Removal Of Paraffin Research Articles

Experimental and numerical investigation on erosion of circular and elliptical immersed tubes in fluidized bed

The erosion of immersed tube is studied by experimental and simulation methods. The results of the ink removal experiment show that the severe erosion is located on both sides of the lower part of the immersed tube. The dense discrete phase model (DDPM) method is adopted to simulate the erosion law of the immersed tube. The results show that the variation law of erosion rate with operating parameters is consistent with that of χ with operating parameters. A new dimensionless parameter of χ is defined to characterize the erosion state of immersed tube, which represents the ratio of particle flow characteristics to particle fluidization characteristics. Particle flow characteristics ( M p ) and particle fluidization degree ( M g ) are directly and inversely proportional to erosion rate respectively. In addition, the erosion rate of EA type elliptical immersed tube is proved to be 53.68% lower than that of circular immersed tube, which is obtained from the simulation results. • The analysis on oily ink removal of the erodible paraffin immersed tube is carried out. • The erosion laws of immersed tubes are simulated by DDPM method. • A new dimensionless parameter is defined to analyze the erosion of immersed tube. • From the numerical simulation, the erosion rate of EA type elliptical immersed tube is 53% lower than that of the circular tube.

Abstract 2039: Confident analyses of proteins in FFPE tissue: Robust, reliable, reproducible sample preparation enables LC-MS based proteomics assays

Abstract Formalin-fixed paraffin-embedded (FFPE) tissue samples are routinely used in clinical histopathology. While FFPE preserves tissue architecture, removal of paraffin is an essential to achieve high quality LC-MS data. Xylol, despite all its efficacy presents severe health risks and xylol removal compromises assay reproducibility. Superior sample preparation ensures removal of unwanted matrix components and provides reliability, reproducibility and robustness of the assays. This is achieved by an optimal combination of lysis by Adaptive Focused Acoustics (AFA®) with a modified protein aggregation capture (PAC) protocol. This method offers robust, reliable, and reproducible separation of paraffin from the tissue and ensures efficient purification of protein. FFPE scrolls were used in a Covaris plate, incubated post-treatment with Tissue Lysis Buffer, and processed with the Covaris LE220-Plus Focused-ultrasonicator for deparaffinization and DNA shearing. PAC protocol was used to extract from FFPE lysate with additional washing steps to ensure paraffin removal. Sera Mag Speed beads (bead:protein 1:5) were used to induce on-bead protein aggregation followed by washing and resuspension in 100 mM Tris-HCl (pH ~ 8) for digestion with Trypsin and LysC. All samples were measured on a 100 min gradient using a Q Exactive HF-X Orbitrap High Resolution Accurate Mass Spectrometer coupled to an EASY-nLC 1200 UHPLC. FFPE scrolls of human adenoma samples were used and processed in single wells. An optimal combination of heating and AFA ensured reversal of crosslinks and paraffin removal from tissue samples. Protein purification was achieved using the PAC protocol. Finally, a tryptic ‘on-bead’ digest was performed resulting in a clean peptide solution that was desalted prior to LC-MS analysis. A comparison of the xylol-free workflow with that of the common xylol deparaffinization resulted in highly similar peptide, protein peptide identification rates, and protein abundance ranges. On average, 3,900 proteins and 20,842 peptides per single run measurement were quantified. From the perspective of monitoring post-translational modifications, a comparison of this method with the xylol-based protocol showed that crosslink reversal was equally efficient in both protocols. Efficiency of tissue lysis and paraffin removal in relation to the thickness of tissue sections were evaluated in 3 and 10 µm thick adenoma sections. While both section types provided enough material for multiple MS measurements, 10 µm thick sections showed a higher spread of peptide yields. The number of protein identifications, however, was found to be similar under both conditions. To further assess the reproducibility of the AFA and PAC workflow, three tissue sections of a colorectal adenoma were used, resulting in quantification of more than 3,500 proteins in each of the tissue replicates. Citation Format: Tom O'Hare, Eugenio Daviso, Debadeep Bhattacharyya, Sameer Vasantgadkar. Confident analyses of proteins in FFPE tissue: Robust, reliable, reproducible sample preparation enables LC-MS based proteomics assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2039.

Synthesizing a Novel Janus Carbon Nano-Onions Modified As a Catalyst Support for Oxygen Reduction Reaction

Carbon materials have been awakening scientific interest for research because it allows chemical functionalization for multiple applications in the sciences, especially in energy applications. Carbon Nano-onions (CNO) are spherical structures composed of multilayers of fullerenes, these layers are connected in a way that shows a shape of an onion. Its development begins with the use of nano-diamonds, a carbon material of strong structure which it forms in a very violent environment. The nano-diamonds are taken to a furnace at a temperature of 1650°C to finally obtain the CNO. Janus particles are receiving increasing attention because of their dual properties, where each side can be functionalized to have distinctive characteristics. The modifications on the surface of these nanoparticles can provide different chemical and physical properties. The interesting properties of Janus nanoparticles are that they have different sizes and shapes which have now been able to be studied in more detail. The purpose of this project is to use asymmetrically modified CNO as a support for metal nanoparticles to avoid agglomeration and, thus, increase their surface area and efficiency. Janus nanoparticles will be designed by a wax-paraffin Pickering emulsion process using CNO on its surface. The deposition of Platinum (Pt) it was carried out by a chemical process using sodium borohydride. The removal process of the paraffin involves a treatment with chloroform and rinse with ethanol to finally obtain the amphiphilic nanoparticle. The differences on the surface of the particles before and after removing the paraffin were observed by Scanning Electron Microscopy. The Energy-Dispersive Spectroscopy was used to validate the elemental information of the particles and assure the deposition of Pt on the surface of the particles. Cyclic Voltammetry was used to characterize the CNO/Pt ink, providing information about the Pt deposition on the surface of the CNO. Oxygen Reduction Reaction experiments were conducted to test the efficiency of this catalyst using the Rotating Ring-Disk Electrode at rotations of 100, 400, 900, 1600, and 2500 rpm. The polarization curves of CNO/Pt and Pt/Vulcan were compared. Thermogravimetric Analysis were used as a characterization technique to corroborate the percent of Pt on the CNO/Pt particles. Characterization techniques as X-ray Diffraction and Raman Spectroscopy were used to validate the deposition of Pt and confirm the removal of paraffin.

Paraffin Removal in the Synthesis of Novel Janus Carbon Nano-Onions

Recent research has demonstrated that Janus nanoparticles provide a novel strategy to prepare catalysts and biomaterials. This is because of the versatility of being able to modify both sides of a particle with different properties. Carbon nano-onions are an excellent material as a support for different applications, such as metal nanoparticles, due to their unique structure and good conductivity. Because of their physical and chemical properties, carbon nano-onions are an ideal material to create Janus nanoparticles for further amphiphillic modifications. This article aims to show a preparatory process to ensure the removal of paraffin efficiently. The main method to be able to create these particles is employing the Pickering emulsion process. Paraffin wax is used as the hydrophobic part of this mixture and serves to block one side of the CNOs to facilitate their modification only on one side. Therefore, its removal is essential to obtain this catalytic nanoparticle.

Three-Dimensional Porous Solar-Driven Interfacial Evaporator for High-Efficiency Steam Generation under Low Solar Flux.

Solar steam generation is critical for many important solar-thermal applications, but is challenging to achieve under low solar flux due to the large evaporation enthalpy of water. Here, we demonstrate a three-dimensional porous solar-driven interfacial evaporator that can generate 100 °C steam under 1 sun illumination with a record high solar-to-steam conversion efficiency of 48%. The high steam generation efficiency is achieved by localizing solar-thermal heating at the evaporation surface and controlling the water supply onto the porous evaporator through tuning its surface wettability, which prevents overheating of the evaporator and thus minimizes conductive, convective, and radiative heat losses from the evaporator. The design of steam outlet located at the sidewall of the evaporator rather than from the solar absorber surface not only facilitates the collection of generated steam, but also avoids potential blockage of solar radiation by the condensing steam. The high-efficiency solar-driven evaporator has been used to generate hot steam for outdoor removal of paraffin on the wall of oil pipelines, offering a promising solution to mitigate the wax deposition issue in petroleum extraction processes.

Abstract P1-20-05: Raman imaging as a tool for the chemical and spatial characterization of breast microcalcifications to improve lesion assessment

Abstract Background: microcalcifications (MC) are common findings on screening mammography and are among the earliest signs of breast cancer. At the same time, from the use of well-known radiographic risk score systems, which include MC assessment, such as Breast Imaging-Reporting and Data System (BI-RADS), only 20% of screened patients are further associated with malignancy. This leads to repeated biopsies and to unnecessary surgeries with discomfort for patients and increased costs for the healthcare system. Furthermore, the definitive diagnosis by histological and immunohistological evaluations is still laborious and time-consuming. Raman Spectroscopy (RS) is a photonic approach capable to provide detailed chemical information of analysed samples without complex tissue preparation or staining. RS has a proven ability to distinguish different crystal structures, including those commonly present in MC. In this context some studies based on RS suggested a correlation between MC chemical features and pathology. On the other hand, previous Raman-based studies mainly investigated the overall MC chemical composition (by single-point scans) while an extensive MC characterization by Raman imaging approaches (mapping) for diagnostic purposes is still lacking. The aim of this study is to assess the usefulness of Raman imaging as a quick and accurate tool for a complete spatial characterization of MC detected on screening mammography and sampled by breast biopsy in order to better distinguish malignant vs. benign lesions. Method: 30 patients with breast calcifications detected on mammography with radiological classification BI-RAD 3-5 where selected and evaluated by core biopsy. 10 μm formalin-fixed paraffin-embedded (FFPE) histological sections obtained from biopsies were dewaxed with a specifically developed protocol that allows the removal of paraffin in less than 15 minutes. All MC present in each tissue section (usually from 2 to >10 per section) were then characterized by a Raman microscope thus obtaining Raman maps with lateral resolution between 5 and 10 μm. After pre-processing steps the Raman maps were analysed by both clustering and multivariate analysis approaches used to produce false-colour images and to perform automated features identification. Results: Our results confirm that hydroxyapatite is the prevalent form of calcium phosphate in MC and that MC composition correlates with lesion malignancy. In addition, thanks to the Raman imaging approach used here, we report for the first time that hydroxyapatite is more homogeneously distributed in malignant lesions and that, on the contrary, benign lesions show a heterogeneous distribution of hydroxyapatite, whitlockite and calcium-carbonate, inside the lesion and in the surrounding tissue. Conclusion: These evidences suggest that the characterization of MC by Raman imaging is a potential tool for the definition of new diagnostic signatures of breast cancer, especially if we consider that these evaluations can be performed by the simple and relative fast scanning of dewaxed slices, without altering the clinical workflow and without the need of staining or antibodies. Further studies with a larger cohort will be done to validate these results. Citation Format: Vanna R, Morasso C, Piccotti F, Sorrentino L, Villani L, Torti E, Bonizzi A, Leporati F, Corsi F. Raman imaging as a tool for the chemical and spatial characterization of breast microcalcifications to improve lesion assessment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-20-05.

Microbial treatment of waxy crude oil for mitigating wax precipitation and improving liquidity

The wax deposition of crude oil during transportation reduces the inner diameter, blocks the pipeline, and eventually increases the cost of petroleum production. This study reports a paraffin-degrading bacterial strain DG2 isolated from petroleum-contaminated soil. The effect of this strain on the removal of paraffin in crude oil was investigated. DG2, identified as Pseudomonas, produced biosurfactant that reduced the surface tension to 35.7 mN/m−1 and exhibited high emulsification activity. Results of gas chromatography–mass spectrometry analysis of the waxy crude oil treated by DG2 showed decreased heavy hydrocarbon fractions (C25 and C32). The degradation test revealed that DG2 reduced the wax appearance temperature by 4.16 °C and the viscosity by 23% of the waxy crude oil. These findings indicate that paraffin-degradation strain DG2 may be used for effective paraffin biodegradation.

Kinetics of microwave heating and drying of drilling fluids and drill cuttings

ABSTRACTDrill cutting decontamination by microwave drying has been studied over the past few years and has proved to be a promising technology. This study aimed to investigate fundamental aspects of kinetics of heating and drying of drilling fluids and drill cuttings by microwaves. The microwave heating curve of cuttings free of fluid, drilling fluids, and pure organic compounds usually used in the formulation of these fluids was evaluated to understand the behavior of each component in microwave cutting decontamination. Furthermore, commercial software was used to describe the heating kinetics of the drilling fluids used in this study. The drying kinetics of cuttings contaminated with these drilling fluids was also studied at three temperatures of control. Some classic models of conventional drying of solids were used to describe the removal kinetics of the liquid components present in contaminated cuttings (water and organic compounds). Important aspects related to the interaction of these components in the drying operation and solid heating, water evaporation, and the drag of organic compounds were investigated. Both drilling fluids showed a very similar kinetic heating. Pure organic bases did not show a significant heating. For the same drying time, the removal of paraffin is more intense than the olefin. In respect to organic component removal from cuttings contaminated with both fluids, the kinetic drying curves are similar. The Page model was the one that best describes the drying operation of drill cuttings contaminated with both drilling fluids. The microwave drying model (MDM) model is proposed in this work as a simple modification in the Henderson–Pabis model: the addition of a third parameter. The incorporation of this parameter enabled a better fit of the experimental data. Computational simulations show an electric field with symmetrical patterns for the two BR-MUL fluids analyzed.

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

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-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission 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.

P66 * AN INVESTIGATION INTO SUBSTRATE EFFECTS AND TISSUE PRE-PROCESSING IN SPECTRAL HISTOPATHOLOGY USING RAMAN SPECTROSCOPY

INTRODUCTION: Raman spectroscopy is a low cost non-destructive and non-invasive technique showing great potential for the diagnosis of cancer. Biobanks are generally comprised of formalin fixed paraffin preserved (FFPP) tissue used in spectroscopic investigations. In order to carry out spectroscopic investigations the tissue needs to undergo a dewaxing process which removes and reduces paraffin contributions in the spectra. The ineffective removal of paraffin from the tissue and substrate contributions can affect the results and quality of the spectra. The aim of this study was to investigate the efficacy of tissue pre-processing steps used in spectral histopathology and the influence of commonly used substrates. METHOD: Nitrocellulose membrane filters of varying density, 8.0 µM, 5.0 µM and 1.2 µM were used to model human tissue. Filters underwent histological processing as clinical biopsies. Sections from each filter were cut at 10 microns and placed on three substrates, CaF2, Low-E and Glass which were subsequently dewaxed using two clearing agents, Histoclear and Xylene in three different bath times, 5 min, 10 min and 15 min. Spectra was collected from three biological replicates of each filter using Raman Spectroscopy with a 785 nm laser. RESULTS: CaF2 had the least background noise; however Glass and Low-E retained the least amount of paraffin whilst Xylene completely removed paraffin under the same bath conditions as Histoclear. CONCLUSION:Glass and Low-E are relatively cheaper and also provide reproducible results. These substrates can allow us to use standard clinical preparation procedures. Whilst Xylene is more effective in paraffin removal it is a hazardous chemical.

Effect of substrate choice and tissue type on tissue preparation for spectral histopathology by Raman microspectroscopy

Raman spectroscopy is a non-destructive, non-invasive, rapid and economical technique which has the potential to be an excellent method for the diagnosis of cancer and understanding disease progression through retrospective studies of archived tissue samples. Historically, biobanks are generally comprised of formalin fixed paraffin preserved tissue and as a result these specimens are often used in spectroscopic research. Tissue in this state has to be dewaxed prior to Raman analysis to reduce paraffin contributions in the spectra. However, although the procedures are derived from histopathological clinical practice, the efficacy of the dewaxing procedures that are currently employed is questionable. Ineffective removal of paraffin results in corruption of the spectra and previous experiments have shown that the efficacy can depend on the dewaxing medium and processing time. The aim of this study was to investigate the influence of commonly used spectroscopic substrates (CaF2, Spectrosil quartz and low-E slides) and the influence of different histological tissue types (normal, cancerous and metastatic) on tissue preparation and to assess their use for spectral histopathology. Results show that CaF2 followed by Spectrosil contribute the least to the spectral background. However, both substrates retain paraffin after dewaxing. Low-E substrates, which exhibit the most intense spectral background, do not retain wax and resulting spectra are not affected by paraffin peaks. We also show a disparity in paraffin retention depending upon the histological identity of the tissue with abnormal tissue retaining more paraffin than normal.

The Comparison of Paraffin Dewaxing Using Methyl Tert-Butyl Ether and Xylene in DNA Extraction from Autopsy Specimens

Formalin fixed and paraffin-embedded tissue specimens collected during surgery or autopsy, are an important source for retrospective diagnosis and identification purposes. Genomic DNA degradation or PCR amplification inhibition are the major cause of DNA amplification failure. Routinely, xylene is used to remove paraffin from paraffinembedded tissue sections. We tested methyl tert-butyl ether (MTBE), as an alternate organic solvent, which is less harmful for organism than xylene. Using different tissues (heart, kidney, liver) from randomly selected autopsies (n=10) we compared performance of MTBE and xylene for removal of paraffin during a preparation process compatible with automated staining equipment. All these extracted DNA samples were amplified and genotyped using human identification Identifiler multiplex. Our experiments points that there is no difference in the range of genotyped microsatellite loci, regardless MTBE or xylene dewaxing. The heart specimen has the highest number of successfully genotyped STR loci, followed by the kidney and the liver. For the genomic template above 260 base pairs of the length no products were obtain from the routinely collected autopsy material. It is worthy of note that MTBE is safer than xylene and according to the current European Community regulations no chemical fume hoods are required for MTBE handling. Thus, MTBE might be preferred to remove paraffin from tissue specimens in forensic or histopathology laboratories not equipped with systems of airborne exposure protection.

Dielectric Characteristics of Hydrated Paraffin

Based on the composite dielectric polarization theory, dielectric permittivity, dissipation factor and loss angle tangent of hydrated paraffin versus temperature and power supply frequency were analyzed. The effect of different water contents in paraffin on its dielectric characteristics was also studied. The more the water content in hydrated paraffin, the greater the dielectric parameters, dissipation factor can reach 1.48 when the water contents was 15%. They can provide references for theoretical researches and industrial applications on removal of paraffin that condense on the interior walls of oil pipes in oil extraction by electric heating method.

Detection of Mycobacterium avium subsp. paratuberculosis in formalin- fixed, paraffin embedded tissues of goats by IS900 polymerase chain reaction

The objective of the present study was to optimise and evaluate a procedure for detection of Mycobacterium avium subsp. paratuberculosis in archived formalin-fixed, paraffin embedded tissue sections from cases of naturally occurring paratuberculosis in goats. A pilot study assessed 3 procedures for extraction of DNA for detection by PCR. The procedure that gave the most consistent results involved removal of paraffin by treatment with xylene and ethanol, disruption of tissue pellets by beating with zirconium/silica beads, extraction of DNA using a DNeasy kit (Qiagen) with overnight proteinase K digestion and final ethanol precipitation. This procedure was used to analyse 82 paraffin embedded tissues (44 small intestine, 38 mesenteric lymph node) with various grades of histological lesions of paratuberculosis and acid-fast bacilli (AFB) loads. The overall sensitivity of the PCR was about 72% of all samples including both paucibacillary and multibacillary lesions. The sensitivity of the assay was 87.5% (42/48) in all paraffin sections having clearly and easily demonstrable AFB. Fifty percent of the tissue sections with rarely detectable AFB were positive by PCR. There was no significant difference (<0.05) between the sensitivity of the PCR analyses carried out on intestinal and mesenteric lymph node tissues. The results of this study suggest that IS900 PCR on formalin-fixed, paraffin embedded histological sections is a practical and important tool for confirming diagnosis of paratuberculosis in goats, where fresh tissues for bacterial culture or PCR are not available due to problem of maintaining a cold chain system.

Non-small cell lung cancer (NSCLC), analysis of healthy, non-smoking, and smoking patients by MALDI imaging mass spectrometry: Proteomic updating perspective.

e21147 Background: Imaging MALDI Mass spectrometry (IMS) was applied to study tumor tissue of NSCLC subtypes adenocarcinoma (ADK). After a previous comparison of healthy (H) and tumor tissues (TT) on slides from frozen biopsies, our objective was : i) to execute an IMS analysis comparing the mass finger print proteome (MFPP) of smoking (S) versus non smoking (NS) patients from formalin-fixed paraffin-embedded tissue microarrays (TMA) followed by a tryptic treatment ii) to apply a large group of statistical methods iii) validate these proteomic. Methods: Specific profiling of tumor proteome by in situ imaging mass spectrometry was performed with a Maldi Tof-Tof apparatus (ULTRAFLEX III Bruker-De).(H) and (TT) cohort were frozen (40 patients). All tissues were controlled and the stages determined by the pathologist diagnosis. TMA were obtained by slides from paraffin blocks with 33 patients. After removal of paraffin and in situ tryptic digestion we analyzed proteic fragments (800 spectrums/group). Results: we observed that the proteome of the two classes (H) and (TT) start to be different only from the stage IB to IV tested. This was confirmed the PCA analysis. The simple and hierarchical clustering corresponding confirm these two distinct classes of proteome and permit to select groups of masses oriented to biomarkers. This was similar the same for TMA, but the proteome of these stages, with IMS, was not the same in the 2 classes (S) and (NS). In each condition were analyzed the under and upper expression to select potential biomarkers. Conclusions: Then main interest of our study is to characterize the proteome of each class analyzed indeed in the under expressed m/z group from the ratio (NS)/(S) we have found 4 tryptic fragments of 1320, 1793, 1838 and 2704 which could be candidates to correspond to fragments of tryptic cleavage in silico of glypican 5 justly under expressed and associated to lung cancer in never smokers (Yafei Li and al. Lancet 2010).

122 IMAGING THE RENAL CELL CARCINOMA PROTEOME

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011122 IMAGING THE RENAL CELL CARCINOMA PROTEOME Todd M. Morgan, Erin H. Seeley, Oluwole Fadare, Richard M. Caprioli, David L. Hachey, and Peter E. Clark Todd M. MorganTodd M. Morgan Nashville, TN More articles by this author , Erin H. SeeleyErin H. Seeley Nashville, TN More articles by this author , Oluwole FadareOluwole Fadare Nashville, TN More articles by this author , Richard M. CaprioliRichard M. Caprioli Nashville, TN More articles by this author , David L. HacheyDavid L. Hachey Nashville, TN More articles by this author , and Peter E. ClarkPeter E. Clark Nashville, TN More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.188AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES No biomarkers for renal cell carcinoma (RCC) are in routine use. However, rapid developments in proteomics offer substantial promise for identifying novel diagnostic and prognostic biomarkers. One barrier to identification of tissue biomarkers is the heterogeneity of protein expression within tumor and normal renal parenchyma. Imaging mass spectrometry (IMS) can provide spectra for every 0.05mm2 area of tissue and therefore reveal the spatial distribution of peptides within a section of tissue. We sought to determine whether this approach could be used to identify and map protein signatures differentially expressed between RCC and normal renal tissue. METHODS We constructed a tissue microarray with two cores each of matched tumor and normal tissue from nephrectomy specimens of 35 patients with clear cell RCC. After mounting sections on a conductive glass slide, removal of paraffin, and antigen retrieval, trypsin digestion was performed directly on the tissue. Samples were analyzed utilizing an AutoFlex Speed matrix assisted laser desorption ionization (MALDI) time-of-flight mass spectrometer (MS). Data analysis was performed with ClinProTools 2.2 and FlexImaging 2.1 software. RESULTS An average of ∼500 peptides was identified in each mass spectrum. Comparison of peptide expression in RCC vs. normal tissue revealed a number of individual peptides that discriminated between malignant and disease-free renal tissue with a high degree of accuracy. For example, receiver operator characteristic (ROC) curve analysis revealed four individual peptides able to identify malignant tissue with >90% accuracy (area under the curve [AUC] 0.91–0.94). Seven peptides demonstrated a classification accuracy of 95% for each 0.05mm2 spot. In toto, 138 of 140 cores (98.6%) were accurately classified (Figure). CONCLUSIONS IMS was able to identify and map specific peptides that accurately distinguished malignant from normal renal tissue, demonstrating its potential as a novel, high-throughput approach to RCC biomarker discovery. Additional work to determine the identity of these differentially expressed proteins may lend insight into the pathogenesis of RCC. Furthermore, given the multiple pathways involved in tumors such as RCC, multiple peptide signatures may offer advantages as both diagnostic and prognostic biomarkers compared to traditional protein biomarkers. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e51 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Todd M. Morgan Nashville, TN More articles by this author Erin H. Seeley Nashville, TN More articles by this author Oluwole Fadare Nashville, TN More articles by this author Richard M. Caprioli Nashville, TN More articles by this author David L. Hachey Nashville, TN More articles by this author Peter E. Clark Nashville, TN More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Genotyping of DNA samples under adverse conditions of Low Copy Number—LCN (formolisados tissue samples and embedded in paraffin)

This paper aims to describe and evaluate a protocol for extraction of DNA (deoxyribonucleic acid) in formalinized tissues and embedded in paraffin for forensics genetic analysis. In outline the method is the removal of paraffin with an organic solvent in 0.3–0.5 mg of the sample of the tissue under study, followed by removal of formaldehyde, rehydration and soon after the extraction of genomic DNA. The extraction is achieved through the stages of cellular lysis, enzymatic digestion of proteins and DNA precipitation in ethanol medium. With the research we can conclude that even when the DNA is present in small quantities in conditions of extreme difficulties in its extraction, as formalinized tissues and embedded in paraffin, the technique of optimizing the extraction of DNA used both to organic extraction as Chelex, for use in the polymerase chain reaction (PCR), and possible the investigation of different samples of human tissue, biological samples, or was obtained under the conditions tested, a DNA with good quality and concentration. The samples were amplified for the mini-STRs loci using the product marketed in multilocus, using a methodology recommended by the supplier and validated for analysis of forensic DNA. Commercial kit was used MiniFiler from Applied Biosystems. The DNA fragments amplified by PCR showed that the extracted DNA had good amplification.

Strategies to improve the adhesion of rubbers to adhesives by means of plasma surface modification

The surface modifications produced by treatment of a synthetic sulfur vulcanized styrene-butadiene rubber with oxidizing (oxygen, air, carbon dioxide) and non oxidizing (nitrogen, argon) RF low pressure plasmas, and by treatment with atmospheric plasma torch have been assessed by ATR-IR and XPS spectroscopy, SEM, and contact angle measurements. The effectiveness of the low pressure plasma treatment depended on the gas atmosphere used to generate the plasma. A lack of relationship between surface polarity and wettability, and peel strength values was obtained, likely due to the cohesive failure in the rubber obtained in the adhesive joints. In general, acceptable adhesion values of plasma treated rubber were obtained for all plasmas, except for nitrogen plasma treatment during 15 minutes due to the creation of low molecular weight moieties on the outermost rubber layer. A toluene wiping of the N2 plasma treated rubber surface for 15 min removed those moieties and increased adhesion was obtained. On the other hand, the treatment of the rubber with atmospheric pressure by means of a plasma torch was proposed. The wettability of the rubber was improved by decreasing the rubber-plasma torch distance and by increasing the duration because a partial removal of paraffin wax from the rubber surface was produced. The rubber surface was oxidized by the plasma torch treatment, and the longer the duration of the plasma torch treatment, the higher the degree of surface oxidation (mainly creation of C–O moieties). However, although the rubber surface was effectively modified by the plasma torch treatment, the adhesion was not greatly improved, due to the migration of paraffin wax to the treated rubber-polyurethane adhesive interface once the adhesive joint was produced. On the other hand, the extended treatment with plasma torch facilitated the migration of zinc stearate to the rubber-adhesive interface, also contributing to deteriorate the adhesion in greater extent. Finally, it has been found that cleaning of SBS rubber in an ultrasonic bath prior to plasma torch treatment produced a partial removal of paraffin waxes from the surface, and thus improved adhesion was obtained.

Nanomanipulation of sections of human tongue squamous cell carcinoma by atomic force microscopy

To investigate the appropriate nanomanipulation of slides of human tongue squamous cell carcinoma tissue and cultured cells by atomic force microscopy (AFM), and develop methods to improve the resolution and contrast of AFM images. Human tongue squamous carcinoma cells of the line Tca8113 were cultured. Cell masses were isolated and collected, and sectioned. Then the white lead and silvery white sections were transferred on mica slides, dried. Eighty slides were used and divided into 2 groups to be immersed into hydrogen dioxide of the concentrations 20% and 30% for 0, 5, 10, 15, 20, 25, 30, and 40 min respectively. Samples of human tongue squamous carcinoma were collected from 15 cases. Every sample was divided into 2 halves. Half of every sample underwent routine paraffin preparation of slide and adhered onto glass, dissolving of paraffin with dimethylbenzene for 0, 10, 15, 30, 45, or 60 min; and half of the sample underwent routine preparation of slides for electron microscopy, the white lead and silvery white sections were transferred on mica slides, dried. The slides were observed by contact mode AFM in air. In the ultrathin sections the metastructure of the cell such as the cell membrane, nuclear membrane and nucleolus could be distinct from each other clearly. The profile of the cell and the nucleus inside could be revealed by AFM after the paraffin sections were treated in dimethylbenzene for 15-30 min. AFM manipulation could be achieved within nanometer range by precise modulation of scanning force, area, angle and so on. The appropriate needle point should contain elastic coefficient > 5.0 N/m. The force exerted on the needle point should be within the range 50-300 nN. After location the scanning direction should be within the range of 20 nm-1 microm. A series of modified techniques of preparing and treating AFM samples, such as adhering the sections onto mica slide, dissolving of epoxy resin by hydrogen dioxide and removal of paraffin by dimethylbenzene, were developed, thus realizing high resolving power AFM imaging, especially for nuclear membrane, and nucleolus, and providing a base of AFM location, dissection, isolation and obtainment at nanometer scale.

Inhibitory effects of anti-glaucoma drugs on corneal epithelial migration in a rabbit organ culture system.

The possible effects of the anti-glaucoma drugs latanoprost, timolol maleate, and nipradilol on corneal epithelial wound healing were investigated in a rabbit organ culture system. Corneal blocks (approximately 2 x 4 mm) isolated from albino Japanese rabbits were cultured for 24 hours in medium containing various concentrations (0.01 to 10 mM) of latanoprost, timolol maleate, or nipradilol. The specimens were then fixed and embedded in paraffin, and thin sections were prepared. After the removal of paraffin, the sections were stained with hematoxylin-eosin and observed under a light microscope. Images were recorded digitally with a CCD camera, and the distance over which the epithelium had migrated down the sides of each block was calculated with a computer-assisted morphometric program. Corneal epithelial migration was inhibited by each anti-glaucoma drug in a dose-dependent manner. The 50% inhibitory doses (ID50) for latanoprost, timolol maleate, and nipradilol were 0.362, 0.585, and 4.89 mM, respectively. Relative to the concentrations present in commercially available eyedrops, ID50 corresponded to concentration ratios for timolol, nipradilol, and latanoprost were of 0.037, 0.64 and 3.1, respectively. The anti-glaucoma drugs examined inhibited corneal epithelial migration in a rabbit organ culture system. However, it is unlikely that the corneal surface would be exposed to these agents at inhibitory concentrations for a prolonged period in the normal clinical setting.