time and from all (15/15) randomly selected oral squamous cell carcinoma. We found that prolonged formalin fixation (>48 h) had a detrimental effect on quantitative RT polymerase chain reaction results that was most marked for MMP-1 and VEGF but less evident for p21 and EGFR. Comparisons of quantitative RT polymerase chain reaction and immunohistochemistry showed that for all markers, except p21, there was good correlation between mRNA and protein levels. p21 mRNA was overexpressed in only one case, but protein levels were elevated in all but one tumor, consistent with the established translational regulation of p21. These results show that RNA can be reliably

  16. Thermophysical properties estimation of paraffin/graphite composite phase change material using an inverse method

    International Nuclear Information System (INIS)

    Lachheb, Mohamed; Karkri, Mustapha; Albouchi, Fethi; Mzali, Foued; Nasrallah, Sassi Ben

    2014-01-01

    Highlights: • Preparation of paraffin/graphite composites by uni-axial compression technique. • Measurement of thermophysical properties of paraffin/graphite using the periodic method. • Measurement of the experimental densities of paraffin/graphite composites. • Prediction of the effective thermal conductivity using analytical models. - Abstract: In this paper, two types of graphite were combined with paraffin in an attempt to improve thermal conductivity of paraffin phase change material (PCM): Synthetic graphite (Timrex SFG75) and graphite waste obtained from damaged Tubular graphite Heat Exchangers. These paraffin/graphite phase change material (PCM) composites are prepared by the cold uniaxial compression technique and the thermophysical properties were estimated using a periodic temperature method and an inverse technique. Results showed that the thermal conductivity and thermal diffusivity are greatly influenced by the graphite addition

  17. Paraffin surfaces for culture-based detection of mycobacteria in environmental samples

    Science.gov (United States)

    Jing, Gaoshan; Eluru, Hima B.; Polaczyk, Amy; Kinkle, Brian; Oerther, Daniel B.; Papautsky, Ian

    2005-02-01

    In this paper, we describe the development of paraffin surfaces for culture-based detection of mycobacteria in environmental samples. The surfaces use the well-described paraffinophilic nature of mycobacteria to rapidly and selectively differentiate them from non-target microorganisms. New methods of depositing and patterning paraffin were developed, including using butanol or a heat gun to generate molten paraffin that was subsequently deposited by spin coating and patterned by wet and dry etching techniques. Using these techniques, 18 mm glass wafers with paraffin films of 10 70 µm were fabricated and subsequently etched to form different patterns. Prototype paraffin surfaces were experimentally tested to demonstrate the concept of rapid and selective detection of mycobacteria in environmental samples collected from a municipal sewage treatment plant. Our successful demonstration of the culture-based detection on paraffin substrates presents an alternative approach for tracking microorganisms in complex environmental samples.

  18. The use of paraffin wax in a new solar cooker with inner and outer reflectors

    OpenAIRE

    Arabacigil Bihter; Yuksel Numan; Avci Atakan

    2015-01-01

    In this paper, the potential use and effectiveness of paraffin wax in a new solar cooker was experimentally investigated during daylight and late evening hours. For these experiments, a cooker having an inner reflecting surface was designed, constructed by filling paraffin wax and metal shavings. The side- and sub-surface temperatures of the paraffin wax in the cooker are measured in the summer months of June and July. The thermal efficiency of the cooker w...

  19. The use of naturally occurring selectively isolated bacteria for inhibiting paraffin deposition

    Energy Technology Data Exchange (ETDEWEB)

    Lazar, I.; Voicu, A.; Dobrota, S.; Petrisor, I.G.; Stefanescu, M.; Sandulescu, L. [Institute of Biology of the Romanian Academy, Spl. Independentei 296, Bucharest (Romania); Nicolescu, C.; Mucenica, D. [PETROSTAR Ploiesti, Bdul Bucuresti 35, Ploiesti (Romania)

    1999-01-01

    One of the most severe problems at any oil fields producing paraffinic oils is that of paraffin depositions. Romania which has a long experience in oil production is also faced with this problem in many oil fields. The microbial treatment, based on the activity of naturally occurring, selectively isolated bacteria, is already proved as an effective alternative to conventional methods to prevent and remove paraffin damage. Using such kind of bacterial products, exciting results for inhibiting paraffin depositions have been obtained. In this paper results concerning the naturally occurring bacteria selectively isolated from hydrocarbon polluted sites as well as from paraffinic oils, semi-solid and solid paraffin depositions are presented. After a laboratory screening, 15 bacterial strains (BS 1-15), three bacterial consortia (BC 1-3) and a Special Bacterial Consortium (SBC1) were selected. For the selection of bacterial consortia, the classical enrichment culture method has been used. The Special Bacterial Consortium resulted from a mixture of BS 1-15 and BC 1-3 following the steps of the classical enrichment culture method. The BS 1-15, BC 1-3 and SBC1 have been tested for their performances in producing biosurfactants and biosolvents as well as for hydrocarbon utilisation. The SBC1 has been tested for its ability in degradation of hydrocarbons contained in several types of paraffinic or non-paraffinic oils, and then for inhibiting paraffin deposition on a `flow equipment` using two types of paraffinic oils. The SBC1 has been also tested for degradation of hydrocarbons contained in semi-solid and solid paraffin depositions. The results obtained could support further applications to prevent and control paraffin depositions

  20. The use of naturally occurring selectively isolated bacteria for inhibiting paraffin deposition

    International Nuclear Information System (INIS)

    Lazar, I.; Voicu, A.; Dobrota, S.; Petrisor, I.G.; Stefanescu, M.; Sandulescu, L.; Nicolescu, C.; Mucenica, D.

    1999-01-01

    One of the most severe problems at any oil fields producing paraffinic oils is that of paraffin depositions. Romania which has a long experience in oil production is also faced with this problem in many oil fields. The microbial treatment, based on the activity of naturally occurring, selectively isolated bacteria, is already proved as an effective alternative to conventional methods to prevent and remove paraffin damage. Using such kind of bacterial products, exciting results for inhibiting paraffin depositions have been obtained. In this paper results concerning the naturally occurring bacteria selectively isolated from hydrocarbon polluted sites as well as from paraffinic oils, semi-solid and solid paraffin depositions are presented. After a laboratory screening, 15 bacterial strains (BS 1-15), three bacterial consortia (BC 1-3) and a Special Bacterial Consortium (SBC1) were selected. For the selection of bacterial consortia, the classical enrichment culture method has been used. The Special Bacterial Consortium resulted from a mixture of BS 1-15 and BC 1-3 following the steps of the classical enrichment culture method. The BS 1-15, BC 1-3 and SBC1 have been tested for their performances in producing biosurfactants and biosolvents as well as for hydrocarbon utilisation. The SBC1 has been tested for its ability in degradation of hydrocarbons contained in several types of paraffinic or non-paraffinic oils, and then for inhibiting paraffin deposition on a 'flow equipment' using two types of paraffinic oils. The SBC1 has been also tested for degradation of hydrocarbons contained in semi-solid and solid paraffin depositions. The results obtained could support further applications to prevent and control paraffin depositions

  1. A Local Composition Model for Paraffinic Solid Solutions

    DEFF Research Database (Denmark)

    Coutinho, A.P. João; Knudsen, Kim; Andersen, Simon Ivar

    1996-01-01

    The description of the solid-phase non-ideality remains the main obstacle in modelling the solid-liquid equilibrium of hydrocarbons. A theoretical model, based on the local composition concept, is developed for the orthorhombic phase of n-alkanes and tested against experimental data for binary sy...... systems. It is shown that it can adequately predict the experimental phase behaviour of paraffinic mixtures. This work extends the applicability of local composition models to the solid phase. Copyright (C) 1996 Elsevier Science Ltd....

  2. Integration of catalyst design and reactor engineering in paraffins dehydrogenation

    Energy Technology Data Exchange (ETDEWEB)

    Sanfilippo, D.; Miracca, I. [Snamprogetti S.p.A., S. Donato Milanese (Italy)

    2005-07-01

    Unfortunately, olefins are not a natural fossil resource. Their production requires sophisticated and costly technologies, highly demanding in terms of investments and energy. Dehydrogenations are applied industrially to light alkanes (propane to propylene for polymers and isobutane to iso-butylene for gasoline and polymers) as well as long linear ones (C{sub 10}-C{sub 14} to linear-alkyl-benzenes) and for the production of styrene from ethylbenzene. The light paraffins dehydrogenation sustains a network of technologies allowing an integrated approach to create value from Natural Gas. (orig.)

  3. The influence of expanded graphite on thermal properties for paraffin/high density polyethylene/chlorinated paraffin/antimony trioxide as a flame retardant phase change material

    International Nuclear Information System (INIS)

    Zhang Ping; Song Lei; Lu Hongdian; Wang Jian; Hu Yuan

    2010-01-01

    The influences of expanded graphite (EG) on the thermal properties of chlorinated paraffin (CP) and antimony trioxide (AT) on phase change material which bases on paraffin/high density polyethylene (HDPE) are studied. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), thermogravimetric analysis-Fourier transform infrared spectrometry (TGA-FTIR), microscale combustion calorimeter (MCC) and cone calorimeter (CONE) were used to evaluate the influence of EG on paraffin/HDPE/CP/AT system. The DSC results indicated that the latent heat value of PCM could be increased when the mass fraction of HDPE was decreased in the PCM, and EG could confine the molecular heat movement of paraffin. EG could improve the thermal stability and increase the char residue at high temperature for paraffin/HDPE/CP/AT hybrid. The volatilized products formed on thermal degradation of paraffin/HDPE/CP/AT with EG showed the release of CO 2 gas was hastened and increased, and the amount of combustible gases were decreased by TGA-FTIR analysis. The MCC and CONE results presented that the flame retardant efficiency of CP/AT could be improved by adding EG in paraffin/HDPE/CP/AT system.

  4. Efficiency of GC-MS method in detection of beeswax adulterated with paraffin

    Directory of Open Access Journals (Sweden)

    Waś Ewa

    2016-06-01

    Full Text Available The efficiency of the gas chromatography - mass detector (GC-MS technique for the detection of beeswax adulterated with paraffin, was evaluated. For this purpose, beeswax samples with paraffin additions (3, 5, 10, 30, 50% were analysed. Since not enough is known about paraffin compositions, and since it is difficult to detect paraffin in beeswax, the aim of our research was also to compare the hydrocarbon composition of different types of paraffin. The analysis showed that the types of paraffin available on the market, differ qualitatively and quantitatively as far as their hydrocarbon compositions are concerned. In all kinds of paraffin, we found homologous series of n-alkanes that were much longer than those in beeswax. In beeswax, the amount of added paraffin that is possible to detect, differs and depends on the kind of paraffin used for adulteration. In this study, the minimum estimated percent that was detected using the GC-MS technique, was 3%. The adulteration is indicated by the presence of hydrocarbons containing over 35 carbon atoms in the molecule, and by the higher contents of n-alkanes (C20H42 - C35H72, in comparison to the concentration of these compounds determined in pure beeswax. We also presented the results of the quality control of commercial beeswax. Based on our results, it can be stated that beeswax adulteration is currently a problem.

  5. Retrospective study of central nervous system lesions and association with Parelaphostrongylus species by histology and specific nested polymerase chain reaction in domestic camelids and wild ungulates.

    Science.gov (United States)

    Dobey, Carrie L; Grunenwald, Caroline; Newman, Shelley J; Muller, Lisa; Gerhold, Richard W

    2014-11-01

    Formalin-fixed, paraffin-embedded tissues from elk (Cervus elaphus), goats, and camelids with case histories and lesions suggestive of Parelaphostrongylus tenuis were examined by histology to characterize lesions that could aid in definitively diagnosing P. tenuis infection. Additionally, sections of paraffin-embedded tissue were used in a nested polymerase chain reaction (nPCR) using Parelaphostrongylus-specific primers to determine how PCR results corresponded with histological findings. Histological changes in brain and spinal cord consisted of linear tracks of hemorrhage; tracks or perivascular accumulations of hemosiderin-laden macrophages; acute foci of axonal degeneration and/or linear glial scars; and perivascular, parenchymal, or meningeal accumulations of eosinophils and/or lymphocytes and plasma cells. Of the 43 samples with histologic lesions consistent with neural larval migrans, 19 were PCR positive; however, only 8 were confirmed Parelaphostrongylus by DNA sequencing. Additionally, 1 goat was identified with a protostrongylid that had a 97% identity to both Parelaphostrongylus odocoilei and a protostrongylid nematode from pampas deer (Ozotoceros bezoarticus celer) from Argentina. None of the histologic lesions individually or in combination correlated statistically to positive molecular tests for the nematode. The results indicate that it is possible to extract Parelaphostrongylus DNA from formalin-fixed, paraffin-embedded tissue, but extended fixation presumably can cause DNA crosslinking. Nested PCR provides another diagnostic tool to identify the cause of neurologic disease in camelids and elk with histologic lesions consistent with neural larval migrans. Furthermore, potential novel protostrongylid DNA was detected from a goat with lesions consistent with P. tenuis infection, suggesting that other neurotropic Parelaphostrongylus species may occur locally. © 2014 The Author(s).

  6. Detection of p62 on Paraffin Sections by Immunohistochemistry.

    Science.gov (United States)

    Watson, Alexander S; Soilleux, Elizabeth J

    2015-08-03

    The study of autophagy in human disease is a rapidly expanding field. Diagnostic paraffin sections of a variety of patient tissues, including bone marrow, are available to researchers-yet are unsuitable for traditional autophagy quantification methods such as western blot or electron microscopy. This protocol outlines the immunohistochemical detection of the protein p62 (sequestosome-1, encoded by the gene SQSTM1)-an indicator of autophagic degradative activity-in slide-mounted paraffin sections such as bone marrow samples cut by a trephine. The p62 protein is an autophagic cargo adaptor, capable of binding to ubiquitylated proteins as well as autophagosome membrane proteins (LC3B and GABA(A) receptor-associated protein [GABARAP] family members) and hypothesized thus to target protein aggregates for lysosomal degradation. p62 itself is degraded by autophagy, remaining at low levels when autophagy is induced, and has been shown to accumulate when autophagy is deficient. Qualitative assessment and comparison of p62 staining between healthy and disease sections or disease subtypes will help target further investigation into the potential roles for autophagy in a variety of disorders. © 2015 Cold Spring Harbor Laboratory Press.

  7. Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma.

    Science.gov (United States)

    Feng, Zipei; Puri, Sachin; Moudgil, Tarsem; Wood, William; Hoyt, Clifford C; Wang, Chichung; Urba, Walter J; Curti, Brendan D; Bifulco, Carlo B; Fox, Bernard A

    2015-01-01

    Adoptive T cell therapy (ACT) has shown great promise in melanoma, with over 50 % response rate in patients where autologous tumor-reactive tumor-infiltrating lymphocytes (TIL) can be cultured and expanded. A major limitation of ACT is the inability to generate or expand autologous tumor-reactive TIL in 25-45 % of patients tested. Methods that successfully identify tumors that are not suitable for TIL generation by standard methods would eliminate the costs of fruitless expansion and enable these patients to receive alternate therapy immediately. Multispectral fluorescent immunohistochemistry with a panel including CD3, CD8, FoxP3, CD163, PD-L1 was used to analyze the tumor microenvironment in 17 patients with melanoma among our 36-patient cohort to predict successful TIL generation. Additionally, we compared tumor fragments and enzymatic digestion of tumor samples for efficiency in generating tumor-reactive TIL. Tumor-reactive TIL were generated from 21/36 (58 %) of melanomas and for 12/13 (92 %) tumors where both enzymatic and fragment methods were compared. TIL generation was successful in 10/13 enzymatic preparations and in 10/13 fragment cultures; combination of both methods resulted in successful generation of autologous tumor-reactive TIL in 12/13 patients. In 17 patients for whom tissue blocks were available, IHC analysis identified that while the presence of CD8(+) T cells alone was insufficient to predict successful TIL generation, the CD8(+) to FoxP3(+) ratio was predictive with a positive-predictive value (PPV) of 91 % and negative-predictive value (NPV) of 86 %. Incorporation of CD163+ macrophage numbers and CD8:PD-L1 ratio did not improve the PPV. However, the NPV could be improved to 100 % by including the ratio of CD8(+):PD-L1(+) expressing cells. This is the first study to apply 7-color multispectral immunohistochemistry to analyze the immune environment of tumors from patients with melanoma. Assessment of the data using unsupervised hierarchical clustering identified tumors from which we were unable to generate TIL. If substantiated, this immune profile could be applied to select patients for TIL generation. Additionally, this biomarker profile may also indicate a pre-existing immune response, and serve as a predictive biomarker of patients who will respond to checkpoint blockade. We postulate that expanding the spectrum of inhibitory cells and molecules assessed using this technique could guide combination immunotherapy treatments and improve response rates.

  8. Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue

    DEFF Research Database (Denmark)

    Gilbert, Marcus T P; Sanchez, Juan J; Haselkorn, Tamara

    2007-01-01

    , multiplex PCR with minisequencing (MPMS), on 92 DNA extractions performed on six archival FFPE samples of variable DNA quality, which date between 9 and 25 years old. On the three extracts with highest quality, we found the assay efficiency to be near 100%. However, the efficiency of the lowest quality...... extracts varied significantly. In this study, we demonstrate that although direct measures of DNA concentration in the extracts provide no useful information with regard to subsequent MPMS success, the success of the assay can be determined to some degree a priori, through initial screening of the DNA...... quality using a simple quantitative real-time PCR (qPCR) assay for nuclear DNA, and/or an assay of the maximum PCR amplifiable size of nuclear DNA. MPMS promises to be of significant use in future genetic studies on FFPE material. It provides a streamlined approach for retrieving a large amount of genetic...

  9. Smart multicore embedded systems

    CERN Document Server

    Bertels, Koen; Karlsson, Sven; Pacull, François

    2014-01-01

    This book provides a single-source reference to the state-of-the-art of high-level programming models and compilation tool-chains for embedded system platforms. The authors address challenges faced by programmers developing software to implement parallel applications in embedded systems, where very often they are forced to rewrite sequential programs into parallel software, taking into account all the low level features and peculiarities of the underlying platforms. Readers will benefit from these authors’ approach, which takes into account both the application requirements and the platform specificities of various embedded systems from different industries. Parallel programming tool-chains are described that take as input parameters both the application and the platform model, then determine relevant transformations and mapping decisions on the concrete platform, minimizing user intervention and hiding the difficulties related to the correct and efficient use of memory hierarchy and low level code generati...

  10. Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin.

    Science.gov (United States)

    Prestes, Suzane Ribeiro; Guerra, Jorge Augusto de Oliveira; Romero, Gustavo Adolfo Sierra; Magalhaes, Laylah Kelre Costa; Santana, Rosa Amelia Gonçalves; Maciel, Marcel Gonçalves; Custódio, Ana; Barbosa, Maria das Graças Vale; Silveira, Henrique

    2015-01-01

    In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species. We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses. Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis. The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

  11. An immunohistochemical study of Flexibacter psychrophilus infection in experimentally and naturally infected rainbow trout (Oncorhynchus mykiss) fry

    DEFF Research Database (Denmark)

    Evensen, O.; Lorenzen, Ellen

    1996-01-01

    An immunohistochemical method is described for the detection of Flexibacter psychrophilus in formalin-fixed, parafiin-wax-embedded fry of rainbow trout. Rabbit antiserum as well as rainbow trout hyperimmune serum were used in the study. The distribution and tissue localization of the bacterium...... was compared in naturally and experimentally (intraperitoneal injections) infected fry by use of immunohistochemistry. This study showed that F. psychrophilus could be detected in paraffin-wax-embedded tissue of rainbow trout fry by immunohistochemistry. The principal immunohistochemical findings in naturally...... and experimentally infected fry showed that there was a localization of bacteria in the monocyte-macrophage system, in skin lesions, and in the retina and the choroid gland of the eye. The dermal changes included superficial or deep ulcers extending to the subcutaneous tissue or the musculature accompanied...

  12. Sealing-free fast-response paraffin/nanoporous gold hybrid actuator

    Science.gov (United States)

    Ye, Xing-Long; Jin, Hai-Jun

    2017-09-01

    Paraffin-based actuators can deliver large actuation strokes and high actuation stress, but often suffer from a low response rate and leaking problems. Here, we report a new paraffin/metal hybrid actuator, which was fabricated by infiltrating nanoporous gold with paraffin. It exhibits a fast actuation rate owing to the high thermal conductivity of the inter-connected metal phase, and requires no external sealing because liquid paraffin can be well confined in nanoscale channels, due to the large capillarity. We found that in this hybrid actuator, the stress generated by actuation is negligibly small when the characteristic size of the nanoporous gold (L) is above ˜70 nm, and increases dramatically with a decreasing size when L paraffin wax—the paraffin in smaller pores can sustain larger tensile stress, and thus the contraction of paraffin during cooling can be translated into larger compression stress and strain energy in a metal framework, leading to a larger actuation stress and energy. We also demonstrate that complex actuation motions can be achieved by incorporating hierarchical-structured nanoporous metal with paraffin.

  13. Paraffin molecule mobility in channel clathrates of urea on spectroscopic NMR relaxation data

    CERN Document Server

    Kriger, Y G; Chekhova, G N

    2001-01-01

    The temperature dependences of the protons spin-lattice relaxation time (T sub I) in the channel clathrates of urea with paraffins are measured. The data on the T sub I are interpreted within the frames of the model of the paraffins molecules and their fragments orientation in the clathrate channels. The dynamics peculiarities are connected with the disproportion effects of these compounds

  14. Corrugated paraffin nanocomposite films as large stroke thermal actuators and self-activating thermal interfaces.

    Science.gov (United States)

    Copic, Davor; Hart, A John

    2015-04-22

    High performance active materials are of rapidly growing interest for applications including soft robotics, microfluidic systems, and morphing composites. In particular, paraffin wax has been used to actuate miniature pumps, solenoid valves, and composite fibers, yet its deployment is typically limited by the need for external volume constraint. We demonstrate that compact, high-performance paraffin actuators can be made by confining paraffin within vertically aligned carbon nanotube (CNT) films. This large-stroke vertical actuation is enabled by strong capillary interaction between paraffin and CNTs and by engineering the CNT morphology by mechanical compression before capillary-driven infiltration of the molten paraffin. The maximum actuation strain of the corrugated CNT-paraffin films (∼0.02-0.2) is comparable to natural muscle, yet the maximum stress is limited to ∼10 kPa by collapse of the CNT network. We also show how a CNT-paraffin film can serve as a self-activating thermal interface that closes a gap when it is heated. These new CNT-paraffin film actuators could be produced by large-area CNT growth, infiltration, and lamination methods, and are attractive for use in miniature systems due to their self-contained design.

  15. Enhanced thermal properties with graphene oxide in the urea-formaldehyde microcapsules containing paraffin PCMs.

    Science.gov (United States)

    Qiao, Zhen; Mao, Jian

    2017-02-01

    In this study, compact urea-formaldehyde microcapsules containing paraffin (UFP) phase change materials (PCMs) were prepared via in situ polymerisation. The thermal conductivity of the PCMs was enhanced without influencing their enthalpy by adding graphene oxide (GO). Two modification methods were investigated: One in which GO is added to the inside of microcapsules, defined as "paraffin/GO@UF composite"; and another in which GO is coated onto the surface of shell, defined as "paraffin@UF/GO composite". The GO sheets were visible in scanning electron microscope (SEM) images of paraffin@UF/GO composite. The thermal conductivity was 0.2236 ± 0.0003 W/(m·K) for UFP particles, was 0.2517 ± 0.0003 W/(m·K) for the paraffin/GO@UF composite (10 wt%), and was 1.0670 ± 0.0020 W/(m·K) for paraffin@UF/GO composite (10 wt%), respectively. The encapsulation efficiency of all samples exceeded 80% (w/w) and all samples exhibited favourable thermal stability and reliability. The IR emissivity of paraffin@UF/GO was lower than that of paraffin/GO@UF when the same GO amount was added to the composite.

  16. High paraffin Kumkol petroleum processing under fuel and lubricant petroleum scheme

    International Nuclear Information System (INIS)

    Nadirov, N.K.; Konaev, Eh.N.

    1997-01-01

    Technological opportunity of high paraffin Kumkol petroleum processing under the fuel and lubricant scheme with production of lubricant materials in short supply, combustible materials and technical paraffin is shown. Mini petroleum block putting into operation on Kumkol deposit is reasonable economically and raises profitableness of hydrocarbon raw material production. (author)

  17. Effects of Chlorinated Paraffin and ZDDP Concentrations on Boundary Lubrication Properties of Mineral and Soybean Oils

    Science.gov (United States)

    The effect of chlorinated paraffin (CP) and zinc di-ethylhexyl dithio phosphate (ZDDP) concentration in polar and non-polar base fluids on boundary lubrication properties was investigated. The non-polar fluid was a solvent refined low sulfur heavy paraffinic mineral oil (150N oil); and the polar fl...

  18. Embedded-monolith armor

    Science.gov (United States)

    McElfresh, Michael W.; Groves, Scott E; Moffet, Mitchell L.; Martin, Louis P.

    2016-07-19

    A lightweight armor system utilizing a face section having a multiplicity of monoliths embedded in a matrix supported on low density foam. The face section is supported with a strong stiff backing plate. The backing plate is mounted on a spall plate.

  19. Isometric embeddings of polyhedra

    Science.gov (United States)

    Minemyer, Barry

    An indefinite metric polyhedron is a triple (X, T, g) where X is a topological space, T is a simplicial triangulation of X with edge set E, and g is a function from E to the reals. g assigns to each k-dimensional simplex S a unique quadratic form on Rk, denoted by G(S). An indefinite metric polyhedron is called a Euclidean polyhedron if the form G(S) is positive definite for every simplex S. Rpq denotes R p + q endowed with the inner product of signature (p, q). Our first result is that every compact n-dimensional indefinite metric polyhedron with vertex set V admits a simplicial isometric embedding into Rqq where q = max{d, 2n + 1} and d = max{deg(v) | v is in V}. We can use the compact case to extend to the non-compact case, but only if we assume that d = max{deg(v) | v is in V} is less than infinity. Specifically, every (non-compact) indefinite metric polyhedron admits a simplicial isometric embedding into Rpp where p = 2q(d3 - d2 + d + 1) and q and d are defined as above. Finally we use results of Akopyan and Greene to prove that every n-dimensional indefinite metric polyhedron admits a piecewise linear isometric embedding into Rn2n. In Chapter 2 we prove that every short (1-Lipschitz) map from an n-dimensional Euclidean polyhedron into EN is epsilon close to a pl isometric embedding (for anyepsilon > 0) provided N ≥ 3n. We can relax the dimensionality of the Euclidean space to 2n + 1 if we allow our map to be continuous instead of pl. These results are extensions of a result due to Akopyan. We provide a detailed proof of Akopyan's Theorem, as the only currently available proof is in Russian. The remaining results in this work are applications of our continuous isometric embedding theorem above. This result is used to prove that every Pro-Euclidean space of rank at most n admits an isometric embedding into E2n + 1. The result, as well as a theorem due to Bridson, also allows for an approximate isometric embedding theorem for geodesic metric spaces with

  20. Experimental study on heat capacity of paraffin/water phase change emulsion

    International Nuclear Information System (INIS)

    Huang, L.; Noeres, P.; Petermann, M.; Doetsch, C.

    2010-01-01

    A paraffin/water phase change emulsion is a multifunctional fluid in which fine paraffin droplets are dispersed in water by a surfactant. This paper presents an experimental study on the heat capacity of an emulsion containing 30 wt.% paraffin in a test rig. The results show that the heat capacity of the emulsion consists of the sensible heat capacity of water and that of the paraffin as well as the latent heat capacity of the paraffin during the phase transition solid-liquid. The emulsion is an attractive alternative to chilled water for comfort cooling applications, because it has a heat capacity of 50 kJ/kg from 5 to 11 deg. C, which is two times as high as that of water in the same temperature range.

  1. Exogenous lipoid pneumonia caused by paraffin in an amateur fire breather.

    Science.gov (United States)

    Weinberg, I; Fridlender, Z G

    2010-05-01

    Paraffin has characteristics that make it popular among fire breathers. To describe a case of paraffin-induced lipoid pneumonia in a fire breather. The patient was evaluated clinically in relation to his occupational history. A 32-year-old man presented with dyspnoea, tachypnoea and non-productive cough of 2 h duration that started immediately following an attempt to blow fire using paraffin as the volatile substance. He was discharged from the emergency ward but returned the next day presenting again with dyspnoea accompanied by mid-sternal pain, fever (38.1 degrees C) and leucocytosis. Chest radiography showed perihilar punctuate infiltrations. A diagnosis of exogenous lipoid pneumonia caused by paraffin was made, and the patient was treated, with full recovery within a week. Fire breathers must be viewed as a population at risk of paraffin-induced lipoid pneumonia.

  2. Paraffin as oxygen vector modulates tyrosine phenol lyase production by Citrobacter freundii MTCC 2424.

    Science.gov (United States)

    Azmi, Wamik; Kumar, Ajay; Dev, Varun

    2013-06-01

    The efficiency of three oxygen-vectors liquid paraffin, silicone oil and n-dodecane in the production of tyrosine phenol lyase (TPL) by Citrobacter freundii MTCC 2424 was evaluated at 4% (v/v) concentration. The liquid paraffin as oxygenvectors was found to exhibit a stimulatory effect on TPL synthesis. The liquid paraffin at 6% (v/v) resulted in 34% increase in the TPL synthesis accompanied by a 13% increase in the production of cell mass at a 10 L scale. This improvement in TPL and cell mass production in the presence of liquid paraffin can be related to the fact that liquid paraffin was capable of maintaining dissolved O2 concentration above 28% throughout the course of the fermentation. Maintenance of the dissolved O2 concentration above 28% could be viewed in terms of an adequate oxygen supply to the rapidly dividing cells of the bacterium, which in turn resulted in enhanced synthesis of TPL and cell mass.

  3. Carbon nanotube/paraffin/montmorillonite composite phase change material for thermal energy storage.

    Science.gov (United States)

    Li, Min; Guo, Qiangang; Nutt, Steven

    2017-04-01

    A composite phase change material (PCM) comprised of organic montmorillonite (OMMT)/paraffin/grafted multi-walled nanotube (MWNT) is synthesized via ultrasonic dispersion and liquid intercalation. The microstructure of the composite PCM has been characterized to determine the phase distribution, and thermal properties (latent heat and thermal conductivity) have been measured by differential scanning calorimetry (DSC) and a thermal constant analyzer. The results show that paraffin molecules are intercalated in the montmorillonite layers and the grafted MWNTs are dispersed in the montmorillonite layers. The latent heat is 47.1 J/g, and the thermal conductivity of the OMMT/paraffin/grafted MWNT composites is 34% higher than that of the OMMT/paraffin composites and 65% higher than that of paraffin.

  4. Preparation and thermal properties of form stable paraffin phase change material encapsulation

    International Nuclear Information System (INIS)

    Liu Xing; Liu Hongyan; Wang Shujun; Zhang Lu; Cheng Hua

    2006-01-01

    Paraffin waxes are cheap and have moderate thermal energy storage density but low thermal conductivity and, hence, require large surface area to be used in energy storage. Form stable paraffin phase change materials (PCM) in which paraffin serves as a latent heat storage material and polyolefins act as a supporting material, because of paraffin leakage, are required to be improved. The form stable paraffin PCM in the present paper was encapsulated in an inorganic silica gel polymer successfully by in situ polymerization. The differential scanning calorimeter (DSC) was used to measure its thermal properties. At the same time, the Washburn equation, which measures the wetting properties of powder materials, was used to test the hydrophilic-lipophilic properties of the PCMs. The result indicated that the enthalpy of the microencapsulated PCMs was reduced little, while their hydrophilic properties were enhanced largely

  5. THERMAL ENERGY STORAGE PROPERTIES OF FORMSTABLE PARAFFIN/RECYCLE BLOCK CONCRETE COMPOSITE PHASE CHANGE MATERIAL

    Directory of Open Access Journals (Sweden)

    PATTARAPORN SUTTAPHAKDEE

    2017-01-01

    Full Text Available In this research, the form-stable composite phase change material was developed by incorporating paraffin on recycle block concrete (RB through the vacuum impregnation method. The compatibility and thermal properties of RB impregnated with paraffin ranging from 0-35 wt% were characterized by Fourier transform infrared spectroscopy (FTIR and differential scanning calorimetry (DSC. Results revealed that paraffin was uniformly absorbed in RB with a good physical compatibility. The optimum adsorption ratio of paraffin in RB was 25 wt% which produced phase transition temperature of 52.85 OC and latent heat of 30.98 J/g. The obtained form-stable paraffin/RB composite PCM had proper latent heat and phase transition temperature and can be applied for thermal energy storage applications such as solar heating and cooling in buildings.

  6. Standard Review Risk Assessment on Medium-chain and Long-chain Chlorinated paraffin PMN submissions by INEOS Chlor Americas

    Science.gov (United States)

    This assessment was conducted under EPA’s TSCA Section 5 New Chemicals Program. EPA is assessing Medium-chain Chlorinated Paraffin (MCCP) and Long-Chain Chlorinated Paraffin (LCCP) chemicals as part of its New Chemicals Review program.

  7. Standard Review Risk Assessment on Medium-chain and Long-chain Chlorinated paraffin PMN submissions by Dover Chemical

    Science.gov (United States)

    This assessment was conducted under EPA’s TSCA Section 5 New Chemicals Program. EPA is assessing Medium-chain Chlorinated Paraffin (MCCP) and Long-Chain Chlorinated Paraffin (LCCP) chemicals as part of its New Chemicals Review program.

  8. Standard Review Risk Assessment on Medium-chain and Long-chain Chlorinated paraffin PMN submissions by Qualice, LLC

    Science.gov (United States)

    This assessment was conducted under EPA’s TSCA Section 5 New Chemicals Program. EPA is assessing Medium-chain Chlorinated Paraffin (MCCP) and Long-Chain Chlorinated Paraffin (LCCP) chemicals as part of its New Chemicals Review program.

  9. Ionic liquids for separation of olefin-paraffin mixtures

    Science.gov (United States)

    Dai, Sheng; Luo, Huimin; Huang, Jing-Fang

    2013-09-17

    The invention is directed to an ionic liquid comprising (i) a cationic portion containing a complex of a silver (I) ion and one or more neutral ligands selected from organoamides, organoamines, olefins, and organonitriles, and (ii) an anionic portion having the chemical formula ##STR00001## wherein m and n are independently 0 or an integer of 1 or above, and p is 0 or 1, provided that when p is 0, the group --N--SO.sub.2--(CF.sub.2).sub.nCF.sub.3 subtended by p is replaced with an oxide atom connected to the shown sulfur atom. The invention is also directed to a method for separating an olefin from an olefin-paraffin mixture by passing the mixture through a layer of the ionic liquid described above.

  10. Round-Robin Test of Paraffin Phase-Change Material

    Science.gov (United States)

    Vidi, S.; Mehling, H.; Hemberger, F.; Haussmann, Th.; Laube, A.

    2015-11-01

    A round-robin test between three institutes was performed on a paraffin phase-change material (PCM) in the context of the German quality association for phase-change materials. The aim of the quality association is to define quality and test specifications for PCMs and to award certificates for successfully tested materials. To ensure the reproducibility and comparability of the measurements performed at different institutes using different measuring methods, a round-robin test was performed. The sample was unknown. The four methods used by the three participating institutes in the round-robin test were differential scanning calorimetry, Calvet calorimetry and three-layer calorimetry. Additionally, T-history measurements were made. The aim of the measurements was the determination of the enthalpy as a function of temperature. The results achieved following defined test specifications are in excellent agreement.

  11. Interaction study of polyisobutylene with paraffins by NMR using the evaluation of spin-lattice relaxation times for hydrogen nuclei

    International Nuclear Information System (INIS)

    Marques, Rosana G.G.; Tavares, Maria I.B.

    2001-01-01

    The evaluation of spin-lattice relaxation times of 1 H for polyisobutylene/paraffin systems, were obtained using the classic inversion recovery technique, and also through Cross Polarization Magic Angle Spinning (CP/MAS) techniques varying the contact time and also by the delayed contact time pulse sequence. NMR results showed that the polyisobutylene/paraffin systems in which high molecular weight paraffins were used, is heterogeneous. However, for paraffins with low molecular weight, the system presents good homogeneity. (author)

  12. Effect of sample preparation techniques on the concentrations and distributions of elements in biological tissues using µSRXRF: a comparative study

    International Nuclear Information System (INIS)

    Al-Ebraheem, A; Dao, E; Desouza, E; McNeill, F E; Farquharson, M J; Li, C; Wainman, B C

    2015-01-01

    Routine tissue sample preparation using chemical fixatives is known to preserve the morphology of the tissue being studied. A competitive method, cryofixation followed by freeze drying, involves no chemical agents and maintains the biological function of the tissue. The possible effects of both sample preparation techniques in terms of the distribution of bio-metals (calcium (Ca), copper (Cu) zinc (Zn), and iron (Fe) specifically) in human skin tissue samples was investigated. Micro synchrotron radiation x-ray fluorescence (μSRXRF) was used to map bio-metal distribution in epidermal and dermal layers of human skin samples from various locations of the body that have been prepared using both techniques. For Ca, Cu and Zn, there were statistically significant differences between the epidermis and dermis using the freeze drying technique (p = 0.02, p < 0.01, and p < 0.01, respectively). Also using the formalin fixed, paraffin embedded technique the levels of Ca, Cu and Zn, were significantly different between the epidermis and dermis layers (p = 0.03, p < 0.01, and p < 0.01, respectively). However, the difference in levels of Fe between the epidermis and dermis was unclear and further analysis was required. The epidermis was further divided into two sub-layers, one mainly composed of the stratum corneum and the other deeper layer, the stratum basale. It was found that the difference between the distribution of Fe in the two epidermal layers using the freeze drying technique resulted in a statistically significant difference (p = 0.012). This same region also showed a difference in Fe using the formalin fixed, paraffin embedded technique (p < 0.01). The formalin fixed, paraffin embedded technique also showed a difference between the deeper epidermal layer and the dermis (p < 0.01). It can be concluded that studies involving Ca, Cu and Zn might show similar results using both sample preparation techniques, however studies involving Fe would need more

  13. Images of paraffin monolayer crystals with perfect contrast: minimization of beam-induced specimen motion

    Science.gov (United States)

    Glaeser, R.M.; McMullan, G.; Faruqi, A.R.; Henderson, R.

    2013-01-01

    Quantitative analysis of electron microscope images of organic and biological two-dimensional crystals has previously shown that the absolute contrast reached only a fraction of that expected theoretically from the electron diffraction amplitudes. The accepted explanation for this is that irradiation of the specimen causes beam-induced charging or movement, which in turn causes blurring of the image due to image or specimen movement. In this paper, we used three different approaches to try to overcome this image-blurring problem for monolayer crystals of paraffin. Our first approach was to use an extreme form of spotscan imaging, in which a single image was assembled on film by the successive illumination of up to 50,000 spots each of diameter around 7nm. The second approach was to use the Medipix II detector with its zero-noise readout to assemble a time-sliced series of images of the same area in which each frame from a movie with up to 400 frames had an exposure of only 500 electrons. In the third approach, we simply used a much thicker carbon support film to increase the physical strength and conductivity of the support. Surprisingly, the first two methods involving dose fractionation respectively in space or time produced only partial improvements in contrast whereas the third approach produced many virtually perfect images, in which the absolute contrast predicted from the electron diffraction amplitudes was observed in the images. We conclude that it is possible to obtain consistently almost perfect images of beam-sensitive specimens if they are attached to an appropriately strong and conductive support, but great care is needed in practice and the problem of how best to image ice-embedded biological structures in the absence of a strong, conductive support film requires more work. PMID:21185452

  14. Effect of fixation on brain and lymphoreticular vCJD prions and bioassay of key positive specimens from a retrospective vCJD prevalence study.

    Science.gov (United States)

    Wadsworth, Jonathan D F; Dalmau-Mena, Inmaculada; Joiner, Susan; Linehan, Jacqueline M; O'Malley, Catherine; Powell, Caroline; Brandner, Sebastian; Asante, Emmanuel A; Ironside, James W; Hilton, David A; Collinge, John

    2011-03-01

    Anonymous screening of lymphoreticular tissues removed during routine surgery has been applied to estimate the UK population prevalence of asymptomatic vCJD prion infection. The retrospective study of Hilton et al (J Pathol 2004; 203: 733-739) found accumulation of abnormal prion protein in three formalin-fixed appendix specimens. This led to an estimated UK prevalence of vCJD infection of ∼1 in 4000, which remains the key evidence supporting current risk reduction measures to reduce iatrogenic transmission of vCJD prions in the UK. Confirmatory testing of these positives has been hampered by the inability to perform immunoblotting of formalin-fixed tissue. Animal transmission studies offer the potential for 'gold standard' confirmatory testing but are limited by both transmission barrier effects and known effects of fixation on scrapie prion titre in experimental models. Here we report the effects of fixation on brain and lymphoreticular human vCJD prions and comparative bioassay of two of the three prevalence study formalin-fixed, paraffin-embedded (FFPE) appendix specimens using transgenic mice expressing human prion protein (PrP). While transgenic mice expressing human PrP 129M readily reported vCJD prion infection after inoculation with frozen vCJD brain or appendix, and also FFPE vCJD brain, no infectivity was detected in FFPE vCJD spleen. No prion transmission was observed from either of the FFPE appendix specimens. The absence of detectable infectivity in fixed, known positive vCJD lymphoreticular tissue precludes interpreting negative transmissions from vCJD prevalence study appendix specimens. In this context, the Hilton et al study should continue to inform risk assessment pending the outcome of larger-scale studies on discarded surgical tissues and autopsy samples.

  15. Preparation and properties of polystyrene encapsulated paraffin wax as possible phase change material in a polypropylene matrix

    Energy Technology Data Exchange (ETDEWEB)

    Mochane, M.J. [Department of Chemistry, University of the Free State (Qwaqwa Campus), Phuthaditjhaba (South Africa); Luyt, A.S., E-mail: LuytAS@qwa.ufs.ac.za [Department of Chemistry, University of the Free State (Qwaqwa Campus), Phuthaditjhaba (South Africa)

    2012-09-20

    Highlights: Black-Right-Pointing-Pointer Polystyrene microcapsules containing about 30 wt% soft paraffin wax were successfully prepared. Black-Right-Pointing-Pointer The presence of microcapsules in polypropylene influenced the morphology and properties of the matrix. Black-Right-Pointing-Pointer The SEBS modifier had little influence on the interaction between polypropylene and microcapsules. - Abstract: The study deals with the preparation and characterization of polystyrene (PS) capsules containing Fischer-Tropsch paraffin wax (PS:wax) as phase change material (PCM) for thermal energy storage embedded in a polypropylene (PP) matrix. Blends of PP/PS:wax were prepared without and with polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene (SEBS) as a modifier. The influence of PS:wax microcapsules on the morphology, as well as thermal and mechanical properties of the PP was investigated. The scanning electron microscopy (SEM) images of the microencapsulated PCM show that the capsules were grouped in irregular spherical agglomerates of size 16-24 {mu}m. However, after melt-blending with PP smaller, perfectly spherical microcapsules were well dispersed in the PP matrix. There was fairly good interaction between the microcapsules and the matrix, even in the absence of SEBS modification. The FTIR spectrum of the microcapsules is almost exactly the same as that of polystyrene, which indicates that the microcapsules were mostly intact and that the FTIR only detected the polystyrene shell. The amount of wax in the PS:wax microcapsules was determined as 20-30% by weight. An increase in PS:wax content resulted in a decrease in the melting peak temperatures of PP. The thermal stability of the blends decreased with an increase in PS:wax microcapsules content as a consequence of the lower thermal stability of both the wax and PS. There was a drop in storage modulus with increasing PS:wax microcapsules content.

  16. Diagnostic accuracy of morphologic identification of filamentous fungi in paraffin embedded tissue sections: Correlation of histological and culture diagnosis

    Directory of Open Access Journals (Sweden)

    Sundaram Challa

    2014-01-01

    Full Text Available Aims and Objectives: The aim was to investigate the correlation between histological and culture diagnosis of filamentous fungi. Materials and Methods: Tissue sections from biopsy samples stained with Hematoxylin and Eosin and special stains from samples of chronic invasive/noninvasive sinusitis and intracranial space occupying lesions during 2005-2011 diagnosed to have infection due to filamentous fungi were reviewed. The histopathology and culture diagnoses were analyzed for correlation and discrepancy. Results: There were 125 samples positive for filamentous fungi on biopsy. Of these 76 (60.8% were submitted for culture and fungi grew in 30 (39.97% samples. There was a positive correlation between histological and culture diagnosis in 25 (83.33% samples that included Aspergillus species (16/19, Zygomycetes species (8/10 and dematiaceous fungi (1/1. The negative yield of fungi was more in Zygomycetes species (20/30 when compared to Aspergillus species (25/44. There was a discrepancy in diagnosis in 5/30 (16.67% samples which included probable dual infection in two, and dematiaceous fungi being interpreted as Aspergillus species in three samples. Conclusion: Histopathology plays a major role in the diagnosis of infection due to filamentous fungi, especially when cultures are not submitted or negative. The discrepancy between histological and culture diagnosis was either due to dematiaceous fungi being interpreted as Aspergillus species or probable dual infection.

  17. The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Haselkorn, Tamara; Bunce, Michael

    2007-01-01

    Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules...

  18. Selective inhibition of paraffin deposition under high flow rate as a function of the crude oil paraffin type and content by fluorescence depolarization: polypropylene and high-density polyethylene.

    Science.gov (United States)

    Quintella, Cristina M; Lima, Angelo M V; Silva, Eduardo B

    2006-04-13

    High-density polyethylene (HDPE) and polypropylene (PP) were studied to identify tailor-made materials for walls of pipelines and ducts for crude paraffinic oil that inhibit paraffin depositions. The interfacial interaction was investigated from 50 to 70 degrees C and as a function of the n-C(36)H(74) paraffin concentration added to the paraffinic crude oil. The static and the dynamic interfacial tensions were observed, respectively, by traditional contact angle measurements and by fluorescence depolarization of the natural fluorescent probes of the crude oil, flowing at a high rate. The static interaction showed a low dependence on the n-paraffin content for both surfaces. For PP, it decreased slightly as the n-paraffin concentration increased, evidencing an increase in the liquid-liquid interaction. The dynamic interfacial tension with PP clearly decreased as temperature and n-paraffin concentration increased, the latter effect being attributed to the PP methyl groups hindering the interaction between the n-paraffin and the PP main chain. For the flow on HDPE, the interaction proved to be highly dependent on the n-paraffin concentration and temperature. It increased as n-paraffin was added and temperature decreased. The former effect is attributed to the alignment of the n-paraffin chains within the high rate flowing liquid and the similar molecular geometry of the n-paraffin and the linear polymer surface (almost without branches), which increases the number of sites available for interaction. PP proved to be more suitable for transportation of crude oil rich in paraffins with more than 36 carbon atoms, while HDPE was more suitable for those with smaller paraffinic chains.

  19. Embedded software verification and debugging

    CERN Document Server

    Winterholer, Markus

    2017-01-01

    This book provides comprehensive coverage of verification and debugging techniques for embedded software, which is frequently used in safety critical applications (e.g., automotive), where failures are unacceptable. Since the verification of complex systems needs to encompass the verification of both hardware and embedded software modules, this book focuses on verification and debugging approaches for embedded software with hardware dependencies. Coverage includes the entire flow of design, verification and debugging of embedded software and all key approaches to debugging, dynamic, static, and hybrid verification. This book discusses the current, industrial embedded software verification flow, as well as emerging trends with focus on formal and hybrid verification and debugging approaches. Includes in a single source the entire flow of design, verification and debugging of embedded software; Addresses the main techniques that are currently being used in the industry for assuring the quality of embedded softw...

  20. Current status and future potential of somatic mutation testing from circulating free DNA in patients with solid tumours.

    Science.gov (United States)

    Aung, K L; Board, R E; Ellison, G; Donald, E; Ward, T; Clack, G; Ranson, M; Hughes, A; Newman, W; Dive, C

    2010-12-01

    Genetic alterations can determine the natural history of cancer and its treatment response. With further advances in DNA sequencing technology, multiple novel genetic alterations will be discovered which could be exploited as prognostic, predictive and pharmacodynamic biomarkers in the development and use of cancer therapeutics. As such, the importance in clinical practice of efficient and robust somatic mutation testing in solid tumours cannot be overemphasized in the current era of personalized medicine. However, significant challenges remain regarding the testing of genetic biomarkers in clinical practice. Reliance on archived formalin fixed, paraffin embedded tumour, obtained from diagnostic biopsies, for testing somatic genetic alterations could restrict the scientific community in asking relevant questions about a patient's cancer biology. Problems inherent with using formalin fixed, archival tissue are well recognized and difficult to resolve. It could be argued that to achieve rapid and efficient incorporation of genetic biomarkers into clinical practice, somatic mutation testing in cancer patients should be simpler, less invasive using a readily available clinical sample, whilst maintaining robustness and reproducibility. In this regard, use of circulating free DNA (cfDNA) from plasma or serum as an alternative and/or additional source of DNA to test cancer specific genetic alterations is an attractive proposition. In light of encouraging results from recent studies, this mini review will discuss the current role and future potential of somatic mutation testing from circulating or cell free DNA derived from the blood of patients with solid tumours.