Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues.

PubMed

Zhang, Fan; Wang, Zhuo-min; Liu, Hong-yu; Bai, Yun; Wei, Sen; Li, Ying; Wang, Min; Chen, Jun; Zhou, Qing-hua

2010-01-01

To analyze gene expression in formalin-fixed, paraffin-embedded lung cancer tissues using modified method. Total RNA from frozen tissues was extracted using TRIZOL reagent. RNA was extracted from formalin-fixed, paraffin-embedded tissues by digestion with proteinase K before the acid-phenol:chloroform extraction and carrier precipitation. We modified this method by using a higher concentration of proteinase K and a longer digestion time, optimized to 16 hours. RT-PCR and real-time RT-PCR were used to check reproducibility and the concordance between frozen and paraffin-embedded samples. The results showed that the RNA extracted from the paraffin-embedded lung tissues had high quality with the most fragment length between 28S and 18S bands (about 1000 to 2000 bases). The housekeeping gene GUSB exhibited low variation of expression in frozen and paraffin-embedded lung tissues, whereas PGK1 had the lowest variation in lymphoma tissues. Furthermore, real-time PCR analysis of the expression of known prognostic genes in non-small cell lung carcinoma (NSCLC) demonstrated an extremely high correlation (r>0.880) between the paired frozen and formalin-fixed, paraffin-embedded specimens. This improved method of RNA extraction is suitable for real-time quantitative RT-PCR, and may be used for global gene expression profiling of paraffin-embedded tissues.

A Comparison of RNA-Seq Results from Paired Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Glioblastoma Tissue Samples

PubMed Central

Esteve-Codina, Anna; Arpi, Oriol; Martinez-García, Maria; Pineda, Estela; Mallo, Mar; Gut, Marta; Carrato, Cristina; Rovira, Anna; Lopez, Raquel; Tortosa, Avelina; Dabad, Marc; Del Barco, Sonia; Heath, Simon; Bagué, Silvia; Ribalta, Teresa; Alameda, Francesc; de la Iglesia, Nuria

2017-01-01

The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved. PMID:28122052

Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System®

PubMed Central

Golubeva, Yelena G.; Smith, Roberta M.; Sternberg, Lawrence R.

2013-01-01

Laser microdissection is an invaluable tool in medical research that facilitates collecting specific cell populations for molecular analysis. Diversity of research targets (e.g., cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, etc.) and varied scientific objectives, however, present challenges toward establishing standard laser microdissection protocols. Sample preparation is crucial for quality RNA, DNA and protein retrieval, where it often determines the feasibility of a laser microdissection project. The majority of microdissection studies in clinical and animal model research are conducted on frozen tissues containing native nucleic acids, unmodified by fixation. However, the variable morphological quality of frozen sections from tissues containing fat, collagen or delicate cell structures can limit or prevent successful harvest of the desired cell population via laser dissection. The CryoJane Tape-Transfer System®, a commercial device that improves cryosectioning outcomes on glass slides has been reported superior for slide preparation and isolation of high quality osteocyte RNA (frozen bone) during laser dissection. Considering the reported advantages of CryoJane for laser dissection on glass slides, we asked whether the system could also work with the plastic membrane slides used by UV laser based microdissection instruments, as these are better suited for collection of larger target areas. In an attempt to optimize laser microdissection slide preparation for tissues of different RNA stability and cryosectioning difficulty, we evaluated the CryoJane system for use with both glass (laser capture microdissection) and membrane (laser cutting microdissection) slides. We have established a sample preparation protocol for glass and membrane slides including manual coating of membrane slides with CryoJane solutions, cryosectioning, slide staining and dissection procedure, lysis and RNA extraction that facilitated efficient dissection and high quality RNA retrieval from CryoJane preparations. CryoJane technology therefore has the potential to facilitate standardization of laser microdissection slide preparation from frozen tissues. PMID:23805281

RNA-Seq-based toxicogenomic assessment of fresh frozen and formalin-fixed tissues yields similar mechanistic insights.

PubMed

Auerbach, Scott S; Phadke, Dhiral P; Mav, Deepak; Holmgren, Stephanie; Gao, Yuan; Xie, Bin; Shin, Joo Heon; Shah, Ruchir R; Merrick, B Alex; Tice, Raymond R

2015-07-01

Formalin-fixed, paraffin-embedded (FFPE) pathology specimens represent a potentially vast resource for transcriptomic-based biomarker discovery. We present here a comparison of results from a whole transcriptome RNA-Seq analysis of RNA extracted from fresh frozen and FFPE livers. The samples were derived from rats exposed to aflatoxin B1 (AFB1 ) and a corresponding set of control animals. Principal components analysis indicated that samples were separated in the two groups representing presence or absence of chemical exposure, both in fresh frozen and FFPE sample types. Sixty-five percent of the differentially expressed transcripts (AFB1 vs. controls) in fresh frozen samples were also differentially expressed in FFPE samples (overlap significance: P < 0.0001). Genomic signature and gene set analysis of AFB1 differentially expressed transcript lists indicated highly similar results between fresh frozen and FFPE at the level of chemogenomic signatures (i.e., single chemical/dose/duration elicited transcriptomic signatures), mechanistic and pathology signatures, biological processes, canonical pathways and transcription factor networks. Overall, our results suggest that similar hypotheses about the biological mechanism of toxicity would be formulated from fresh frozen and FFPE samples. These results indicate that phenotypically anchored archival specimens represent a potentially informative resource for signature-based biomarker discovery and mechanistic characterization of toxicity. Copyright © 2014 John Wiley & Sons, Ltd.

Filter paper-assisted cell transfer (FaCT) technique: A novel cell-sampling technique for intraoperative diagnosis of central nervous system tumors.

PubMed

Kawamura, Jumpei; Kamoshida, Shingo; Shimakata, Takaaki; Hayashi, Yurie; Sakamaki, Kuniko; Denda, Tamami; Kawai, Kenji; Kuwao, Sadahito

2017-04-01

Intraoperative diagnosis of central nervous system (CNS) tumors provides critical guidance to surgeons in the determination of surgical resection margins and treatment. The techniques and preparations used for the intraoperative diagnosis of CNS tumors include frozen sectioning and cytologic methods (squash smear and touch imprint). Cytologic specimens, which do not have freezing artifacts, are important as an adjuvant tool to frozen sections. However, if the amount of submitted tissue samples is limited, then it is difficult to prepare both frozen sections and squash smears or touch imprint specimens from a single sample at the same time. Therefore, the objective of this study was to derive cells directly from filter paper on which tumor samples are placed. The authors established the filter paper-assisted cell transfer (FaCT) smear technique, in which tumor cells are transferred onto a glass slide directly from the filter paper sample spot after the biopsy is removed. Cell yields and diagnostic accuracy of the FaCT smears were assessed in 40 CNS tumors. FaCT smears had ample cell numbers and well preserved cell morphology sufficient for cytologic diagnosis, even if the submitted tissues were minimal. The overall diagnostic concordance rates between frozen sections and FaCT smears were 90% and 87.5%, respectively (no significant differences). When combining FaCT smears with frozen sections, the diagnostic concordance rate rose to 92.5%. The current results suggest that the FaCT smear technique is a simple and effective processing method that has significant value for intraoperative diagnosis of CNS tumors. Cancer Cytopathol 2017;125:277-282. © 2016 American Cancer Society. © 2017 American Cancer Society.

Frozen section analysis of margins for head and neck tumor resections: reduction of sampling errors with a third histologic level.

PubMed

Olson, Stephen M; Hussaini, Mohammad; Lewis, James S

2011-05-01

Frozen section analysis is an essential tool for assessing margins intra-operatively to assure complete resection. Many institutions evaluate surgical defect edge tissue provided by the surgeon after the main lesion has been removed. With the increasing use of transoral laser microsurgery, this method is becoming even more prevalent. We sought to evaluate error rates at our large academic institution and to see if sampling errors could be reduced by the simple method change of taking an additional third section on these specimens. All head and neck tumor resection cases from January 2005 through August 2008 with margins evaluated by frozen section were identified by database search. These cases were analyzed by cutting two levels during frozen section and a third permanent section later. All resection cases from August 2008 through July 2009 were identified as well. These were analyzed by cutting three levels during frozen section (the third a 'much deeper' level) and a fourth permanent section later. Error rates for both of these periods were determined. Errors were separated into sampling and interpretation types. There were 4976 total frozen section specimens from 848 patients. The overall error rate was 2.4% for all frozen sections where just two levels were evaluated and was 2.5% when three levels were evaluated (P=0.67). The sampling error rate was 1.6% for two-level sectioning and 1.2% for three-level sectioning (P=0.42). However, when considering only the frozen section cases where tumor was ultimately identified (either at the time of frozen section or on permanent sections) the sampling error rate for two-level sectioning was 15.3 versus 7.4% for three-level sectioning. This difference was statistically significant (P=0.006). Cutting a single additional 'deeper' level at the time of frozen section identifies more tumor-bearing specimens and may reduce the number of sampling errors.

Determination of the methylation status of MGMT in different regions within glioblastoma multiforme.

PubMed

Hamilton, Mark G; Roldán, Gloria; Magliocco, Anthony; McIntyre, John B; Parney, Ian; Easaw, Jacob C

2011-04-01

Epigenetic silencing of the MGMT gene through promoter methylation correlates with improved survival in Glioblastoma Multiforme (GBM) patients receiving concurrent chemoradiotherapy. Although the clinical benefit is primarily seen in patients with methylated MGMT promoter, some unmethylated patients also respond to Temozolomide. One possible explanation may be intratumoral heterogeneity. This study was designed to assess the methylation status of the MGMT promoter in different areas of GBM and determine if methylation status varied depending on the fixation technique (paraffin-embedding versus fresh frozen) used to store tissue. Using intraoperative navigation, biopsies were obtained from three distinct regions: the enhancing outer area, the non-enhancing inner core, and an area immediately outside the enhancing region. Only patients with GBM were included for evaluation and analysis. Samples taken from each area were divided with half stored by flash freezing and the other half stored using paraffin fixation. Methylation Specific-PCR (MS-PCR) was used for analysis of MGMT promoter methylation. Thirteen patients were included. Ten were male with a median age of 62 years. In each patient, samples were taken from the enhancing rim and the necrotic centre. However, it was not considered safe or feasible to obtain samples from the area immediately adjacent to the enhancing tumor rim in one case. All patients were homogeneous for methylation status throughout their tumor and tissue taken adjacent to it when frozen tissue was used. However, four patients had discrepancies in the MGMT promoter status between the frozen and paraffin-embedded blocks and one patient was not homogeneous within the tumor when paraffin-embedded tissue was used. MGMT promoter methylation status was homogeneous in all GBM tumors. Our observation that methylation status varied depending if the DNA was extracted from paraffin-embedded versus frozen tissue is concerning. Although the reason for this is unclear, we postulate that the timing from resection to fixation or the process of fixation itself may potentially alter methylation status in paraffin-embedded tumors.

Marine Mammal Necropsy: An Introductory Guide for Stranding Responders and Field Biologists

DTIC Science & Technology

2007-09-01

the researcher or lab for required tissues and proper sample storage protocols (chill, fix, freeze and/or place in viral transport media). The most...tissues and fluids such as: liver, kidney, serum, aqueous humor, stom- ach contents, intestinal contents, feces, and urine . Tissue samples can be stored...refer to the Figure (2-1) for further explanation on frozen sample storage . The first label is written in black Sharpie on a 1 - 2 square inch piece of

Studies on the quantitative autoradiography. III. Quantitative comparison of a novel tissue-mold measurement technique "paste-mold method," to the semiquantitative whole body autoradiography (WBA), using the same animals.

PubMed

Motoji, N; Hamai, Y; Niikura, Y; Shigematsu, A

1995-01-01

A novel preparation technique, so called "Paste Mold," was devised for organ and tissue distribution studies. This is the most powerful by joining with autoradioluminography (ARLG), which was established and validated recently in the working group of Forum '93 of Japanese Society for study of xenobiotics. A small piece (10-50 mg) of each organ or tissue was available for measuring its radioactive concentration and it was sampled from the remains of frozen carcass used for macroautoradiography (MARG). The solubilization of the frozen pieces was performed with mixing a suitable volume of gelatine and strong alkaline solution prior to mild heating kept at 40 degrees C for a few hours. After that, the tissue paste was molded in template pattern to form the small plates. The molded plates were contacted with Imaging plate (IP) for recording their radioactive concentration. The recorded IP was processed by BAS2000. The molded plate was formed in thickness of 200 microns, so called infinit thickness against soft beta rays, and therefore the resulting relative intensities, represented by (PSL-BG)/S values, indicated practically responsible ratio of the radioactive concentration in organs and tissues, without any calibulation for beta-self absorption coefficiency. On the other hand, the left half body of the frozen carcass was used for making whole body autoradiography (WBA) before the Paste-Mold preparation. Comparison was performed for difference in (PSL-BG)/S values of organs and tissues between frozen and dried sections. A good concordance in relative intensities, (PSL-BG)/S by the Paste-Mold preparation was given with those by the frozen sections rather than dried sections.(ABSTRACT TRUNCATED AT 250 WORDS)

The value of frozen cartilage tissues without cryoprotection for genetic conservation.

PubMed

Cetinkaya, Gaye; Hatipoglu, Ibrahim; Arat, Sezen

2014-02-01

Animal tissues frozen without cryoprotection are thought to be inappropriate for use as a donor for somatic cell nuclear transfer (SCNT) studies. Cells in tissues that have been frozen without a cryoprotectant are commonly thought to be dead or to have lost genomic integrity. However, in this study we show that the frozen auricular cartilage tissues of anatolian buffalo contain a considerable number of viable healthy cells. The cells in auricular cartilage tissues are resistant to cryo-injury at -80°C. Primary cell cultures were established from defrosted ear tissues which were frozen without cryoprotectant. The growth and functional characteristics of primary cell cultures are characterized according to cell growth curve, cell cycle analysis, karyotype and GAG synthesis. The results indicate that frozen cartilage tissues could be valuable materials for the conservation of species and SCNT technology. Copyright © 2013 Elsevier Inc. All rights reserved.

Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics.

PubMed

Munchel, Sarah; Hoang, Yen; Zhao, Yue; Cottrell, Joseph; Klotzle, Brandy; Godwin, Andrew K; Koestler, Devin; Beyerlein, Peter; Fan, Jian-Bing; Bibikova, Marina; Chien, Jeremy

2015-09-22

Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes.

The Pyrolytic Profile of Lyophilized and Deep-Frozen Compact Part of the Human Bone

PubMed Central

Lodowska, Jolanta; Wolny, Daniel; Kurkiewicz, Sławomir; Węglarz, Ludmiła

2012-01-01

Background. Bone grafts are used in the treatment of nonunion of fractures, bone tumors and in arthroplasty. Tissues preserved by lyophilization or deep freezing are used as implants nowadays. Lyophilized grafts are utilized in the therapy of birth defects and bone benign tumors, while deep-frozen ones are applied in orthopedics. The aim of the study was to compare the pyrolytic pattern, as an indirect means of the analysis of organic composition of deep-frozen and lyophilized compact part of the human bone. Methods. Samples of preserved bone tissue were subjected to thermolysis and tetrahydroammonium-hydroxide- (TMAH-) associated thermochemolysis coupled with gas chromatography and mass spectrometry (Py-GC/MS). Results. Derivatives of benzene, pyridine, pyrrole, phenol, sulfur compounds, nitriles, saturated and unsaturated aliphatic hydrocarbons, and fatty acids (C12–C20) were identified in the pyrolytic pattern. The pyrolyzates were the most abundant in derivatives of pyrrole and nitriles originated from proteins. The predominant product in pyrolytic pattern of the investigated bone was pyrrolo[1,2-α]piperazine-3,6-dione derived from collagen. The content of this compound significantly differentiated the lyophilized graft from the deep-frozen one. Oleic and palmitic acid were predominant among fatty acids of the investigated samples. The deep-frozen implants were characterized by higher percentage of long-chain fatty acids than lyophilized grafts. PMID:22619606

Optical redox imaging of fixed unstained tissue slides to identify biomarkers for breast cancer diagnosis/prognosis: feasibility study

NASA Astrophysics Data System (ADS)

Xu, He N.; Tchou, Julia; Li, Yusheng; Feng, Min; Zhang, Paul; Quinn, William J.; Baur, Joseph A.; Li, Lin Z.

2018-02-01

We previously showed that optical redox imaging (ORI) of snap-frozen breast biopsies by the Chance redox scanner readily discriminates cancer from normal tissue. Moreover, indices of redox heterogeneity differentiate among tumor xenografts with different metastatic potential. These observations suggest that ORI of fluorescence of NADH and oxidized flavoproteins (Fp) may provide diagnostic/prognostic value for clinical applications. In this work, we investigate whether ORI of formalin-fixed-paraffin-embedded (FFPE) unstained clinical tissue slides of breast tumors is feasible and comparable to ORI of snap-frozen tumors. If ORI of FFPE is validated, it will enhance the versatility of ORI as a novel diagnostic/prognostic assay as FFPE samples are readily available. ORI of fixed tissue slides was performed using a fluorescence microscope equipped with a precision automated stage and appropriate optical filters. We developed a vignette correction algorithm to remove the tiling effect of stitched-images. The preliminary data from imaging fixed slides of breast tumor xenografts showed intratumor redox heterogeneity patterns similar to that of the frozen tissues imaged by the Chance redox scanner. From ORI of human breast tissue slides we identified certain redox differences among normal, ductal carcinoma in situ, and invasive carcinoma. We found paraformaldehyde fixation causes no change in NADH signals but enhances Fp signals of fresh muscle fibers. We also investigated the stability of the fluorescence microscope and reproducibility of tissue slide fluorescence signals. We plan to validate the diagnostic/prognostic value of ORI using clinically annotated breast cancer sample set from patients with long-term follow-up data.

A Comparison of Raman Spectral Features of Frozen and Deparaffinized Tissues in Neuroblastoma and Ganglioneuroma

NASA Astrophysics Data System (ADS)

Devpura, Suneetha; Thakur, Jagdish S.; Poulik, Janet M.; Rabah, Raja; Naik, Vaman M.; Naik, Ratna

2012-02-01

We have investigated the cellular regions in neuroblastoma and ganglioneuroma using Raman spectroscopy and compared their spectral characteristics with those of normal adrenal gland. Thin sections from both frozen and deparaffinized tissues, obtained from the same tissue specimen, were studied in conjunction with the pathological examination of the tissues. We found a significant difference in the spectral features of frozen sections of normal adrenal gland, neuroblastoma, and ganglioneuroma when compared to deparaffinized tissues. The quantitative analysis of the Raman data using chemometric methods of principal component analysis and discriminant function analysis obtained from the frozen tissues show a sensitivity and specificity of 100% each. The biochemical identification based on the spectral differences shows that the normal adrenal gland tissues have higher levels of carotenoids, lipids, and cholesterol compared to the neuroblastoma and ganglioneuroma frozen tissues. However, deparaffinized tissues show complete removal of these biochemicals in adrenal tissues. This study demonstrates that Raman spectroscopy combined with chemometric methods can successfully distinguish neuroblastoma and ganglioneuroma at cellular level.

Preparation and Immunoaffinity Depletion of Fresh Frozen Tissue Homogenates for Mass Spectrometry-Based Proteomics in the Context of Drug Target/Biomarker Discovery.

PubMed

Prieto, DaRue A; Chan, King C; Johann, Donald J; Ye, Xiaoying; Whitely, Gordon; Blonder, Josip

2017-01-01

The discovery of novel drug targets and biomarkers via mass spectrometry (MS)-based proteomic analysis of clinical specimens has proven to be challenging. The wide dynamic range of protein concentration in clinical specimens and the high background/noise originating from highly abundant proteins in tissue homogenates and serum/plasma encompass two major analytical obstacles. Immunoaffinity depletion of highly abundant blood-derived proteins from serum/plasma is a well-established approach adopted by numerous researchers; however, the utilization of this technique for immunodepletion of tissue homogenates obtained from fresh frozen clinical specimens is lacking. We first developed immunoaffinity depletion of highly abundant blood-derived proteins from tissue homogenates, using renal cell carcinoma as a model disease, and followed this study by applying it to different tissue types. Tissue homogenate immunoaffinity depletion of highly abundant proteins may be equally important as is the recognized need for depletion of serum/plasma, enabling more sensitive MS-based discovery of novel drug targets, and/or clinical biomarkers from complex clinical samples. Provided is a detailed protocol designed to guide the researcher through the preparation and immunoaffinity depletion of fresh frozen tissue homogenates for two-dimensional liquid chromatography, tandem mass spectrometry (2D-LC-MS/MS)-based molecular profiling of tissue specimens in the context of drug target and/or biomarker discovery.

A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors.

PubMed

Espinal, Allyson C; Wang, Dan; Yan, Li; Liu, Song; Tang, Li; Hu, Qiang; Morrison, Carl D; Ambrosone, Christine B; Higgins, Michael J; Sucheston-Campbell, Lara E

2017-02-28

DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples. For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER- tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types. Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER-) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort. FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues.

Avian toxicologic diagnosis

USGS Publications Warehouse

Sigurdson, C.J.; Franson, J.C.; Fudge, A.M.

2000-01-01

This chapter describes the sources and pathophysiology of some potential poisons that affect birds and summarizes useful laboratory tests. The diagnosis of poisoning in birds, as in mammals, requires a complete and accurate history, careful observation of clinical signs, and a thorough necropsy evaluation. Appropriate sample handling and analysis, based on consultation with the diagnostic toxicologist, are critical (Table 19--1). Veterinary toxicology laboratories are becoming increasingly specialized, with only certain laboratories capable of analyzing for drug residues or anticoagulants, for example. Although a local laboratory may not be able to fulfill a specific test request, they may recommend an alternative laboratory or may be willing to forward the sample. As a general rule in suspect poisoning cases, large tissue samples of liver, kidney, brain, and subcutaneous fat and of crop, proventriculus, and ventriculus contents should be collected at necropsy and frozen. Appropriate samples should be submitted frozen, with the remainder held in the freezer for possible later testing. A second set of tissues should be placed in 10% formalin for histopathologic examination.

Freezing without Ice Crystal Damage: Semithin and Ultrathin Frozen Sections of Ethanol-Infiltrated Tissue for Microscopy, with Applications to Immunocytochemistry

NASA Astrophysics Data System (ADS)

Christensen, A. Kent; Lowry, Terry B.

1995-10-01

Ethanol (ethyl alcohol) has long been a standard reagent used in preparing tissues for light and electron microscopy. After fixation, tissues are usually dehydrated with ethanol before being embedded in paraffin or plastic. In this study we show that the ethanol-infiltrated tissue can be frozen and sectioned directly without embedding. When tissue impregnated with ethanol is cooled below about [minus sign]117°C with liquid nitrogen, the ethanol solidifies without appreciable crystallization. The frozen tissue can then be sectioned in a commercial cryoultramicrotome that is set at [minus sign]155 to [minus sign]170°C to produce semithin frozen sections (0.5 to 3 [mu]m thick) for light microscopy or ultrathin frozen sections (50 to 100 nm thick) for electron microscopy. Sections are picked up and mounted on glass slides or EM grids by means that are in current use for ice ultrathin frozen sectioning. Because there is no apparent freezing damage, the morphology in these ethanol frozen sections of unembedded tissue appears generally quite good, often resembling that obtained by conventional EM techniques. Examples are provided that illustrate the use of this material for immunocytochemistry at the light and electron microscope levels.

Ecological-Evaluation of Organotin-Contaminated Sediment.

DTIC Science & Technology

1985-07-01

the potential for bioaccumulation of cadmium, chromium, copper, mercury , silver, pesticides, PCBs, petroleum hydrocarbons, and organotins RESULTS The...tissues were frozen for subsequent bioaccumulation estimates. Tissues and sediment samples were analyzed for cadmium, chromium, copper, mercury , silver...spectroscopy; mercury was analyzed by cold vapor atomic absorption spectroscopy. Pesticides, PCBs, and petroleum hydrocarbons were measured by gas

Long-term stability and temporal trends of organic contaminants in four collections of mussel tissue frozen standard reference materials.

PubMed

Schantz, Michele M; Pugh, Rebecca S; Pol, Stacy S Vander; Wise, Stephen A

2015-04-01

The stability of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and chlorinated pesticides in frozen mussel tissue Standard Reference Materials (SRMs) stored at -80 °C was assessed by analyzing samples of SRM 1974, SRM 1974a, and SRM 1974b Organics in Mussel Tissue (Mytilus edulis) periodically over 25 y, 20 y, and 12 y, respectively. The most recent analyses were performed during the certification of the fourth release of this material, SRM 1974c. Results indicate the concentrations of these persistent organic pollutants have not changed during storage at -80 °C. In addition, brominated diphenyl ethers (BDEs) were quantified in each of the materials during this study. The stability information is important for on-going monitoring studies collecting large quantities of samples for future analyses (i.e., formally established specimen banking programs). Since all four mussel tissue SRMs were prepared from mussels collected at the same site in Dorchester Bay, MA, USA, the results provide a temporal trend study for these contaminants over a 17 year period (1987 to 2004).

Preservation of Multiple Mammalian Tissues to Maximize Science Return from Ground Based and Spaceflight Experiments.

PubMed

Choi, Sungshin; Ray, Hami E; Lai, San-Huei; Alwood, Joshua S; Globus, Ruth K

2016-01-01

Even with recent scientific advancements, challenges posed by limited resources and capabilities at the time of sample dissection continue to limit the collection of high quality tissues from experiments that can be conducted only infrequently and at high cost, such as in space. The resources and time it takes to harvest tissues post-euthanasia, and the methods and duration of long duration storage, potentially have negative impacts on sample quantity and quality, thereby limiting the scientific outcome that can be achieved. The goals of this study were to optimize methods for both sample recovery and science return from rodent experiments, with possible relevance to both ground based and spaceflight studies. The first objective was to determine the impacts of tissue harvest time post-euthanasia, preservation methods, and storage duration, focusing on RNA quality and enzyme activities in liver and spleen as indices of sample quality. The second objective was to develop methods that will maximize science return by dissecting multiple tissues after long duration storage in situ at -80°C. Tissues of C57Bl/6J mice were dissected and preserved at various time points post-euthanasia and stored at -80°C for up to 11 months. In some experiments, tissues were recovered from frozen carcasses which had been stored at -80°C up to 7 months. RNA quantity and quality was assessed by measuring RNA Integrity Number (RIN) values using an Agilent Bioanalyzer. Additionally, the quality of tissues was assessed by measuring activities of hepatic enzymes (catalase, glutathione reductase and GAPDH). Fresh tissues were collected up to one hour post-euthanasia, and stored up to 11 months at -80°C, with minimal adverse effects on the RNA quality of either livers or RNAlater-preserved spleens. Liver enzyme activities were similar to those of positive controls, with no significant effect observed at any time point. Tissues dissected from frozen carcasses that had been stored for up to 7 months at -80°C had variable results, depending on the specific tissue analyzed. RNA quality of liver, heart, and kidneys were minimally affected after 6-7 months of storage at -80°C, whereas RNA degradation was evident in tissues such as small intestine, bone, and bone marrow when they were collected from the carcasses frozen for 2.5 months. These results demonstrate that 1) the protocols developed for spaceflight experiments with on-orbit dissections support the retrieval of high quality samples for RNA expression and some protein analyses, despite delayed preservation post-euthanasia or prolonged storage, and 2) many additional tissues for gene expression analysis can be obtained by dissection even following prolonged storage of the tissue in situ at -80°C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to spaceflight experiments that entail on-orbit sample recovery by astronauts.

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas.

PubMed

Mohapatra, Gayatry; Engler, David A; Starbuck, Kristen D; Kim, James C; Bernay, Derek C; Scangas, George A; Rousseau, Audrey; Batchelor, Tracy T; Betensky, Rebecca A; Louis, David N

2011-04-01

Array comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA). Because diffuse malignant gliomas are often sampled by small biopsies, formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis; FFPE tissues are also needed to study the intratumoral heterogeneity that characterizes these neoplasms. In this paper, we present a combination of evaluations and technical advances that provide strong support for the ready use of oligonucleotide aCGH on FFPE diffuse gliomas. We first compared aCGH using bacterial artificial chromosome (BAC) arrays in 45 paired frozen and FFPE gliomas, and demonstrate a high concordance rate between FFPE and frozen DNA in an individual clone-level analysis of sensitivity and specificity, assuring that under certain array conditions, frozen and FFPE DNA can perform nearly identically. However, because oligonucleotide arrays offer advantages to BAC arrays in genomic coverage and practical availability, we next developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. To demonstrate utility in FFPE tissues, we applied this approach to biphasic anaplastic oligoastrocytomas and demonstrate CNA differences between DNA obtained from the two components. Therefore, BAC and oligonucleotide aCGH can be sensitive and specific tools for detecting CNAs in FFPE DNA, and novel labeling techniques enable the routine use of oligonucleotide arrays for FFPE DNA. In combination, these advances should facilitate genome-wide analysis of rare, small and/or histologically heterogeneous gliomas from FFPE tissues.

Precision of Multiple Reaction Monitoring Mass Spectrometry Analysis of Formalin-Fixed, Paraffin-Embedded Tissue

PubMed Central

2012-01-01

We compared the reproducibility of multiple reaction monitoring (MRM) mass spectrometry-based peptide quantitation in tryptic digests from formalin-fixed, paraffin-embedded (FFPE) and frozen clear cell renal cell carcinoma tissues. The analyses targeted a candidate set of 114 peptides previously identified in shotgun proteomic analyses, of which 104 were detectable in FFPE and frozen tissue. Although signal intensities for MRM of peptides from FFPE tissue were on average 66% of those in frozen tissue, median coefficients of variation (CV) for measurements in FFPE and frozen tissues were nearly identical (18–20%). Measurements of lysine C-terminal peptides and arginine C-terminal peptides from FFPE tissue were similarly reproducible (19.5% and 18.3% median CV, respectively). We further evaluated the precision of MRM-based quantitation by analysis of peptides from the Her2 receptor in FFPE and frozen tissues from a Her2 overexpressing mouse xenograft model of breast cancer and in human FFPE breast cancer specimens. We obtained equivalent MRM measurements of HER2 receptor levels in FFPE and frozen mouse xenografts derived from HER2-overexpressing BT474 cells and HER2-negative Sum159 cells. MRM analyses of 5 HER2-positive and 5 HER-negative human FFPE breast tumors confirmed the results of immunohistochemical analyses, thus demonstrating the feasibility of HER2 protein quantification in FFPE tissue specimens. The data demonstrate that MRM analyses can be performed with equal precision on FFPE and frozen tissues and that lysine-containing peptides can be selected for quantitative comparisons, despite the greater impact of formalin fixation on lysine residues. The data further illustrate the feasibility of applying MRM to quantify clinically important tissue biomarkers in FFPE specimens. PMID:22530795

A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors

PubMed Central

Yan, Li; Liu, Song; Tang, Li; Hu, Qiang; Morrison, Carl D.; Ambrosone, Christine B.; Higgins, Michael J.; Sucheston-Campbell, Lara E.

2017-01-01

Background DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples. Results For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER– tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types. Materials and Methods Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER−) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort. Conclusions FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues. PMID:28118602

Time-resolved fluorescence (TRF) and diffuse reflectance spectroscopy (DRS) for margin analysis in breast cancer.

PubMed

Shalaby, Nourhan; Al-Ebraheem, Alia; Le, Du; Cornacchi, Sylvie; Fang, Qiyin; Farrell, Thomas; Lovrics, Peter; Gohla, Gabriela; Reid, Susan; Hodgson, Nicole; Farquharson, Michael

2018-03-01

One of the major problems in breast cancer surgery is defining surgical margins and establishing complete tumor excision within a single surgical procedure. The goal of this work is to establish instrumentation that can differentiate between tumor and normal breast tissue with the potential to be implemented in vivo during a surgical procedure. A time-resolved fluorescence and reflectance spectroscopy (tr-FRS) system is used to measure fluorescence intensity and lifetime as well as collect diffuse reflectance (DR) of breast tissue, which can subsequently be used to extract optical properties (absorption and reduced scatter coefficient) of the tissue. The tr-FRS data obtained from patients with Invasive Ductal Carcinoma (IDC) whom have undergone lumpectomy and mastectomy surgeries is presented. A preliminary study was conducted to determine the validity of using banked pre-frozen breast tissue samples to study the fluorescence response and optical properties. Once the validity was established, the tr-FRS system was used on a data-set of 40 pre-frozen matched pair cases to differentiate between tumor and normal breast tissue. All measurements have been conducted on excised normal and tumor breast samples post surgery. Our results showed the process of freezing and thawing did not cause any significant differences between fresh and pre-frozen normal or tumor breast tissue. The tr-FRS optical data obtained from 40 banked matched pairs showed significant differences between normal and tumor breast tissue. The work detailed in the main study showed the tr-FRS system has the potential to differentiate malignant from normal breast tissue in women undergoing surgery for known invasive ductal carcinoma. With further work, this successful outcome may result in the development of an accurate intraoperative real-time margin assessment system. Lasers Surg. Med. 50:236-245, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

An efficient field and laboratory workflow for plant phylotranscriptomic projects1

PubMed Central

Yang, Ya; Moore, Michael J.; Brockington, Samuel F.; Timoneda, Alfonso; Feng, Tao; Marx, Hannah E.; Walker, Joseph F.; Smith, Stephen A.

2017-01-01

Premise of the study: We describe a field and laboratory workflow developed for plant phylotranscriptomic projects that involves cryogenic tissue collection in the field, RNA extraction and quality control, and library preparation. We also make recommendations for sample curation. Methods and Results: A total of 216 frozen tissue samples of Caryophyllales and other angiosperm taxa were collected from the field or botanical gardens. RNA was extracted, stranded mRNA libraries were prepared, and libraries were sequenced on Illumina HiSeq platforms. These included difficult mucilaginous tissues such as those of Cactaceae and Droseraceae. Conclusions: Our workflow is not only cost effective (ca. $270 per sample, as of August 2016, from tissue to reads) and time efficient (less than 50 h for 10–12 samples including all laboratory work and sample curation), but also has proven robust for extraction of difficult samples such as tissues containing high levels of secondary compounds. PMID:28337391

Impact of Collection and Storage of Lung Tumor Tissue on Whole Genome Expression Profiling

PubMed Central

Freidin, Maxim B.; Bhudia, Neesa; Lim, Eric; Nicholson, Andrew G.; Cookson, William O.; Moffatt, Miriam F.

2012-01-01

Gene expression profiling could assist in revealing biomarkers of lung cancer prognosis and progression. The handling of biological samples may strongly influence global gene expression, a fact that has not been addressed in many studies. We sought to investigate the changes in gene expression that may occur as a result of sample processing time and conditions. Using Illumina Human WG-6 arrays, we quantified gene expression in lung carcinoma samples from six patients obtained at chest opening before and immediately after lung resection with storage in RNAlater [T1a(CO) and T1b(LR)], after receipt of the sample for histopathology, placed in RNAlater [T2a(HP)]; snap frozen [T2b(HP.SF)]; or snap frozen and stored for 1 week [T2c(HP.SFA)], as well as formalin-fixed, paraffin-embedded (FFPE) block samples. Sampling immediately after resection closely represented the tissue obtained in situ, with only 1% of genes differing more than twofold [T1a(CO) versus T1b(LR)]. Delaying tissue harvest for an average of 30 minutes from the operating theater had a significant impact on gene expression, with approximately 25% of genes differing between T1a(CO) and T2a(HP). Many genes previously identified as lung cancer biomarkers were altered during this period. Examination of FFPE specimens showed minimal correlation with fresh samples. This study shows that tissue collection immediately after lung resection with conservation in RNAlater is an optimal strategy for gene expression profiling. PMID:22240448

A Study of Some Problems in Network Information Theory

ERIC Educational Resources Information Center

Kamath, Sudeep Uday

2013-01-01

Red snapper, "Lutjanus campechanus," were sampled with hook and line at natural (n = 33) and artificial (n = 27) reef sites in the northern Gulf of Mexico from 2009-2011. Stomachs (n = 708) were extracted and their contents preserved for gut content analysis, and muscle tissue samples (n = 200) were dissected and frozen for stable…

Validation of cold chain shipping environment for transport of allografts as part of a human tissue bank returns policy.

PubMed

Rooney, P; Eagle, M J; Kearney, J N

2015-12-01

Human tissue is shipped to surgeons in the UK in either a freeze-dried or frozen state. To ensure quality and safety of the tissue, frozen tissue must be shipped in insulated containers such that tissue is maintained at an appropriate temperature. UK Blood Transfusion Service regulations state "Transportation systems must be validated to show maintenance of the required storage temperature" and also state that frozen, non-cryopreserved tissue "must be transported… at -20 °C or lower" (Guidelines for the Blood Transfusion Services in the United Kingdom, 8th Edn. 2013). To maintain an expiry date for frozen tissue longer than 6 months, the tissue must be maintained at a temperature of -40 °C or below. The objective of this study was to evaluate and validate the capability of a commercially available insulated polystyrene carton (XPL10), packed with dry ice, to maintain tissue temperature below -40 °C. Tissue temperature of a single frozen femoral head or a single frozen Achilles tendon, was recorded over a 4-day period at 37 °C, inside a XPL10 carton with dry ice as refrigerant. The data demonstrate that at 37 °C, the XPL10 carton with 9.5 kg of dry ice maintained femoral head and tendon tissue temperature below -55 °C for at least 48 h; tissue temperature did not rise above -40 °C until at least 70 h. Data also indicated that at a storage temperature lower than 37 °C, tissue temperature was maintained for longer periods.

Handbook of Human Tissue Sources. A National Resource of Human Tissue Samples

DTIC Science & Technology

1999-01-01

be frozen and thawed and still be viable for artificial insemination procedures or implan- tation. The newest type of human tissue storage for future...use is the storage of umbilical cord blood. SPERM, OVUM, AND EMBRYO BANKS Artificial insemination or donor insemination (DI) is a procedure to...anonymous human sperm for use in artificial insemination ; long-term semen storage for men facing the possibility of steril- ization, reduction in fertility

Tissue Fixation and Processing for the Histological Identification of Lipids.

PubMed

Carriel, Víctor; Campos, Fernando; Aneiros-Fernández, José; Kiernan, John A

2017-01-01

Lipids are a heterogeneous group of substances characterized by their solubility in organic solvents and insolubility in water. Lipids can be found as normal components of different tissues and organs, and they can be affected by several pathological conditions. The histochemical identification of lipids plays an important role in histopathological diagnosis and research, but successful staining depends on adequate fixation and processing of the tissue. Here we describe methods to fix and process tissue samples for the histochemical identification of lipids in frozen or paraffin-embedded tissues.

The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings

PubMed Central

Shabihkhani, Maryam; Lucey, Gregory M.; Wei, Bowen; Mareninov, Sergey; Lou, Jerry J.; Vinters, Harry V.; Singer, Elyse J.; Cloughesy, Timothy F.; Yong, William H.

2014-01-01

Well preserved frozen biospecimens are ideal for evaluating the genome, transcriptome, and proteome. While papers reviewing individual aspects of frozen biospecimens are available, we present a current overview of experimental data regarding procurement, storage, and quality assurance that can inform the handling of frozen biospecimens. Frozen biospecimen degradation can be influenced by factors independent of the collection methodology including tissue type, premortem agonal changes, and warm ischemia time during surgery. Rapid stabilization of tissues by snap freezing immediately can mitigate artifactually altered gene expression and, less appreciated, protein phosphorylation profiles. Collection protocols may be adjusted for specific tissue types as cellular ischemia tolerance varies widely. If data is not available for a particular tissue type, a practical goal is snap freezing within 20 minutes. Tolerance for freeze-thaw events is also tissue type dependent. Tissue storage at −80°C can preserve DNA and protein for years but RNA can show degradation at 5 years. For −80°C freezers, aliquots frozen in RNAlater or similar RNA stabilizing solutions is a consideration. It remains unresolved as to whether storage at −150°C provides significant advantages relative to −80°C. Histologic quality assurance of tissue biospecimens is typically performed at the time of surgery but should also be conducted on the aliquot to be distributed because of tissue heterogeneity. Biobanking protocols for blood and its components are highly dependent on intended use and multiple collection tube types may be needed. Additional quality assurance testing should be dictated by the anticipated downstream applications. PMID:24424103

Mass spectrometric imaging and laser desorption ionization (LDI) with ice as a matrix using femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Berry, Jamal Ihsan

The desorption of biomolecules from frozen aqueous solutions on metal substrates with femtosecond laser pulses is presented for the first time. Unlike previous studies using nanosecond pulses, this approach produces high quality mass spectra of biomolecules repeatedly and reproducibly. This novel technique allows analysis of biomolecules directly from their native frozen environments. The motivation for this technique stems from molecular dynamics computer simulations comparing nanosecond and picosecond heating of water overlayers frozen on Au substrates which demonstrate large water cluster formation and ejection upon substrate heating within ultrashort timescales. As the frozen aqueous matrix and analyte molecules are transparent at the wavelengths used, the laser energy is primarily absorbed by the substrate, causing rapid heating and explosive boiling of the ice overlayer, followed by the ejection of ice clusters and the entrained analyte molecule. Spectral characteristics at a relatively high fluence of 10 J/cm 2 reveal the presence of large molecular weight metal clusters when a gold substrate is employed, with smaller cluster species observed from frozen aqueous solutions on Ag, Cu, and Pb substrates. The presence of the metal clusters is indicative of an evaporative cooling mechanism which stabiles cluster ion formation and the ejection of biomolecules from frozen aqueous solutions. Solvation is necessary as the presence of metal clusters and biomolecular ion signals are not observed from bare metal substrates in absence of the frozen overlayer. The potential for mass spectrometric imaging with femtosecond LDI of frozen samples is also presented. The initial results for the characterization of peptides and peptoids linked to combinatorial beads frozen in ice and the assay of frozen brain tissue from the serotonin transporter gene knockout mouse via LDI imaging are discussed. Images of very good quality and resolution are obtained with 400 nm, 200 fs pulses at a fluence of 1.25 J/cm2 . An attractive feature of this technique is that images are acquired within minutes for large sample areas. Additionally, the images obtained with femtosecond laser desorption are high in lateral resolution with the laser capable of being focused to a spot size of 30 mum. Femtosecond laser desorption from ice is unique in that unlike matrix assisted laser desorption ionization mass spectrometry, it does not employ an organic UV absorbing matrix to desorb molecular ions. Instead, the laser energy is absorbed by the metal substrate causing explosive boiling and ejection of the frozen overlayer. This approach is significant in that femtosecond laser desorption possess the potential of analyzing and assaying biomolecules directly from their frozen native environments. This technique was developed to compliment existing ToF-SIMS imaging capability for analysis of tissue and cells, as well as other biological systems of interest.

Traumatic Hemothorax Blood Contains Elevated Levels of Microparticles that are Prothrombotic but Inhibit Platelet Aggregation.

PubMed

Mitchell, Thomas A; Herzig, Maryanne C; Fedyk, Chriselda G; Salhanick, Marc A; Henderson, Aaron T; Parida, Bijaya K; Prat, Nicolas J; Dent, Daniel L; Schwacha, Martin G; Cap, Andrew P

2017-06-01

Autotransfusion of shed blood from traumatic hemothorax is an attractive option for resuscitation of trauma patients in austere environments. However, previous analyses revealed that shed hemothorax (HX) blood is defibrinated, thrombocytopenic, and contains elevated levels of D-dimer. Mixing studies with normal pooled plasma demonstrated hypercoagulability, evoking concern for potentiation of acute traumatic coagulopathy. We hypothesized that induction of coagulopathic changes by shed HX blood may be due to increases in cellular microparticles (MP) and that these may also affect recipient platelet function. Shed HX blood was obtained from 17 adult trauma patients under an Institutional Review Board approved prospective observational protocol. Blood samples were collected every hour up to 4 h after thoracostomy tube placement. The corresponding plasma was isolated and frozen for analysis. The effects of shed HX frozen plasma (HFP) and isolated HX microparticles (HMP) on coagulation and platelet function were assessed through mixing studies with platelet-rich plasma at various dilutions followed by analysis with thromboelastometry (ROTEM), platelet aggregometry (Multiplate), enzyme-linked immunosorbent assays, and flow cytometry. Furthermore, HFP was assessed for von Willebrand factor antigen levels and multimer content, and plasma-free hemoglobin. ROTEM analysis demonstrated that diluted HFP and isolated HMP samples decreased clotting time, clotting formation time, and increased α angle, irrespective of sample concentrations, when compared with diluted control plasma. Isolated HMP inhibited platelet aggregation in response to adenosine diphosphate, arachidonic acid, and collagen. HFP contained elevated levels of fibrin-degradation products and tissue factor compared with control fresh frozen plasma samples. MP concentrations in HFP were significantly increased and enriched in events positive for phosphatidylserine, tissue factor, CD235, CD45, CD41a, and CD14. von Willebrand factor (vWF) multimer analysis revealed significant loss of high molecular weight multimers in HFP samples. Plasma-free hemoglobin levels were 8-fold higher in HFP compared with fresh frozen plasma. HFP induces plasma hypercoagulability that is likely related to increased tissue factor and phosphatidylserine expression originating from cell-derived MP. In contrast, platelet dysfunction is induced by HMP, potentially aggravated by depletion of high molecular weight multimers of vWF. Thus, autologous transfusion of shed traumatic hemothorax blood may induce a range of undesirable effects in patients with acute traumatic coagulopathy.

Deep Sequencing to Identify the Causes of Viral Encephalitis

PubMed Central

Chan, Benjamin K.; Wilson, Theodore; Fischer, Kael F.; Kriesel, John D.

2014-01-01

Deep sequencing allows for a rapid, accurate characterization of microbial DNA and RNA sequences in many types of samples. Deep sequencing (also called next generation sequencing or NGS) is being developed to assist with the diagnosis of a wide variety of infectious diseases. In this study, seven frozen brain samples from deceased subjects with recent encephalitis were investigated. RNA from each sample was extracted, randomly reverse transcribed and sequenced. The sequence analysis was performed in a blinded fashion and confirmed with pathogen-specific PCR. This analysis successfully identified measles virus sequences in two brain samples and herpes simplex virus type-1 sequences in three brain samples. No pathogen was identified in the other two brain specimens. These results were concordant with pathogen-specific PCR and partially concordant with prior neuropathological examinations, demonstrating that deep sequencing can accurately identify viral infections in frozen brain tissue. PMID:24699691

The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane.

PubMed

Russo, Alessandra; Bonci, Paola; Bonci, Paolo

2012-06-01

The aim of this work is to quantify the total protein and growth factors content in a tissue-suspension obtained from processed human amniotic membrane (hAM). hAM was collected, frozen, freeze dried, powdered and sterilized by γ-irradiation. At each step of the process, samples were characterized for the total protein amounts by a Bradford protein assay and for the growth factor concentrations by ELISA test of the tissue suspensions. Frozen-hAM samples show higher release of total proteins and specific growth factors in the tissue suspension in comparison with freeze-dried hAM. We observed that even if the protein extraction is hindered once the tissue is dried, the powdering process allows a greater release in the tissue suspension of total proteins and growth factors after tissue re-solubilization in comparison with only the freeze-drying process (+91 ± 13% for EGF, +16 ± 4% for HGF, +11 ± 5% for FGF, +16 ± 9% for TGF-β1), and a greater release of EGF (85 ± 10%) in comparison with only the freezing process, because proteins become much readily solubilized in the solution. According with these results, we describe a protocol to obtain a new sterile biological product from hAM tissue, with well-known effects of thermal, mechanical and physical processes on the total protein and grow factors contents.

Development of a compact terahertz time-domain spectrometer for the measurement of the optical properties of biological tissues.

PubMed

Wilmink, Gerald J; Ibey, Bennett L; Tongue, Thomas; Schulkin, Brian; Laman, Norman; Peralta, Xomalin G; Roth, Caleb C; Cerna, Cesario Z; Rivest, Benjamin D; Grundt, Jessica E; Roach, William P

2011-04-01

Terahertz spectrometers and imaging systems are currently being evaluated as biomedical tools for skin burn assessment. These systems show promise, but due to their size and weight, they have restricted portability, and are impractical for military and battlefield settings where space is limited. In this study, we developed and tested the performance of a compact, light, and portable THz time-domain spectroscopy (THz-TDS) device. Optical properties were collected with this system from 0.1 to 1.6 THz for water, ethanol, and several ex vivo porcine tissues (muscle, adipose, skin). For all samples tested, we found that the index of refraction (n) decreases with frequency, while the absorption coefficient (μ(a)) increases with frequency. Muscle, adipose, and frozen/thawed skin samples exhibited comparable n values ranging between 2.5 and 2.0, whereas the n values for freshly harvested skin were roughly 40% lower. Additionally, we found that the freshly harvested samples exhibited higher μ(a) values than the frozen/thawed skin samples. Overall, for all liquids and tissues tested, we found that our system measured optical property values that were consistent with those reported in the literature. These results suggest that our compact THz spectrometer performed comparable to its larger counterparts, and therefore may be a useful and practical tool for skin health assessment.

Tissue spray ionization mass spectrometry for rapid recognition of human lung squamous cell carcinoma

NASA Astrophysics Data System (ADS)

Wei, Yiping; Chen, Liru; Zhou, Wei; Chingin, Konstantin; Ouyang, Yongzhong; Zhu, Tenggao; Wen, Hua; Ding, Jianhua; Xu, Jianjun; Chen, Huanwen

2015-05-01

Tissue spray ionization mass spectrometry (TSI-MS) directly on small tissue samples has been shown to provide highly specific molecular information. In this study, we apply this method to the analysis of 38 pairs of human lung squamous cell carcinoma tissue (cancer) and adjacent normal lung tissue (normal). The main components of pulmonary surfactants, dipalmitoyl phosphatidylcholine (DPPC, m/z 757.47), phosphatidylcholine (POPC, m/z 782.52), oleoyl phosphatidylcholine (DOPC, m/z 808.49), and arachidonic acid stearoyl phosphatidylcholine (SAPC, m/z 832.43), were identified using high-resolution tandem mass spectrometry. Monte Carlo sampling partial least squares linear discriminant analysis (PLS-LDA) was used to distinguish full-mass-range mass spectra of cancer samples from the mass spectra of normal tissues. With 5 principal components and 30 - 40 Monte Carlo samplings, the accuracy of cancer identification in matched tissue samples reached 94.42%. Classification of a tissue sample required less than 1 min, which is much faster than the analysis of frozen sections. The rapid, in situ diagnosis with minimal sample consumption provided by TSI-MS is advantageous for surgeons. TSI-MS allows them to make more informed decisions during surgery.

In Vitro Tissue Differentiation using Dynamics of Tissue Mechanical Properties

NASA Astrophysics Data System (ADS)

Lin, Wei-Chiang; Phillips, Paul J.

2002-03-01

Dynamics of tissue mechanical properties of various human tissue types were studied at macroscopic as well as microscopic level in vitro. This study was conducted to enable the development of a feedback system based on dynamics of tissue mechanical properties for intraoperative guidance for tumor treatment (e.g., RF ablation of liver tumor) and noninvasive tumor localization. Human liver tissues, including normal, cancerous, and cirrhotic tissues, were obtained from patients receiving liver transplant or tumor resection at Vanderbilt University Medical Center with the approval of the Vanderbilt Institutional Review Board. Tissue samples, once resected from the patients, were snap-frozen using liquid nitrogen and stored at -70 oC. Measurements of the mechanical properties of these tissue samples were conducted at the University of Tennessee at Knoxville. Dynamics of tissue mechanical properties were measured from both native and thermally coagulated tissue samples at macroscopic and microscopic level. Preliminary results suggest the dynamics of mechanical properties of normal liver tissues are very different from those of cancerous liver tissues. The correlation between the dynamics of mechanical properties at macroscopic level and those at microscopic level is currently under investigation.

Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas.

PubMed

Butler, Alexandra E; Matveyenko, Aleksey V; Kirakossian, David; Park, Johanna; Gurlo, Tatyana; Butler, Peter C

Laser capture microdissection (LCM) is a powerful method to isolate specific populations of cells for subsequent analysis such as gene expression profiling, for example, microarrays or ribonucleic (RNA)-Seq. This technique has been applied to frozen as well as formalin-fixed, paraffin-embedded (FFPE) specimens with variable outcomes regarding quality and quantity of extracted RNA. The goal of the study was to develop the methods to isolate high-quality RNA from islets of Langerhans and pancreatic duct glands (PDG) isolated by LCM. We report an optimized protocol for frozen sections to minimize RNA degradation and maximize recovery of expected transcripts from the samples using quantitative real-time polymerase chain reaction (RT-PCR) by adding RNase inhibitors at multiple steps during the experiment. This technique reproducibly delivered intact RNA (RIN values 6-7). Using quantitative RT-PCR, the expected profiles of insulin, glucagon, mucin6 (Muc6), and cytokeratin-19 (CK-19) mRNA in PDGs and pancreatic islets were detected. The described experimental protocol for frozen pancreas tissue might also be useful for other tissues with moderate to high levels of intrinsic ribonuclease (RNase) activity.

Development and Translation of a Tissue- Engineered Disc in a Preclinical Rodent Model

DTIC Science & Technology

2011-10-01

samples were stored frozen, lyophilized, papain digested and assayed for collagen, GAG, and DNA content. Likewise, media in both shaken and static...construct dynamic and equilibrium properties. Total dsDNA, sulfated glycosaminoglycan (s-GAG), and collagen content was determined after papain

The Impact of Repeated Freeze-Thaw Cycles on the Quality of Biomolecules in Four Different Tissues.

PubMed

Ji, Xiaoli; Wang, Min; Li, Lingling; Chen, Fang; Zhang, Yanyang; Li, Qian; Zhou, Junmei

2017-10-01

High-quality biosamples are valuable resources for biomedical research. However, some tissues are stored without being sectioned into small aliquots and have to undergo repeated freeze-thaw cycles throughout prolonged experimentation. Little is known regarding the effects of repeated freeze-thaw cycles on the quality of biomolecules in tissues. The aim of this study was to evaluate the impact of repeated freeze-thaw (at room temperature or on ice) cycles on biomolecules and gene expression in four different types of tissues. Each fresh tissue was sectioned into seven aliquots and snap-frozen before undergoing repeated freeze-thaw cycles at room temperature or on ice. Biomolecules were extracted and analyzed. Both relative and absolute quantification were used to detect the changes in gene expression. The results indicated that the impact of repeated freeze-thaw cycles on RNA integrity varied by tissue type. Gene expression, including the housekeeping gene, was affected in RNA-degraded samples according to absolute quantification rather than relative quantification. Furthermore, our results suggest that thawing on ice could protect RNA integrity compared with thawing at room temperature. No obvious degradation of protein or DNA was observed with repeated freeze-thaw cycles either at room temperature or on ice. This research provides ample evidence for the necessity of sectioning fresh tissues into small aliquots before snap-freezing, thus avoiding degradation of RNA and alteration of gene expression resulting from repeated freeze-thaw cycles. For frozen tissue samples that were already in storage and had to be used repeatedly during their lifecycle, thawing on ice or sectioned at ultralow temperature is recommended.

Histologic and ultrastructural evaluation of fresh and frozen-thawed human ovarian xenografts in nude mice.

PubMed

Nisolle, M; Casanas-Roux, F; Qu, J; Motta, P; Donnez, J

2000-07-01

To compare histologic and ultrastructural characteristics of fresh and frozen-thawed human ovarian cortical tissue grafted into nude mice. Experimental prospective study. An academic research environment. Ovarian biopsy specimens were obtained from 13 women undergoing laparoscopy for tubal ligation or infertility. Forty nude mice. A minilaparotomy was performed to place fresh and frozen-thawed ovarian grafts subcutaneously (sc) or intraperitoneally (ip). Removal of the ovarian grafts was performed at 24 days. [1] the follicular population, [2] fibrosis, [3] vascularization of the grafted tissue, and [4] ultrastructural evaluation. A greater fibrosis relative surface area was noted in frozen-thawed transplanted tissue than in fresh transplants. Regardless of this fibrosis, a similar follicular density was observed in fresh and frozen-thawed ovarian tissue 24 days after transplantation. Active angiogenesis was proved by both immunohistochemical study of the vascular endothelial growth factor and morphometric study of the vascular network. Normal ultrastructural characteristics were noted in frozen-thawed ovarian biopsies. Angiogenesis allows implantation of the graft even if it has been cryopreserved and thawed similarly to implantation of fresh tissue. The greater fibrosis observed in grafts after cryopreservation and implantation does not seem to affect the primordial and primary ovocyte population and their ultrastructural characteristics, but further studies must be conducted to prove that after cryopreservation and transplantation, ovocytes may achieve full maturation and fertilization.

Lack of concordance in microarray gene expression responses to Phenobarbital in companion aged FFPE and Frozen liver samples

EPA Science Inventory

Despite the immense potential value of public and private biorepositories, direct utilization of archival tissues for molecular profiling has been limited. A major reason for this limited use is the difficulty in obtaining reliable transcriptomic profiles from formalin-fixed par...

Effects of different freezing methods on calcium enriched papaya (Carica papaya L.).

PubMed

Lovera, Nancy N; Ramallo, Laura; Salvadori, Viviana O

2018-06-01

The effect of calcium impregnation on drip loss, colour, mechanical properties, sensory perception and freezing time on frozen-thawed papaya was studied, evaluating different freezing methods: cryogenic, tunnel and household freezer freezing. Osmotic dehydration as pre-treatment was also evaluated. Freezing in liquid nitrogen was considered an inappropriate method for papaya preservation due to cracking. Calcium impregnation and osmotic dehydration increased tissue firmness and decreased freezing time (freezing time for fresh, calcium impregnated and osmo-dehydrated fruit was 23, 17 and 5 min in a tunnel and 118, 83 and 60 min in a household freezer, respectively). Calcium lactate was the most effective way to protect tissue's firmness before and after a freeze-thaw cycle (maximum stress values approx. 300-400% of the raw tissue for tunnel freezing and 260% for household freezer). Microstructure analysis showed better tissue integrity retention in papaya samples impregnated with calcium lactate than in those with calcium gluconate, after a freezing-thawing cycle, in agreement with the drip loss results. In spite of these results, consumers preferred frozen papaya without pre-treatment or impregnated with calcium gluconate.

Automated cell disruption is a reliable and effective method of isolating RNA from fresh snap-frozen normal and malignant oral mucosa samples.

PubMed

Van der Vorst, Sébastien; Dekairelle, Anne-France; Irenge, Léonid; Hamoir, Marc; Robert, Annie; Gala, Jean-Luc

2009-01-01

This study compared automated vs. manual tissue grinding in terms of RNA yield obtained from oral mucosa biopsies. A total of 20 patients undergoing uvulectomy for sleep-related disorders and 10 patients undergoing biopsy for head and neck squamous cell carcinoma were enrolled in the study. Samples were collected, snap-frozen in liquid nitrogen, and divided into two parts of similar weight. Sample grinding was performed on one sample from each pair, either manually or using an automated cell disruptor. The performance and efficacy of each homogenization approach was compared in terms of total RNA yield (spectrophotometry, fluorometry), mRNA quantity [densitometry of specific TP53 amplicons and TP53 quantitative reverse-transcribed real-time PCR (qRT-PCR)], and mRNA quality (functional analysis of separated alleles in yeast). Although spectrophotometry and fluorometry results were comparable for both homogenization methods, TP53 expression values obtained by amplicon densitometry and qRT-PCR were significantly and consistently better after automated homogenization (p<0.005) for both uvula and tumor samples. Functional analysis of separated alleles in yeast results was better with the automated technique for tumor samples. Automated tissue homogenization appears to be a versatile, quick, and reliable method of cell disruption and is especially useful in the case of small malignant samples, which show unreliable results when processed by manual homogenization.

Analysis of human tissues by total reflection X-ray fluorescence. Application of chemometrics for diagnostic cancer recognition

NASA Astrophysics Data System (ADS)

Benninghoff, L.; von Czarnowski, D.; Denkhaus, E.; Lemke, K.

1997-07-01

For the determination of trace element distributions of more than 20 elements in malignant and normal tissues of the human colon, tissue samples (approx. 400 mg wet weight) were digested with 3 ml of nitric acid (sub-boiled quality) by use of an autoclave system. The accuracy of measurements has been investigated by using certified materials. The analytical results were evaluated by using a spreadsheet program to give an overview of the element distribution in cancerous samples and in normal colon tissues. A further application, cluster analysis of the analytical results, was introduced to demonstrate the possibility of classification for cancer diagnosis. To confirm the results of cluster analysis, multivariate three-way principal component analysis was performed. Additionally, microtome frozen sections (10 μm) were prepared from the same tissue samples to compare the analytical results, i.e. the mass fractions of elements, according to the preparation method and to exclude systematic errors depending on the inhomogeneity of the tissues.

Isochoric and isobaric freezing of fish muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Năstase, Gabriel; Department of Building Services, University of Transilvania, Braşov, Braşov, 500152; Lyu, Chenang

We have recently shown that, a living organism, which succumbs to freezing to −4 °C in an isobaric thermodynamic system (constant atmospheric pressure), can survive freezing to −4 °C in an isochoric thermodynamic system (constant volume). It is known that the mechanism of cell damage in an isobaric system is the freezing caused increase in extracellular osmolality, and, the consequent cell dehydration. An explanation for the observed survival during isochoric freezing is the thermodynamic modeling supported hypothesis that, in the isochoric frozen solution the extracellular osmolality is comparable to the cell intracellular osmolality. Therefore, cells in the isochoric frozen organism do not dehydrate, andmore » the tissue maintains its morphological integrity. Comparing the histology of: a) fresh fish white muscle, b) fresh muscle frozen to −5 °C in an isobaric system and c) fresh muscle frozen to −5 °C I in an isochoric system, we find convincing evidence of the mechanism of cell dehydration during isobaric freezing. In contrast, the muscle tissue frozen to −5 °C in an isochoric system appears morphologically identical to fresh tissue, with no evidence of dehydration. This is the first experimental evidence in support of the hypothesis that in isochoric freezing there is no cellular dehydration and therefore the morphology of the frozen tissue remains intact. - Highlights: • Preservation of fish muscle at, subfreezing temperatures, in an isochoric system, is demonstrated. • Experiments were performed to an average pressure of 41.3 MPa and temperatures of −5 °C. • Isochoric subfreezing temperature is a new preservation method that does not require the.use of cryoprotectants. • No cellular dehydration and therefore the morphology of the frozen tissue remains intact.« less

Microscopic fluorescence spectral analysis of basal cell carcinomas

NASA Astrophysics Data System (ADS)

He, Qingli; Lui, Harvey; Zloty, David; Cowan, Bryce; Warshawski, Larry; McLean, David I.; Zeng, Haishan

2007-05-01

Background and Objectives. Laser-induced autofluorescence (LIAF) is a promising tool for cancer diagnosis. This method is based on the differences in autofluorescence spectra between normal and cancerous tissues, but the underlined mechanisms are not well understood. The objective of this research is to study the microscopic origins and intrinsic fluorescence properties of basal cell carcinoma (BCC) for better understanding of the mechanism of in vivo fluorescence detection and margin delineation of BCCs on skin patients. A home-made micro- spectrophotometer (MSP) system was used to image the fluorophore distribution and to measure the fluorescence spectra of various microscopic structures and regions on frozen tissue sections. Materials and Methods. BCC tissue samples were obtained from 14 patients undergoing surgical resections. After surgical removal, each tissue sample was immediately embedded in OCT medium and snap-frozen in liquid nitrogen. The frozen tissue block was then cut into 16-μm thickness sections using a cryostat microtome and placed on microscopic glass slides. The sections for fluorescence study were kept unstained and unfixed, and then analyzed by the MSP system. The adjacent tissue sections were H&E stained for histopathological examination and also served to help identify various microstructures on the adjacent unstained sections. The MSP system has all the functions of a conventional microscope, plus the ability of performing spectral analysis on selected micro-areas of a microscopic sample. For tissue fluorescence analysis, 442nm He-Cd laser light is used to illuminate and excite the unstained tissue sections. A 473-nm long pass filter was inserted behind the microscope objective to block the transmitted laser light while passing longer wavelength fluorescence signal. The fluorescence image of the sample can be viewed through the eyepieces and also recorded by a CCD camera. An optical fiber is mounted onto the image plane of the photograph port of the microscope to collect light from a specific micro area of the sample. The collected light is transmitted via the fiber to a disperserve type CCD spectrometer for spectral analysis. Results. The measurement results showed significant spectral differences between normal and cancerous tissues. For normal tissue regions, the spectral results agreed with our previous findings on autofluorescence of normal skin sections. For the cancerous regions, the epidermis showed very weak fluorescence signal, while the stratum corneum exhibited fluorescence emissions peaking at about 510 nm. In the dermis, the basal cell island and a band of surrounding areas showed very weak fluorescence signal, while distal dermis above and below the basal cell island showed greater fluorescence signal but with different spectral shapes. The very weak autofluorescence from the basal cell island and its surrounding area may be attributed to their degenerative properties that limited the production of collagens. Conclusions. The obtained microscopic results very well explain the in vivo fluorescence properties of BCC lesions in that they have decreased fluorescence intensity compared to the surrounding normal skin. The intrinsic spectra of various microstructures and the microscopic fluorescence images (corresponding fluorophore distribution in tissue) obtained in this study will be used for further theoretical modeling of in vivo fluorescence spectroscopy and imaging of skin cancers.

Effect of sample handling on thiamine and thiaminolytic activity in alewife

USGS Publications Warehouse

Wright, G.M.; Brown, S.B.; Brown, L.R.; Moore, K.; Villella, M.; Zajicek, J.L.; Tillitt, D.E.; Fitzsimons, J.D.; Honeyfield, D.C.

2005-01-01

Alewives Alosa pseudoharengus were collected to evaluate handling and processing conditions that may affect the measurement of their thiamine-thiaminase content. Fish were captured by otter trawl, and reference samples of live fish were quick-frozen on dry ice immediately following capture. Other samples were placed on wet ice (4??C) or held in ambient lake water (21.5??C) for periods of up to 5 h before freezing. Total thiamine levels for reference samples averaged 26 nmol/g and consisted of 66, 15, and 19% thiamine pyrophosphate (TPP), thiamine monophosphate (TMP), and unphosphorylated thiamine (Th), respectively. After 120 min at either 4??C or 21.5??C, total thiamine concentrations were lower. At 21.5??C, the TPP proportion had decreased by 30 min and the proportion as Th increased after 60 min. In the groups sampled after 5 h, total thiamine concentrations were not altered but the proportion of TPP was lower and that of Th was higher than in reference samples. The stability of thiamine in thawed muscle samples from previously frozen alewives was poor (40% loss by 1 h at 22??C and 30% loss by 2 h at 4??C). Thiaminase activity averaged 5,975 pmol??g wet weight -1??min-1 in reference samples. In fresh-caught alewives, thiaminase activities were remarkably consistent throughout the sampling period. At 4??C, thiaminase activity in muscle tissue from previously frozen alewives was stable for the entire investigation period. At 25??C, the activity initially increased by 40% after 60 min but then decreased to 50% of initial value after 5 h. We conclude that sampling times greater than 25 min could cause some changes in the various thiamine forms and net loss in total thiamine. The thiamine content in previously frozen alewife samples is highly labile, requiring low temperatures during processing for analysis. ?? Copyright by the American Fisheries Society 2005.

Pathobiological investigation of naturally infected canine rabies cases from Sri Lanka.

PubMed

Beck, S; Gunawardena, P; Horton, D L; Hicks, D J; Marston, D A; Ortiz-Pelaez, A; Fooks, A R; Núñez, A

2017-04-12

The recommended screening of rabies in 'suspect' animal cases involves testing fresh brain tissue. The preservation of fresh tissue however can be difficult under field conditions and formalin fixation provides a simple alternative that may allow a confirmatory diagnosis. The occurrence and location of histopathological changes and immunohistochemical (IHC) labelling for rabies in formalin fixed paraffin embedded (FFPE) canine brain is described in samples from 57 rabies suspect cases from Sri-Lanka. The presence of Negri bodies and immunohistochemical detection of rabies virus antigen were evaluated in the cortex, hippocampus, cerebellum and brainstem. The effect of autolysis and artefactual degeneration of the tissue was also assessed. Rabies was confirmed in 53 of 57 (93%) cases by IHC. IHC labelling was statistically more abundant in the brainstem. Negri bodies were observed in 32 of 53 (60.4%) of the positive cases. Although tissue degradation had no effect on IHC diagnosis, it was associated with an inability to detect Negri bodies. In 13 cases, a confirmatory Polymerase chain reaction (PCR) testing for rabies virus RNA was undertaken by extracting RNA from fresh frozen tissue, and also attempted using FFPE samples. PCR detection using fresh frozen samples was in agreement with the IHC results. The PCR method from FFPE tissues was suitable for control material but unsuccessful in our field cases. Histopathological examination of the brain is essential to define the differential diagnoses of behaviour modifying conditions in rabies virus negative cases, but it is unreliable as the sole method for rabies diagnosis, particularly where artefactual change has occurred. Formalin fixation and paraffin embedding does not prevent detection of rabies virus via IHC labelling even where artefactual degeneration has occurred. This could represent a pragmatic secondary assay for rabies diagnosis in the field because formalin fixation can prevent sample degeneration. The brain stem was shown to be the site with most viral immunoreactivity; supporting recommended sampling protocols in favour of improved necropsy safety in the field. PCR testing of formalin fixed tissue may be successful in certain circumstances as an alternative test.

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas

PubMed Central

Mohapatra, Gayatry; Engler, David A.; Starbuck, Kristen D.; Kim, James C.; Bernay, Derek C.; Scangas, George A.; Rousseau, Audrey; Batchelor, Tracy T.; Betensky, Rebecca A.; Louis, David N.

2010-01-01

Molecular genetic analysis of cancer is rapidly evolving as a result of improvement in genomic technologies and the growing applicability of such analyses to clinical oncology. Array based comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA), particularly in solid tumors, and has been applied to the study of malignant gliomas. In the clinical setting, however, gliomas are often sampled by small biopsies and thus formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis, especially for rare types of gliomas. Moreover, the biological basis for the marked intratumoral heterogeneity in gliomas is most readily addressed in FFPE material. Therefore, for gliomas, the ability to use DNA from FFPE tissue is essential for both clinical and research applications. In this study, we have constructed a custom bacterial artificial chromosome (BAC) array and show excellent sensitivity and specificity for detecting CNAs in a panel of paired frozen and FFPE glioma samples. Our study demonstrates a high concordance rate between CNAs detected in FFPE compared to frozen DNA. We have also developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. This labeling technique was applied to a panel of biphasic anaplastic oligoastrocytomas (AOA) to identify genetic changes unique to each component. Together, results from these studies suggest that BAC and oligonucleotide aCGH are sensitive tools for detecting CNAs in FFPE DNA, and can enable genome-wide analysis of rare, small and/or histologically heterogeneous gliomas. PMID:21080181

A new device for the efficient pulverisation and extraction of myocardial biopsies for high energy phosphate analysis.

PubMed

Speir, E H; Sullivan, J; Patterson, R E

1985-07-01

We developed a new device for processing frozen myocardial biopsies. Frozen samples of 20 to 50 mg were dropped into a 25 ml stainless steel centrifuge tube held in a custom-made aluminium container precooled in liquid nitrogen. A stainless steel pestle attached to a stainless steel disk was driven by a modified heavy-duty staple gun to pulverise the tissue rapidly at low temperatures. The tissue powder was extracted with 0.3N PCA at 0 degree C in the centrifuge tube which was then transferred to a Sorvall super-speed centrifuge. Values for adenosine triphosphate (ATP) were 5.6 +/- 0.7 mumol . g-1 wet weight (mean +/- SD). Creatine phosphate (CP) yield was 12.2 +/- 3 mumol . g-1 wet weight. The % recovery of an added internal standard for ATP was 86 +/- 18% and for CP 90 +/- 16% with the new method.

Isochoric and isobaric freezing of fish muscle.

PubMed

Năstase, Gabriel; Lyu, Chenang; Ukpai, Gideon; Şerban, Alexandru; Rubinsky, Boris

2017-04-01

We have recently shown that, a living organism, which succumbs to freezing to -4 °C in an isobaric thermodynamic system (constant atmospheric pressure), can survive freezing to -4 °C in an isochoric thermodynamic system (constant volume). It is known that the mechanism of cell damage in an isobaric system is the freezing caused increase in extracellular osmolality, and, the consequent cell dehydration. An explanation for the observed survival during isochoric freezing is the thermodynamic modeling supported hypothesis that, in the isochoric frozen solution the extracellular osmolality is comparable to the cell intracellular osmolality. Therefore, cells in the isochoric frozen organism do not dehydrate, and the tissue maintains its morphological integrity. Comparing the histology of: a) fresh fish white muscle, b) fresh muscle frozen to -5 °C in an isobaric system and c) fresh muscle frozen to -5 °C I in an isochoric system, we find convincing evidence of the mechanism of cell dehydration during isobaric freezing. In contrast, the muscle tissue frozen to -5 °C in an isochoric system appears morphologically identical to fresh tissue, with no evidence of dehydration. This is the first experimental evidence in support of the hypothesis that in isochoric freezing there is no cellular dehydration and therefore the morphology of the frozen tissue remains intact. Copyright © 2017 Elsevier Inc. All rights reserved.

Optical properties of cells with melanin

NASA Astrophysics Data System (ADS)

Rohde, Barukh; Coats, Israel; Krueger, James; Gareau, Dan

2014-02-01

The optical properties of pigmented lesions have been studied using diffuse reflectance spectroscopy in a noninvasive configuration on optically thick samples such as skin in vivo. However, it is difficult to un-mix the effects of absorption and scattering with diffuse reflectance spectroscopy techniques due to the complex anatomical distributions of absorbing and scattering biomolecules. We present a device and technique that enables absorption and scattering measurements of tissue volumes much smaller than the optical mean-free path. Because these measurements are taken on fresh-frozen sections, they are direct measurements of the optical properties of tissue, albeit in a different hydration state than in vivo tissue. Our results on lesions from 20 patients including melanomas and nevi show the absorption spectrum of melanin in melanocytes and basal keratinocytes. Our samples consisted of fresh frozen sections that were unstained. Fitting the spectrum as an exponential decay between 500 and 1100 nm [mua = A*exp(-B*(lambda-C)) + D], we report on the fit parameters of and their variation due to biological heterogeneity as A = 4.20e4 +/- 1.57e5 [1/cm], B = 4.57e-3 +/- 1.62e-3 [1/nm], C = 210 +/- 510 [nm] , D = 613 +/- 534 [1/cm]. The variability in these results is likely due to highly heterogeneous distributions of eumelanin and pheomelanin.

Impact of Ischemia and Procurement Conditions on Gene Expression in Renal Cell Carcinoma

PubMed Central

Liu, Nick W.; Sanford, Thomas; Srinivasan, Ramaprasad; Liu, Jack L.; Khurana, Kiranpreet; Aprelikova, Olga; Valero, Vladimir; Bechert, Charles; Worrell, Robert; Pinto, Peter A.; Yang, Youfeng; Merino, Maria; Linehan, W. Marston; Bratslavsky, Gennady

2013-01-01

Purpose Previous studies have shown that ischemia alters gene expression in normal and malignant tissues. There are no studies that evaluated effects of ischemia in renal tumors. This study examines the impact of ischemia and tissue procurement conditions on RNA integrity and gene expression in renal cell carcinoma. Experimental Design Ten renal tumors were resected without renal hilar clamping from 10 patients with renal clear cell carcinoma. Immediately after tumor resection, a piece of tumor was snap frozen. Remaining tumor samples were stored at 4C, 22C and 37C and frozen at 5, 30, 60, 120, and 240 minutes. Histopathologic evaluation was performed on all tissue samples, and only those with greater than 80% tumor were selected for further analysis. RNA integrity was confirmed by electropherograms and quantitated using RIN index. Altered gene expression was assessed by paired, two-sample t-test between the zero time point and aliquots from various conditions obtained from the same tumor. Results One hundred and forty microarrays were performed. Some RNA degradation was observed 240 mins after resection at 37C. The expression of over 4,000 genes was significantly altered by ischemia times or storage conditions. The greatest gene expression changes were observed with longer ischemia time and warmer tissue procurement conditions. Conclusion RNA from kidney cancer remains intact for up to 4 hours post surgical resection regardless of storage conditions. Despite excellent RNA preservation, time after resection and procurement conditions significantly influence gene expression profiles. Meticulous attention to pre-acquisition variables is of paramount importance for accurate tumor profiling. PMID:23136194

Effect of cryopreservation and transplantation on the expression of kit ligand and anti-Mullerian hormone in human ovarian tissue.

PubMed

David, Anu; Van Langendonckt, Anne; Gilliaux, Sébastien; Dolmans, Marie-Madeleine; Donnez, Jacques; Amorim, Christiani A

2012-04-01

Although cryopreservation and transplantation of ovarian tissue represent a promising alternative to safeguard fertility in cancer patients, low recovery rates of oocytes aspirated from antral follicles and a significant number of empty follicles have been observed in women with transplanted frozen-thawed ovarian tissue. In order to understand how freezing and/or grafting may affect follicular development, the follicular expression of kit ligand (KL) and anti-Müllerian hormone (AMH), two key factors activating and inhibiting follicle growth, were assessed after long-term grafting in severe combined immunodeficient (SCID) mice. Ovarian biopsies from eight patients were used for fresh and frozen-thawed tissue xenografting in 13 SCID mice for a period of 28 weeks, including 2 weeks of gonadotrophin stimulation. KL, AMH and proliferating cell nuclear antigen (PCNA) immunostaining were quantified before and after grafting in the two treatment groups (fresh and frozen-thawed grafted ovarian tissue). Lower expression of KL was found in primordial and primary follicles after grafting of both fresh and frozen-thawed tissue. Consistent expression of AMH was found in most growing follicles at a similar rate in both graft types. In fresh and frozen-thawed grafts, 13-14% of primordial follicles were PCNA-positive, indicating a similar maintenance of quiescent follicles despite follicle activation. Grafting and/or gonadotrophin stimulation appear to affect the follicular expression of KL, which may alter oocyte quality. AMH expression in growing follicles after ovarian tissue transplantation may be one of the factors contributing to the preservation of resting follicles in 28-week-old grafts.

RNA quality in fresh-frozen gastrointestinal tumor specimens-experiences from the tumor and healthy tissue bank TU Dresden.

PubMed

Zeugner, Silke; Mayr, Thomas; Zietz, Christian; Aust, Daniela E; Baretton, Gustavo B

2015-01-01

The term "pre-analytics" summarizes all procedures concerned with specimen collection or processing as well as logistical aspects like transport or storage of tissue specimens. All or these variables as well as tissue-specific characteristics affect sample quality. While certain parameters like warm ischemia or tissue-specific characteristics cannot be changed, other parameters can be assessed and optimized. The aim of this study was to determine RNA quality by assessing the RIN values of specimens from different organs and to assess the influence of vacuum preservation. Samples from the GI tract, in general, appear to have lower RNA quality when compared to samples from other organ sites. This may be due to the digestive enzymes or bacterial colonization. Processing time in pathology does not significantly influence RNA quality. Tissue preservation with a vacuum sealer leads to preserved RNA quality over an extended period of time and offers a feasible alternative to minimize the influence of transport time into pathology.

Next-gen tissue: preservation of molecular and morphological fidelity in prostate tissue.

PubMed

Gillard, Marc; Tom, Westin R; Antic, Tatjana; Paner, Gladell P; Lingen, Mark W; VanderWeele, David J

2015-01-01

Personalization of cancer therapy requires molecular evaluation of tumor tissue. Traditional tissue preservation involves formalin fixation, which degrades the quality of nucleic acids. Strategies to bank frozen prostate tissue can interfere with diagnostic studies. PAXgene is an alternative fixative that preserves protein and nucleic acid quality. Portions of prostates obtained from autopsy specimens were fixed in either 10% buffered formalin or PAXgene, and processed and embedded in paraffin. Additional sections were immediately embedded in OCT and frozen. DNA and RNA were extracted from the formalin-fixed, PAXgene-fixed, or frozen tissue. Quantitative PCR was used to compare the quality of DNA and RNA obtained from all three tissue types. In addition, 5 μm sections were cut from specimens devoid of cancer and from prostate cancer specimens obtained at prostatectomy and fixed in PAXgene. They were either stained with hematoxylin and eosin or interrogated with antibodies for p63, PSA and p504. Comparable tissue morphology was observed in both the formalin and PAXgene-fixed specimens. Similarly, immunohistochemical expression of the P63, PSA and P504 proteins was comparable between formalin and PAXgene fixation techniques. DNA from the PAXgene-fixed tissue was of similar quality to that from frozen tissue. RNA was also amplified with up to 8-fold greater efficiency in the PAXgene fixed tissue compared to the formalin-fixed tissue. Prostate specimens fixed with PAXgene have preserved histologic morphology, stain appropriately, and have preserved quality of nucleic acids. PAXgene fixation facilitates the use of prostatectomy tissue for molecular biology techniques such as next-generation sequencing.

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.

PubMed

Mohr, Annika; Lüder Ripoli, Florenza; Hammer, Susanne Conradine; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Kiełbowicz, Zdzisław; Murua Escobar, Hugo; Nolte, Ingo

2016-01-01

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.

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA.

PubMed

Lundblom, Klara; Macharia, Alex; Lebbad, Marianne; Mohammed, Adan; Färnert, Anna

2011-08-08

Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.

Role of Mitochondrial Inheritance on Prostate Cancer Outcome in African American Men. Addendum

DTIC Science & Technology

2016-11-01

DNA sequencing technique developed by our collaborator using single amplicon long-range PCR that permits deep coverage (10,000-20,000X on average) of...the mitochondrial genome. We have sequenced 652 samples derived from frozen fully using this technology. The additional DNA samples derived from...paraffin embedded (FFPE) tissue were more challenging, but have now been sequenced . Mapping of DNA variants in our sequenced genomes to mitochondrial

FROZEN THIN SECTIONS OF FRESH TISSUE FOR ELECTRON MICROSCOPY, WITH A DESCRIPTION OF PANCREAS AND LIVER

PubMed Central

Christensen, A. Kent

1971-01-01

A simple method has been developed that allows frozen thin sections of fresh-frozen tissue to be cut on a virtually unmodified ultramicrotome kept at room temperature. A bowl-shaped Dewar flask with a knifeholder in its depths replaces the stage of the microtome; a bar extends down into the bowl from the microtome's cutting arm and bears the frozen tissue near its lower end. When the microtome is operated, the tissue passes a glass or diamond knife in the depths of the bowl as in normal cutting. The cutting temperature is maintained by flushing the bowl with cold nitrogen gas, and can be set anywhere from about -160°C up to about -30°C. The microtome is set for a cutting thickness of 540–1000 A. Sections are picked up from the dry knife edge, and are placed on membrane-coated grids, flattened with the polished end of a copper rod, and either dried in nitrogen gas or freeze-dried. Throughout the entire process the tissue is kept cold and does not come in contact with any solvent. The morphology seen in frozen thin sections of rat pancreas and liver generally resembles that in conventional preparations, although freezing damage and low contrast limit the detail that can be discerned. Among unusual findings is a frequent abundance of mitochondrial granules in material prepared by this method. PMID:4942776

The influence of low temperatures on dynamic mechanical properties of animal bone

NASA Astrophysics Data System (ADS)

Mardas, Marcin; Kubisz, Leszek; Mielcarek, Slawomir; Biskupski, Piotr

2009-01-01

Different preservation methods are currently used in bone banks, even though their effects on allograft quality are not fully understood. Freezing is one of the most popular methods of preservation in tissue banking. Yet, there is not a lot of data on dynamic mechanical properties of frozen bone. Material used in this study was femoral bones from adult bovine that were machine cut and frozen to the temperature 140°C. Both elastic modulus and loss modulus were measured at 1, 3, 5, 10, and 20 Hz in the temperature range of 30-200°C. Differences between frozen and control samples were observed. The frequency increase always led to the increase in elastic modulus values and decrease in loss modulus values. Freezing reduced the elastic modulus values of about 25% and the loss modulus values of about 45% when measured at 20°C.

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

PubMed

Wimmer, Isabella; Tröscher, Anna R; Brunner, Florian; Rubino, Stephen J; Bien, Christian G; Weiner, Howard L; Lassmann, Hans; Bauer, Jan

2018-04-20

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

Freezing/Thawing without Cryoprotectant Damages Native but not Decellularized Porcine Renal Tissue

PubMed Central

Poornejad, Nafiseh; Frost, Timothy S; Scott, Daniel R; Elton, Brinden B; Reynolds, Paul R; Roeder, Beverly L; Cook, Alonzo D

2015-01-01

abstract Whole organ decellularization of porcine renal tissue and recellularization with a patient's own cells would potentially overcome immunorejection, which is one of the most significant problems with allogeneic kidney transplantation. However, there are obstacles to achieving this goal, including preservation of the decellularized extracellular matrix (ECM), identifying the proper cell types, and repopulating the ECM before transplantation. Freezing biological tissue is the best option to avoid spoilage; however, it may damage the structure of the tissue or disrupt cellular membranes through ice crystal formation. Cryoprotectants have been used to repress ice formation during freezing, although cell toxicity can still occur. The effect of freezing/thawing on native (n = 10) and decellularized (n = 10) whole porcine kidneys was studied without using cryoprotectants. Results showed that the elastic modulus of native kidneys was reduced by a factor of 22 (P < 0.0001) by freezing/thawing or decellularization, while the elastic modulus for decellularized ECM was essentially unchanged by the freezing/thawing process (p = 0.0636). Arterial pressure, representative of structural integrity, was also reduced by a factor of 52 (P < 0.0001) after freezing/thawing for native kidneys, compared to a factor of 43 (P < 0.0001) for decellularization and a factor of 4 (P < 0.0001) for freezing/thawing decellularized structures. Both freezing/thawing and decellularization reduced stiffness, but the reductions were not additive. Investigation of the microstructure of frozen/thawed native and decellularized renal tissues showed increased porosity due to cell removal and ice crystal formation. Orcein and Sirius staining showed partial damage to elastic and collagen fibers after freezing/thawing. It was concluded that cellular damage and removal was more responsible for reducing stiffness than fibril destruction. Cell viability and growth were demonstrated on decellularized frozen/thawed and non-frozen samples using human renal cortical tubular epithelial (RCTE) cells over 12 d. No adverse effect on the ability to recellularize after freezing/thawing was observed. It is recommended that porcine kidneys be frozen prior to decellularization to prevent contamination, and after decellularization to prevent protein denaturation. Cryoprotectants may still be necessary, however, during storage and transportation after recellularization. PMID:25730294

A preliminary result of three-dimensional microarray technology to gene analysis with endoscopic ultrasound-guided fine-needle aspiration specimens and pancreatic juices

PubMed Central

2010-01-01

Background Analysis of gene expression and gene mutation may add information to be different from ordinary pathological tissue diagnosis. Since samples obtained endoscopically are very small, it is desired that more sensitive technology is developed for gene analysis. We investigated whether gene expression and gene mutation analysis by newly developed ultra-sensitive three-dimensional (3D) microarray is possible using small amount samples from endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimens and pancreatic juices. Methods Small amount samples from 17 EUS-FNA specimens and 16 pancreatic juices were obtained. After nucleic acid extraction, the samples were amplified with labeling and analyzed by the 3D microarray. Results The analyzable rate with the microarray was 46% (6/13) in EUS-FNA specimens of RNAlater® storage, and RNA degradations were observed in all the samples of frozen storage. In pancreatic juices, the analyzable rate was 67% (4/6) in frozen storage samples and 20% (2/10) in RNAlater® storage. EUS-FNA specimens were classified into cancer and non-cancer by gene expression analysis and K-ras codon 12 mutations were also detected using the 3D microarray. Conclusions Gene analysis from small amount samples obtained endoscopically was possible by newly developed 3D microarray technology. High quality RNA from EUS-FNA samples were obtained and remained in good condition only using RNA stabilizer. In contrast, high quality RNA from pancreatic juice samples were obtained only in frozen storage without RNA stabilizer. PMID:20416107

Ensuring the Safety and Security of Frozen Lung Cancer Tissue Collections through the Encapsulation of Dried DNA.

PubMed

Washetine, Kevin; Kara-Borni, Mehdi; Heeke, Simon; Bonnetaud, Christelle; Félix, Jean-Marc; Ribeyre, Lydia; Bence, Coraline; Ilié, Marius; Bordone, Olivier; Pedro, Marine; Maitre, Priscilla; Tanga, Virginie; Gormally, Emmanuelle; Mossuz, Pascal; Lorimier, Philippe; Marquette, Charles Hugo; Mouroux, Jérôme; Cohen, Charlotte; Lassalle, Sandra; Long-Mira, Elodie; Clément, Bruno; Dagher, Georges; Hofman, Véronique; Hofman, Paul

2018-06-11

Collected specimens for research purposes may or may not be made available depending on their scarcity and/or on the project needs. Their protection against degradation or in the event of an incident is pivotal. Duplication and storage on a different site is the best way to assure their sustainability. The conservation of samples at room temperature (RT) by duplication can facilitate their protection. We describe a security system for the collection of non-small cell lung cancers (NSCLC) stored in the biobank of the Nice Hospital Center, France, by duplication and conservation of lyophilized (dried), encapsulated DNA kept at RT. Therefore, three frozen tissue collections from non-smoking, early stage and sarcomatoid carcinoma NSCLC patients were selected for this study. DNA was extracted, lyophilized and encapsulated at RT under anoxic conditions using the DNAshell technology. In total, 1974 samples from 987 patients were encapsulated. Six and two capsules from each sample were stored in the biobanks of the Nice and Grenoble (France) Hospitals, respectively. In conclusion, DNA maintained at RT allows for the conservation, duplication and durability of collections of interest stored in biobanks. This is a low-cost and safe technology that requires a limited amount of space and has a low environmental impact.

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

PubMed

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.

INFLUENCE OF INTRAMUSCULAR FAT LEVEL ON ORGANOLEPTIC, PHYSICAL, AND CHEMICAL CHARACTERISTICS OF IRRADIATED PORK. II. LOW-TEMPERATURE LONG-TIME PRE-IRRADIATION HEAT TREATMENT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bray, R.W.; Weckel, K.G.; Evans, G.W.

1964-02-01

Pork muscle (longissimus dorsi) was graded in three marbling levels by both visual appraisal and ether-extraction analysis of total fat. A low- temperature (121 deg F, gradually increasing to 191 deg F), long-time (19-hr) heat treatment was used for enzyme inactivation. Samples were packed under vacuum in rigid containers and irradiated to 4.5 Mrad with gamma radiation. Irradiated and frozen control samples were evaluated at intervals over a 180-day period. The heat treatment caused excessive connective-tissue breakdown as evidenced by a soft, dry texture. Marbling level had no significant effect on consumer-panel judgments of irradiated or frozen control samples (servedmore » plain, without bread). Lower degrees of marbling were preferred in irradiated sandwich items. Irradiated samples were less preferred than frozen control samples in both plain and sandwich form. Hunter L (color) values a/sub L/ (redness), hue, and saturation attributes were increased by irradiation treatment. Hunter L and b/sub L/ (yellowness) values of highly marbled irradiated sample-s were elevated. Highly marbled samples displayed greater tenderness qualities, as evidenced by mechanical tenderness measurements. Expressible moisture values were decreased by marbling degree and radiation treatment and increased by advancing storage time. As a measure of oxidation rancidity, thiobarbituric acid values were increased by increasing levels of marbling and advancing storage time, but were not influenced by preservation method. No significant differences in pH values due to marbling level or preservation method were detected. Bacteriologic counts of randomly selected irradiated samples indicated that they were commercially sterile (average of 57 colonies/g of sample). Methylene blue stains of colonles revealed Micrococcus. Frozen (control) samples contained moderate numbers of colonies (av. 2300 colonies per g of sample). Micrococcus colonies were again predominant, but a few colonles of film yeasts were also seen. Irradiated samples were negative for thermophilic anaerobic spores producing gas. Control samples were positive, containing gram-negative rods, Escherichia coli, and gram- positive cocci. (BBB)« less

Use of cryostat sections from snap-frozen nervous tissue for combining stereological estimates with histological, cellular, or molecular analyses on adjacent sections.

PubMed

Schmitz, C; Dafotakis, M; Heinsen, H; Mugrauer, K; Niesel, A; Popken, G J; Stephan, M; Van de Berg, W D; von Hörsten, S; Korr, H

2000-10-01

Adequate tissue preparation is essential for both modern stereological and immunohistochemical investigations. However, combining these methodologies in a single study presents a number of obstacles pertaining to optimal histological preparation. Tissue shrinkage and loss of nuclei/nucleoli from the unprotected section surfaces of unembedded tissue used for immunohistochemistry may be problematic with regard to adequate stereological design. In this study, frozen cryostat sections from hippocampal and cerebellar regions of two rat strains and cerebellar and cerebral regions from a human brain were analyzed to determine the potential impact of these factors on estimates of neuron number obtained using the optical disector. Neuronal nuclei and nucleoli were clearly present in thin sections of snap-frozen rat (3 microm) and human (6 microm) tissue, indicating that neuronal nuclei/nucleoli are not unavoidably lost from unprotected section surfaces of unembedded tissue. In order to quantify the potential impact of any nuclear loss, optical fractionator estimates of rat hippocampal pyramidal cells in areas CA1-3 and cerebellar granule and Purkinje cells were made using minimal (1 microm) upper guard zones. Estimates did not differ from data reported previously in the literature. This data indicates that cryostat sections of snap-frozen nervous tissue may successfully be used for estimating total neuronal numbers using optical disectors.

Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers

PubMed Central

Exner, Ruth; Pulverer, Walter; Diem, Martina; Spaller, Lisa; Woltering, Laura; Schreiber, Martin; Wolf, Brigitte; Sonntagbauer, Markus; Schröder, Fabian; Stift, Judith; Wrba, Fritz; Bergmann, Michael; Weinhäusel, Andreas; Egger, Gerda

2015-01-01

Background: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. Methods: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples, respectively. The CpG island methylator phenotype (CIMP) was assessed by MethyLight in FFPE material from 78 patients with pT2 and pT3 rectal adenocarcinoma. Results: We identified and confirmed two novel three-gene signatures in fresh frozen samples that can distinguish tumours from adjacent tissue as well as from blood with a high sensitivity and specificity of up to 1 and an AUC of 1. In addition, methylation of individual CIMP markers was associated with specific clinical parameters such as tumour stage, therapy or patients' age. Methylation of CDKN2A was a negative prognostic factor for overall survival of patients. Conclusions: The newly defined methylation markers will be suitable for early disease detection and monitoring of rectal cancer. PMID:26335606

Downregulation of external death receptor genes FAS and DR5 in colorectal cancer samples positive for human papillomavirus infection.

PubMed

Karbasi, Ashraf; Borhani, Nasim; Daliri, Karim; Kazemi, Bahram; Manoochehri, Mehdi

2015-06-01

Human papillomaviruses (HPV) have frequently been detected in colorectal cancer tumor samples, and may play a role in the pathogenesis of colorectal cancer. This study was designed to investigate the presence of DNA and RNA for the high-risk HPV genotypes 16 and 18 in samples of colorectal cancer tumors and adjacent normal tissues. We also investigated the expression of proapoptotic genes in HPV-positive colorectal tumors compared to normal tissue samples. Samples of tumoral and adjacent normal tissues were fresh-frozen, and HPV DNA was identified by nested and semiquantitative PCR. Real time PCR was used to quantitatively compare the expression of HPV-18 E6 and nine proapoptotic genes in HPV-positive tumors and samples of adjacent normal tissue. HPV-16 DNA was found in 10.5% of the tumor samples, and HPV-18 DNA was found in 23.6% of the samples. Real time PCR results showed lower expression of the E6 gene in HPV-positive tumors than in adjacent normal tissue. The expression of two proapoptotic genes, FAS and DR5, was significantly lower in tumor samples than in adjacent normal tissues. HPV infection, especially HPV-18, may play a role in colorectal cancer tumorigenesis by downregulating death receptor genes and interfering with the extrinsic pathway of apoptosis. Copyright © 2015 Elsevier GmbH. All rights reserved.

The changes in crosslink contents in tissues after formalin fixation.

PubMed

Abe, Masashi; Takahashi, Masaaki; Horiuchi, Kentaro; Nagano, Akira

2003-07-01

The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage. Pyridinoline (Pyr) is a stable and nonreducible crosslink of collagen. Pentosidine (Pen) is a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety of tissues. We used HPLC to compare the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with their concentrations in the frozen tissues. Pyr and Pen were detected in both the formalin-fixed yellow ligament and the cartilage, and their concentrations were not significantly affected by or related to the duration of formalin fixation. Des and Isodes were detected in the formalin-fixed yellow ligament but in significantly lower amounts compared to the frozen samples. We concluded that crosslinks of collagen were preserved in formalin, but crosslinks of elastin were not preserved in it. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered elastin crosslinks.

Sample processing, protocol, and statistical analysis of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) of protein, cell, and tissue samples.

PubMed

Barreto, Goncalo; Soininen, Antti; Sillat, Tarvo; Konttinen, Yrjö T; Kaivosoja, Emilia

2014-01-01

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is increasingly being used in analysis of biological samples. For example, it has been applied to distinguish healthy and osteoarthritic human cartilage. This chapter discusses ToF-SIMS principle and instrumentation including the three modes of analysis in ToF-SIMS. ToF-SIMS sets certain requirements for the samples to be analyzed; for example, the samples have to be vacuum compatible. Accordingly, sample processing steps for different biological samples, i.e., proteins, cells, frozen and paraffin-embedded tissues and extracellular matrix for the ToF-SIMS are presented. Multivariate analysis of the ToF-SIMS data and the necessary data preprocessing steps (peak selection, data normalization, mean-centering, and scaling and transformation) are discussed in this chapter.

Comparison of methods of preserving tissues for pesticide analysis

USGS Publications Warehouse

Stickel, W.H.; Stickel, L.F.; Dyrland, R.A.; Hughes, D.L.

1984-01-01

Formalin preservation, freezing, spoiling followed by freezing, and phenoxyethanol were compared in terms of concentrations of DDT, DDD, DDE, endrin, and hepatachlor epoxide measured in brain, liver and carcass of birds fed dietary dosages of pesticides and in spiked egg homogenate. Phenoxyethanol proved to be an unsatisfactory preservative; the amount of 'extractable lipid' was excessive, and measurements of concentrations in replicates were erratic. Concentrations of residues in formalin-preserved and frozen samples did not differ significantly in any tissue. Percentage lipid in brains and eggs, however, were significantly lower in formalin-preserved samples. Samples of muscle and liver that had been spoiled before freezing yielded less DDD, and muscle samples yielded more DDT than formalin-preserved samples. The authors conclude that formalin preservation is a satisfactory method for preservation of field samples and that the warming and spoiling of samples that may occur unavoidably in the field will not result in misleading analytical results.

Cryo-planing of frozen-hydrated samples using cryo triple ion gun milling (CryoTIGM™).

PubMed

Chang, Irene Y T; Joester, Derk

2015-12-01

Cryo-SEM is a high throughput technique for imaging biological ultrastructure in its most pristine state, i.e. without chemical fixation, embedding, or drying. Freeze fracture is routinely used to prepare internal surfaces for cryo-SEM imaging. However, the propagation of the fracture plane is highly dependent on sample properties, and the resulting surface frequently shows substantial topography, which can complicate image analysis and interpretation. We have developed a broad ion beam milling technique, called cryogenic triple ion gun milling (CryoTIGM™ ['krī-ə-,tīm]), for cryo-planing frozen-hydrated biological specimens. Comparing sample preparation by CryoTIGM™ and freeze fracture in three model systems, Baker's yeast, mouse liver tissue, and whole sea urchin embryos, we find that CryoTIGM™ yields very large (∼700,000 μm(2)) and smooth sections that present ultrastructural details at similar or better quality than freeze-fractured samples. A particular strength of CryoTIGM™ is the ability to section samples with hard-soft contrast such as brittle calcite (CaCO3) spicules in the sea urchin embryo. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantitative analysis of the effect of environmental-scanning electron microscopy on collagenous tissues.

PubMed

Lee, Woowon; Toussaint, Kimani C

2018-05-31

Environmental-scanning electron microscopy (ESEM) is routinely applied to various biological samples due to its ability to maintain a wet environment while imaging; moreover, the technique obviates the need for sample coating. However, there is limited research carried out on electron-beam (e-beam) induced tissue damage resulting from using the ESEM. In this paper, we use quantitative second-harmonic generation (SHG) microscopy to examine the effects of e-beam exposure from the ESEM on collagenous tissue samples prepared as either fixed, frozen, wet or dehydrated. Quantitative SHG analysis of tissues, before and after ESEM e-beam exposure in low-vacuum mode, reveals evidence of cross-linking of collagen fibers, however there are no structural differences observed in fixed tissue. Meanwhile wet-mode ESEM appears to radically alter the structure from a regular fibrous arrangement to a more random fiber orientation. We also confirm that ESEM images of collagenous tissues show higher spatial resolution compared to SHG microscopy, but the relative tradeoff with collagen specificity reduces its effectiveness in quantifying collagen fiber organization. Our work provides insight on both the limitations of the ESEM for tissue imaging, and the potential opportunity to use as a complementary technique when imaging fine features in the non-collagenous regions of tissue samples.

Cryoelectrolysis—electrolytic processes in a frozen physiological saline medium

PubMed Central

Lugnani, Franco; Macchioro, Matteo

2017-01-01

Background Cryoelectrolysis is a new minimally invasive tissue ablation surgical technique that combines the ablation techniques of electrolytic ablation with cryosurgery. The goal of this study is to examine the hypothesis that electrolysis can take place in a frozen aqueous saline solution. Method To examine the hypothesis we performed a cryoelectrolytic ablation protocol in which electrolysis and cryosurgery are delivered simultaneously in a tissue simulant made of physiological saline gel with a pH dye. We measured current flow, voltage and extents of freezing and pH dye staining. Results Using optical measurements and measurements of currents, we have shown that electrolysis can occur in frozen physiological saline, at high subzero freezing temperatures, above the eutectic temperature of the frozen salt solution. It was observed that electrolysis occurs when the tissue resides at high subzero temperatures during the freezing stage and essentially throughout the entire thawing stage. We also found that during thawing, the frozen lesion temperature raises rapidly to high subfreezing values and remains at those values throughout the thawing stage. Substantial electrolysis occurs during the thawing stage. Another interesting finding is that electro-osmotic flows affect the process of cryoelectrolysis at the anode and cathode, in different ways. Discussion The results showing that electrical current flow and electrolysis occur in frozen saline solutions imply a mechanism involving ionic movement in the fluid concentrated saline solution channels between ice crystals, at high subfreezing temperatures. Temperatures higher than the eutectic are required for the brine to be fluid. The particular pattern of temperature and electrical currents during the thawing stage of frozen tissue, can be explained by the large amounts of energy that must be removed at the outer edge of the frozen lesion because of the solid/liquid phase transformation on that interface. Conclusion Electrolysis can occur in a frozen domain at high subfreezing temperature, probably above the eutectic. It appears that the most effective period for delivering electrolytic currents in cryoelectrolysis is during the high subzero temperatures stage while freezing and immediately after cooling has stopped, throughout the thawing stage. PMID:28123904

Cryoelectrolysis-electrolytic processes in a frozen physiological saline medium.

PubMed

Lugnani, Franco; Macchioro, Matteo; Rubinsky, Boris

2017-01-01

Cryoelectrolysis is a new minimally invasive tissue ablation surgical technique that combines the ablation techniques of electrolytic ablation with cryosurgery. The goal of this study is to examine the hypothesis that electrolysis can take place in a frozen aqueous saline solution. To examine the hypothesis we performed a cryoelectrolytic ablation protocol in which electrolysis and cryosurgery are delivered simultaneously in a tissue simulant made of physiological saline gel with a pH dye. We measured current flow, voltage and extents of freezing and pH dye staining. Using optical measurements and measurements of currents, we have shown that electrolysis can occur in frozen physiological saline, at high subzero freezing temperatures, above the eutectic temperature of the frozen salt solution. It was observed that electrolysis occurs when the tissue resides at high subzero temperatures during the freezing stage and essentially throughout the entire thawing stage. We also found that during thawing, the frozen lesion temperature raises rapidly to high subfreezing values and remains at those values throughout the thawing stage. Substantial electrolysis occurs during the thawing stage. Another interesting finding is that electro-osmotic flows affect the process of cryoelectrolysis at the anode and cathode, in different ways. The results showing that electrical current flow and electrolysis occur in frozen saline solutions imply a mechanism involving ionic movement in the fluid concentrated saline solution channels between ice crystals, at high subfreezing temperatures. Temperatures higher than the eutectic are required for the brine to be fluid. The particular pattern of temperature and electrical currents during the thawing stage of frozen tissue, can be explained by the large amounts of energy that must be removed at the outer edge of the frozen lesion because of the solid/liquid phase transformation on that interface. Electrolysis can occur in a frozen domain at high subfreezing temperature, probably above the eutectic. It appears that the most effective period for delivering electrolytic currents in cryoelectrolysis is during the high subzero temperatures stage while freezing and immediately after cooling has stopped, throughout the thawing stage.

Biomonitoring of Environmental Status and Trends (BEST) Program: Field Procedures for Assessing the Exposure of Fish to Environmental Contaminants

USGS Publications Warehouse

Schmitt, Christopher J.; Blazer, Vicki; Dethloff, Gail M.; Tillitt, Donald E.; Gross, Timothy S.; Bryant, Wade L.; DeWeese, L. Rod; Smith, Stephen B.; Goede, Ronald W.; Bartish, Timothy M.; Kubiak, Timothy J.

1999-01-01

This document describes procedures used to collect information, tissues, and fluids for documenting the exposure of fish to environmental contaminants. For the procedures described here, fish are captured (preferably by electrofishing) and held alive until processing (generally <1 h). Fish are weighed, measured, and examined for grossly visible external lesions and pathologies. A blood sample is collected by caudal veinipuncture using a needle and syringe. The fish is subdued and it's abdominal cavity opened. The internal organs are dissected from the fish for examination. The sex of the fish is determined by direct observation of its gonads. The liver is weighed (most species) and cut into small cubes and flash-frozen in cryogenic vials, which are stored and shipped in dry ice or liquid nitrogen. Additional liver cubes plus all grossly visible anomalies are preserved for histopathology. The gonads and spleen are weighed, and samples are preserved for histopathology. The kidneys are examined, and histopathology samples collected. A gill sample is also collected and preserved. All remaining tissues are returned to the carcass, which is wrapped in foil, labeled for chemical analysis, and chilled. Individual fish carcasses are composited by station, species, and gender; frozen; and shipped to the analytical laboratory. Procedures are also described for record keeping; processing blood to obtain serum and plasma; flash-freezing samples; cleaning equipment; and preventing the transport of living organisms among waterways. A list of necessary equipment and supplies is also provided.

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA

PubMed Central

2011-01-01

Background Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. Methods High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. Results High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. Conclusions High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots. PMID:21824391

Effect of freezing on the rheological, chemical and colour properties of Serpa cheese.

PubMed

Alvarenga, Nuno; Canada, João; Sousa, Isabel

2011-02-01

The effect of freezing on the properties of a raw ewes'-milk semi-soft cheese (Serpa cheese) was studied using small amplitude oscillatory (SAOS) and texture measurements, colour and chemical parameters. The freezing was introduced at three different stages of the ripening process (28, 35 and 42 days), and the cheeses were maintained frozen for 12 months. Cheeses were submitted to a slow or fast freezing method, and to different storage temperatures: -10 and -20°C (three replicates for each set conditions). Chemical data showed that only the proteolysis indicators exhibited differences between frozen and non-frozen samples; frozen samples showed higher values of NPN than the non-frozen samples, indicating that the freezing process did not prevent the secondary proteolysis of cheese. Frozen samples showed a significantly (P<0·05) stronger structure than the non-frozen, as indicated by hardness. However, the differences between the frozen and non-frozen samples were not significantly for storage modulus (G' 1Hz) and loss tangent (tan δ 1Hz) (P>0·05). Freezing affected mainly colour parameters: frozen samples were more luminous, and more yellow-green. The results allowed us to conclude that the damages caused by freezing to cheese properties could be minimized if this type of storage is introduced at the end of ripening (42 d) using a freezing temperature of -20°C.

A new use for long-term frozen brain tissue: Golgi impregnation

PubMed Central

Melendez-Ferro, Miguel; Perez-Costas, Emma; Roberts, Rosalinda C.

2009-01-01

The study of dendritic spine shape and number has become a standard in the analysis of synaptic transmission anomalies since a considerable number of neuropsychiatric and neurological diseases have their foundation in alterations in these structures. One of the best ways to study possible alterations of dendritic spines is the use of Golgi impregnation. Although usually the Golgi method implies the use of fresh or fixed tissue, here we report the use of Golgi-Cox for the staining of human and animal brain tissue kept frozen for long periods of time. We successfully applied the Golgi-Cox method to human brain tissue stored for up to 15 years in a freezer. The technique produced reliable and reproducible impregnation of dendrites and dendritic spines in different cortical areas. We also applied the same technique to rat brain frozen for up to one year, obtaining the same satisfactory results. The fact that Golgi-Cox can be successfully applied to this type of tissue adds a new value for hundreds of frozen human or animal brains kept in the freezers of the laboratories, that otherwise would not be useful for anything else. Researchers other than neuroanatomists, i.e. in fields such as biochemistry and molecular biology can also benefit from a simple and reliable technique that can be applied to tissue left from their primary experiments. PMID:18789970

Immunogold Staining of Ultrathin Thawed Cryosections for Transmission Electron Microscopy (TEM).

PubMed

Skepper, Jeremy N; Powell, Janet M

2008-06-01

INTRODUCTIONA pre-embedding method of immunochemical staining is used if antigens are damaged by resin embedding, or if the best preservation of membranes is required. Applying immunogold reagents to sections of lightly fixed tissue, free of embedding medium, can be a very sensitive method of immunochemical staining. Cells or tissues are fixed as strongly as possible and then treated with a cryoprotectant, which is usually a mixture of sucrose and polyvinylpyrrolidone (PVP). They are frozen onto pins in liquid nitrogen and sectioned at approximately -100°C. The frozen sections are thaw-mounted on to Formvar/nickel film grids and the cryoprotectant is removed by floating the grids on drops of phosphate-buffered saline (PBS). The immunogold staining is performed on the unembedded sections, which are subsequently contrast counterstained and infiltrated with a mixture of methylcellulose and uranyl acetate. In this protocol, samples are sectioned at low temperature, thaw-mounted onto film grids, immunochemically stained, contrast counterstained, and embedded/encapsulated in situ on the grid before viewing by transmission electron microscopy (TEM).

Validation of a robust proteomic analysis carried out on formalin-fixed paraffin-embedded tissues of the pancreas obtained from mouse and human.

PubMed

Kojima, Kyoko; Bowersock, Gregory J; Kojima, Chinatsu; Klug, Christopher A; Grizzle, William E; Mobley, James A

2012-11-01

A number of reports have recently emerged with focus on extraction of proteins from formalin-fixed paraffin-embedded (FFPE) tissues for MS analysis; however, reproducibility and robustness as compared to flash frozen controls is generally overlooked. The goal of this study was to identify and validate a practical and highly robust approach for the proteomics analysis of FFPE tissues. FFPE and matched frozen pancreatic tissues obtained from mice (n = 8) were analyzed using 1D-nanoLC-MS(MS)(2) following work up with commercially available kits. The chosen approach for FFPE tissues was found to be highly comparable to that of frozen. In addition, the total number of unique peptides identified between the two groups was highly similar, with 958 identified for FFPE and 1070 identified for frozen, with protein identifications that corresponded by approximately 80%. This approach was then applied to archived human FFPE pancreatic cancer specimens (n = 11) as compared to uninvolved tissues (n = 8), where 47 potential pancreatic ductal adenocarcinoma markers were identified as significantly increased, of which 28 were previously reported. Further, these proteins share strongly overlapping pathway associations to pancreatic cancer that include estrogen receptor α. Together, these data support the validation of an approach for the proteomic analysis of FFPE tissues that is straightforward and highly robust, which can also be effectively applied toward translational studies of disease. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue

PubMed Central

2015-01-01

Objectives To translocation (externalization) of phosphatidylserine lead at least the five negative effects observed during cells cryopreservation: hypoxia, increasing of intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species (ROS) and lipid peroxidation. The aim of this study was to test the intensiveness of the phosphatidylserine translocation immediately after thawing and after 45 d xenografting of human ovarian tissue, which was either frozen just after operative removal from patient or cooled before cryopreservation to 5°C for 24 h and then frozen. Materials and Methods Ovarian fragments from twelve patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following four groups. Pieces of Group 1 (n=30) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n=30) after operation were cooled to 5°C for 24 h, then frozen after 24 h pre-cooling to 5°C, thawed and just after thawing their quality was analyzed. Group 3 pieces (n=30) were frozen immediately after operation without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was analyzed. Group 4 pieces (n=30) were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was analyzed. The effectiveness of the pre-freezing cooling of tissuewas evaluated by the development of follicles (histology) and by intensiveness of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide). Results For groups 1, 2, 3 and 4 the mean densities of follicles per 1 mm3 was 19.0, 20.2, 12.9, and 12.2, respectively (P1-2, 3-4 >0.1). For these groups, 99%, 98%, 88% and 90% preantral follicles, respectively were morphologically normal (P1-2, 3-4 >0.1). The FACS analysis showed significantly decreased intensiveness of translocation of phosphatidylserine after pre-cooling of frozen tissue (46.3% and 33.6% in Groups 2 and 4, respectively), in contrast with tissue frozen without pre-cooling (77.1% and 60.2 % in Groups 1 and 3, respectively, P1, 3-2, 4 <0.05). Conclusions Long time (24 h) cooling of ovarian tissue to 5°C before cryopreservation decreased translocation of phosphatidylserine that evidences about increases the viability of the cells in the tissue after thawing. PMID:26083026

Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue.

PubMed

Isachenko, Vladimir; Todorov, Plamen; Isachenko, Evgenia; Rahimi, Gohar; Tchorbanov, Andrey; Mihaylova, Nikolina; Manoylov, Iliyan; Mallmann, Peter; Merzenich, Markus

2015-01-01

To translocation (externalization) of phosphatidylserine lead at least the five negative effects observed during cells cryopreservation: hypoxia, increasing of intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species (ROS) and lipid peroxidation. The aim of this study was to test the intensiveness of the phosphatidylserine translocation immediately after thawing and after 45 d xenografting of human ovarian tissue, which was either frozen just after operative removal from patient or cooled before cryopreservation to 5°C for 24 h and then frozen. Ovarian fragments from twelve patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following four groups. Pieces of Group 1 (n=30) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n=30) after operation were cooled to 5°C for 24 h, then frozen after 24 h pre-cooling to 5°C, thawed and just after thawing their quality was analyzed. Group 3 pieces (n=30) were frozen immediately after operation without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was analyzed. Group 4 pieces (n=30) were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was analyzed. The effectiveness of the pre-freezing cooling of tissuewas evaluated by the development of follicles (histology) and by intensiveness of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide). For groups 1, 2, 3 and 4 the mean densities of follicles per 1 mm3 was 19.0, 20.2, 12.9, and 12.2, respectively (P1-2, 3-4 >0.1). For these groups, 99%, 98%, 88% and 90% preantral follicles, respectively were morphologically normal (P1-2, 3-4 >0.1). The FACS analysis showed significantly decreased intensiveness of translocation of phosphatidylserine after pre-cooling of frozen tissue (46.3% and 33.6% in Groups 2 and 4, respectively), in contrast with tissue frozen without pre-cooling (77.1% and 60.2 % in Groups 1 and 3, respectively, P1, 3-2, 4 <0.05). Long time (24 h) cooling of ovarian tissue to 5°C before cryopreservation decreased translocation of phosphatidylserine that evidences about increases the viability of the cells in the tissue after thawing.

Infrared micro-spectral imaging: distinction of tissue types in axillary lymph node histology

PubMed Central

Bird, Benjamin; Miljkovic, Milos; Romeo, Melissa J; Smith, Jennifer; Stone, Nicholas; George, Michael W; Diem, Max

2008-01-01

Background Histopathologic evaluation of surgical specimens is a well established technique for disease identification, and has remained relatively unchanged since its clinical introduction. Although it is essential for clinical investigation, histopathologic identification of tissues remains a time consuming and subjective technique, with unsatisfactory levels of inter- and intra-observer discrepancy. A novel approach for histological recognition is to use Fourier Transform Infrared (FT-IR) micro-spectroscopy. This non-destructive optical technique can provide a rapid measurement of sample biochemistry and identify variations that occur between healthy and diseased tissues. The advantage of this method is that it is objective and provides reproducible diagnosis, independent of fatigue, experience and inter-observer variability. Methods We report a method for analysing excised lymph nodes that is based on spectral pathology. In spectral pathology, an unstained (fixed or snap frozen) tissue section is interrogated by a beam of infrared light that samples pixels of 25 μm × 25 μm in size. This beam is rastered over the sample, and up to 100,000 complete infrared spectra are acquired for a given tissue sample. These spectra are subsequently analysed by a diagnostic computer algorithm that is trained by correlating spectral and histopathological features. Results We illustrate the ability of infrared micro-spectral imaging, coupled with completely unsupervised methods of multivariate statistical analysis, to accurately reproduce the histological architecture of axillary lymph nodes. By correlating spectral and histopathological features, a diagnostic algorithm was trained that allowed both accurate and rapid classification of benign and malignant tissues composed within different lymph nodes. This approach was successfully applied to both deparaffinised and frozen tissues and indicates that both intra-operative and more conventional surgical specimens can be diagnosed by this technique. Conclusion This paper provides strong evidence that automated diagnosis by means of infrared micro-spectral imaging is possible. Recent investigations within the author's laboratory upon lymph nodes have also revealed that cancers from different primary tumours provide distinctly different spectral signatures. Thus poorly differentiated and hard-to-determine cases of metastatic invasion, such as micrometastases, may additionally be identified by this technique. Finally, we differentiate benign and malignant tissues composed within axillary lymph nodes by completely automated methods of spectral analysis. PMID:18759967

Effect of multiple cycles of freeze-thawing on the RNA quality of lung cancer tissues.

PubMed

Yu, Keke; Xing, Jie; Zhang, Jie; Zhao, Ruiying; Zhang, Ye; Zhao, Lanxiang

2017-09-01

RNA degradation is a major problem in tissue banking. We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematoxylin and eosin staining. RNA extraction from 60 lung cancer tissue samples subjected to various freeze/thaw cycles was performed using the RNeasy Plus isolation kit. RNA integrity was assessed by using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Furthermore, RNA from different groups was used for fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene mutation to verify whether it can be used for research or clinical testing. Highly variable RIN values were observed among the samples, which showed no correlation with the number of freeze/thaw cycles conducted. However, after 3 freeze/thaw cycles (each thaw event lasted for 10 min), an increasing number of changes in peak intensity in RINs were observed. After 5 freeze/thaw cycles, RNA integrity decreased to approximately 35%. After 3 freeze/thaw cycles, the RNA could still be used for RT-PCR analysis of EML4-ALK fusion gene mutations; whereas those subjected to 5 freeze/thaw cycles could not. Limited (<3) freeze/thaw cycles did not adversely affect the quality of RNA extracted from tumor tissues and subsequent RT-PCR analysis. Our data could be utilized in the establishment of a standardized procedure for tissue biospecimen collection and storage.

Molecular Epidemiology of Ovarian Cancer

DTIC Science & Technology

2006-07-01

Testing of hypotheses relating to androgen exposure ( polycystic ovary syndrome, hirsutism, acne etc): (a) Analysis by histologic subtype (b) Analysis by...having primary epithelial cancer of the ovary , peritoneum or fallopian tube, of whom 1092 returned a questionnaire, 1022 gave a blood sample and we...increasing recognition that invasive mucinous tumours of the ovary are usually secondary neoplasms (Lee et al). Table 4: Total number of AOCS frozen tissue

NASA's Rodent Research Project: Validation of Capabilities for Conducting Long Duration Experiments in Space

NASA Technical Reports Server (NTRS)

Choi, Sungshin Y.; Cole, Nicolas; Reyes, America; Lai, San-Huei; Klotz, Rebecca; Beegle, Janet E.; Wigley, Cecilia L.; Pletcher, David; Globus, Ruth K.

2015-01-01

Research using rodents is an essential tool for advancing biomedical research on Earth and in space. Prior rodent experiments on the Shuttle were limited by the short flight duration. The International Space Station (ISS) provides a new platform for conducting rodent experiments under long duration conditions. Rodent Research (RR)-1 was conducted to validate flight hardware, operations, and science capabilities that were developed at the NASA Ames Research Center. Twenty C57BL6J adult female mice were launched on Sept 21, 2014 in a Dragon Capsule (SpaceX-4), then transferred to the ISS for a total time of 21-22 days (10 commercial mice) or 37 days (10 validation mice). Tissues collected on-orbit were either rapidly frozen or preserved in RNAlater at -80C (n2group) until their return to Earth. Remaining carcasses on-orbit were rapidly frozen for dissection post-flight. The three controls groups at Kennedy Space Center consisted of: Basal mice euthanized at the time of launch, Vivarium controls housed in standard cages, and Ground Controls (GC) housed in flight hardware within an environmental chamber. Upon return to Earth, there were no differences in body weights between Flight (FLT) and GC at the end of the 37 days in space. Liver enzyme activity levels of FLT mice and all control mice were similar in magnitude to those of the samples that were processed under optimal conditions in the laboratory. Liver samples dissected on-orbit yielded high quality RNA (RIN8.99+-0.59, n7). Liver samples dissected post-flight from the intact, frozen FLT carcasses yielded RIN of 7.27 +- 0.52 (n6). Additionally, wet weights of various tissues were measured. Adrenal glands and spleen showed no significant differences in FLT compared to GC although thymus and livers weights were significantly greater in FLT compared to GC. Over 3,000 tissue aliquots collected post-flight from the four groups of mice were deposited into the Ames Life Science Data Archives for future Biospecimen Sharing Program. Together, the RR validation flight successfully demonstrates the capability to support long-duration experimentation on the ISS to achieve both basic science and biomedical objectives.

The Japanese Society of Pathology Guidelines on the handling of pathological tissue samples for genomic research: Standard operating procedures based on empirical analyses.

PubMed

Kanai, Yae; Nishihara, Hiroshi; Miyagi, Yohei; Tsuruyama, Tatsuhiro; Taguchi, Kenichi; Katoh, Hiroto; Takeuchi, Tomoyo; Gotoh, Masahiro; Kuramoto, Junko; Arai, Eri; Ojima, Hidenori; Shibuya, Ayako; Yoshida, Teruhiko; Akahane, Toshiaki; Kasajima, Rika; Morita, Kei-Ichi; Inazawa, Johji; Sasaki, Takeshi; Fukayama, Masashi; Oda, Yoshinao

2018-02-01

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed "The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research" based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about -180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine. © 2018 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

RT-PCR analysis of RNA extracted from Bouin-fixed and paraffin-embedded lymphoid tissues.

PubMed

Gloghini, Annunziata; Canal, Barbara; Klein, Ulf; Dal Maso, Luigino; Perin, Tiziana; Dalla-Favera, Riccardo; Carbone, Antonino

2004-11-01

In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]- and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA. To explore the possibility for giving accurate real time quantitative RT-PCR results, cDNA obtained from matched frozen, Bouin-fixed and formalin-fixed neoplastic samples (two diffuse large cell lymphomas, one plasmacytoma) was tested for the following target genes: CD40, Aquaporin-3, BLIMP1, IRF4, Syndecan-1. Delta threshold cycle (DeltaC(T)) values for Bouin-fixed and formalin-fixed paraffin-embedded tissues and their correlation with those for frozen samples showed an extremely high correlation (r > 0.90) for all of the tested genes. These results show that the method of RNA extraction we propose is suitable for giving accurate real time quantitative RT-PCR results.

Rapid demonstration of Legionella pneumophila in unembedded tissue. An adaptation of the Giménez stain.

PubMed

Greer, P W; Chandler, F W; Hicklin, M D

1980-06-01

The Giménez stain, originally developed for demonstrating rickettsiae, readily stained the Legionnaires' disease bacterium (Legionella pneumophila) in frozen tissue sections and smears of fresh or formalin-fixed lung tissue from patients who had confirmed Legionnaires' disease. With the Giménez procedure, the bacterium stained bright red against a blue-green background. The tissue Gram procedures also stained L. pneumophila in frozen sections and smears, but the staining reaction was weak, and these stains were neither as sensitive nor a consistent as the Giménez procedure.

Evaluation of the branched-chain DNA assay for measurement of RNA in formalin-fixed tissues.

PubMed

Knudsen, Beatrice S; Allen, April N; McLerran, Dale F; Vessella, Robert L; Karademos, Jonathan; Davies, Joan E; Maqsodi, Botoul; McMaster, Gary K; Kristal, Alan R

2008-03-01

We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93-100%) than for qPCR (82.4-95%). Correlations between qPCR(FROZEN), the gold standard, and bDNA(FFPE) ranged from 0.60 to 0.94, similar to those from qPCR(FROZEN) and qPCR(FFPE). Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management.

An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis

PubMed Central

Karsten, Stanislav L.; Van Deerlin, Vivianna M. D.; Sabatti, Chiara; Gill, Lisa H.; Geschwind, Daniel H.

2002-01-01

Archival formalin-fixed, paraffin-embedded and ethanol-fixed tissues represent a potentially invaluable resource for gene expression analysis, as they are the most widely available material for studies of human disease. Little data are available evaluating whether RNA obtained from fixed (archival) tissues could produce reliable and reproducible microarray expression data. Here we compare the use of RNA isolated from human archival tissues fixed in ethanol and formalin to frozen tissue in cDNA microarray experiments. Since an additional factor that can limit the utility of archival tissue is the often small quantities available, we also evaluate the use of the tyramide signal amplification method (TSA), which allows the use of small amounts of RNA. Detailed analysis indicates that TSA provides a consistent and reproducible signal amplification method for cDNA microarray analysis, across both arrays and the genes tested. Analysis of this method also highlights the importance of performing non-linear channel normalization and dye switching. Furthermore, archived, fixed specimens can perform well, but not surprisingly, produce more variable results than frozen tissues. Consistent results are more easily obtainable using ethanol-fixed tissues, whereas formalin-fixed tissue does not typically provide a useful substrate for cDNA synthesis and labeling. PMID:11788730

Comparison of different histological protocols for the preservation and quantification of the intestinal mucus layer in pigs.

PubMed

Röhe, Ilen; Hüttner, Friedrich Joseph; Plendl, Johanna; Drewes, Barbara; Zentek, Jürgen

2018-02-05

The histological characterization of the intestinal mucus layer is important for many scientific experiments investigating the interaction between intestinal microbiota, mucosal immune response and intestinal mucus production. The aim of this study was to examine and compare different fixation protocols for displaying and quantifying the intestinal mucus layer in piglets and to test which histomorphological parameters may correlate with the determined mucus layer thickness. Jejunal and colonal tissue samples of weaned piglets (n=10) were either frozen in liquid nitrogen or chemically fixed using methacarn solution. The frozen tissue samples were cryosectioned and subsequently postfixed using three different postfixatives: paraformaldehyde vapor, neutrally buffered formalin solution and ethanol solution. After dehydration, methacarn fixed tissues were embedded in paraffin wax. Both sections of cryopreserved and methacarn fixed tissue samples were stained with Alcian blue (AB)-PAS followed by the microscopically determination of the mucus layer thickness. Different pH values of the Alcian Blue staining solution and two mucus layer thickness measuring methods were compared. In addition, various histomorphological parameters of methacarn fixed tissue samples were evaluated including the number of goblet cells and the mucin staining area. Cryopreservation in combination with chemical postfixation led to mucus preservation in the colon of piglets allowing mucus thickness measurements. Mucus could be only partly preserved in cryosections of the jejunum impeding any quantitative description of the mucus layer thickness. The application of different postfixations, varying pH values of the AB solution and different mucus layer measuring methods led to comparable results regarding the mucus layer thickness. Methacarn fixation proved to be unsuitable for mucus depiction as only mucus patches were found in the jejunum or a detachment of the mucus layer from the epithelium was observed in the colon. Correlation analyses revealed that the proportion of the mucin staining area per crypt area (relative mucin staining) measured in methacarn fixed tissue samples corresponded to the colonal mucus layer thickness determined in cryopreserved tissue samples. In conclusion, the results showed that cryopreservation using liquid nitrogen followed by chemical postfixation and AB-PAS staining led to a reliable mucus preservation allowing a mucus thickness determination in the colon of pigs. Moreover, the detected relative mucin staining area may serve as a suitable histomorphological parameter for the assessment of the intestinal mucus layer thickness. The findings obtained in this study can be used for the implementation of an improved standard for the histological description of the mucus layer in the colon of pigs.

Efficient Recovery of Fluoroquinolone-Susceptible and Fluoroquinolone-Resistant Escherichia coli Strains From Frozen Samples

PubMed Central

Lautenbach, Ebbing; Santana, Evelyn; Lee, Abby; Tolomeo, Pam; Black, Nicole; Babson, Andrew; Perencevich, Eli N.; Harris, Anthony D.; Smith, Catherine A.; Maslow, Joel

2010-01-01

We assessed the rate of recovery of fluoroquinolone-resistant and fluoroquinolone-susceptible Escherichia coli isolates from culture of frozen perirectal swab samples compared with the results for culture of the same specimen before freezing. Recovery rates for these 2 classes of E. coli were 91% and 83%, respectively. The majority of distinct strains recovered from the initial sample were also recovered from the frozen sample. The strains that were not recovered were typically present only in low numbers in the initial sample. These findings emphasize the utility of frozen surveillance samples. PMID:18279070

Efficient recovery of fluoroquinolone-susceptible and fluoroquinolone-resistant Escherichia coli strains from frozen samples.

PubMed

Lautenbach, Ebbing; Santana, Evelyn; Lee, Abby; Tolomeo, Pam; Black, Nicole; Babson, Andrew; Perencevich, Eli N; Harris, Anthony D; Smith, Catherine A; Maslow, Joel

2008-04-01

We assessed the rate of recovery of fluoroquinolone-resistant and fluoroquinolone-susceptible Escherichia coli isolates from culture of frozen perirectal swab samples compared with the results for culture of the same specimen before freezing. Recovery rates for these 2 classes of E. coli were 91% and 83%, respectively. The majority of distinct strains recovered from the initial sample were also recovered from the frozen sample. The strains that were not recovered were typically present only in low numbers in the initial sample. These findings emphasize the utility of frozen surveillance samples.

Effects of freezing and thawing on texture, microstructure and cell wall composition changes in papaya tissues.

PubMed

Phothiset, Suphatta; Charoenrein, Sanguansri

2014-01-30

During storage, frozen fruit may be thawed and refrozen many times before consumption, which may be extremely damaging to the texture of the frozen fruit and reverse the advantage of fast freezing. The effects of freezing and thawing on texture, microstructure and cell wall composition changes in papaya tissues were investigated. The frozen-thawed papayas had an increase in drip loss and a decrease in firmness with increasing number of freeze-thaw cycles. Light microscopy showed irregular shapes and cell damage in parenchyma cells of frozen-thawed papayas, whereas transmission electron microscopy showed loss of cell wall materials in middle lamella. Moreover, destruction of cell wall was observed after being subjected to five freeze-thaw cycles. These changes related with a significant decrease in alcohol-insoluble solids, Na₂CO₃- and 24% KOH-soluble fractions and an increase in the water-, EDTA- and 4% KOH-soluble fractions. This was due to a decrease in the molecular mass of pectic and hemicellulosic polymers in frozen-thawed papayas using high-performance size-exclusion chromatography. The freezing and thawing processes caused fine structural damage and cell wall composition changes which contributed to a loss of drip volume and firmness of papaya tissues. © 2013 Society of Chemical Industry.

Validation of the Lung Subtyping Panel in Multiple Fresh-Frozen and Formalin-Fixed, Paraffin-Embedded Lung Tumor Gene Expression Data Sets.

PubMed

Faruki, Hawazin; Mayhew, Gregory M; Fan, Cheng; Wilkerson, Matthew D; Parker, Scott; Kam-Morgan, Lauren; Eisenberg, Marcia; Horten, Bruce; Hayes, D Neil; Perou, Charles M; Lai-Goldman, Myla

2016-06-01

Context .- A histologic classification of lung cancer subtypes is essential in guiding therapeutic management. Objective .- To complement morphology-based classification of lung tumors, a previously developed lung subtyping panel (LSP) of 57 genes was tested using multiple public fresh-frozen gene-expression data sets and a prospectively collected set of formalin-fixed, paraffin-embedded lung tumor samples. Design .- The LSP gene-expression signature was evaluated in multiple lung cancer gene-expression data sets totaling 2177 patients collected from 4 platforms: Illumina RNAseq (San Diego, California), Agilent (Santa Clara, California) and Affymetrix (Santa Clara) microarrays, and quantitative reverse transcription-polymerase chain reaction. Gene centroids were calculated for each of 3 genomic-defined subtypes: adenocarcinoma, squamous cell carcinoma, and neuroendocrine, the latter of which encompassed both small cell carcinoma and carcinoid. Classification by LSP into 3 subtypes was evaluated in both fresh-frozen and formalin-fixed, paraffin-embedded tumor samples, and agreement with the original morphology-based diagnosis was determined. Results .- The LSP-based classifications demonstrated overall agreement with the original clinical diagnosis ranging from 78% (251 of 322) to 91% (492 of 538 and 869 of 951) in the fresh-frozen public data sets and 84% (65 of 77) in the formalin-fixed, paraffin-embedded data set. The LSP performance was independent of tissue-preservation method and gene-expression platform. Secondary, blinded pathology review of formalin-fixed, paraffin-embedded samples demonstrated concordance of 82% (63 of 77) with the original morphology diagnosis. Conclusions .- The LSP gene-expression signature is a reproducible and objective method for classifying lung tumors and demonstrates good concordance with morphology-based classification across multiple data sets. The LSP panel can supplement morphologic assessment of lung cancers, particularly when classification by standard methods is challenging.

Evaporation process in histological tissue sections for neutron autoradiography.

PubMed

Espector, Natalia M; Portu, Agustina; Santa Cruz, Gustavo A; Saint Martin, Gisela

2018-05-01

The analysis of the distribution and density of nuclear tracks forming an autoradiography in a nuclear track detector (NTD) allows the determination of 10 B atoms concentration and location in tissue samples from Boron Neutron Capture Therapy (BNCT) protocols. This knowledge is of great importance for BNCT dosimetry and treatment planning. Tissue sections studied with this technique are obtained by cryosectioning frozen tissue specimens. After the slicing procedure, the tissue section is put on the NTD and the sample starts drying. The thickness varies from its original value allowing more particles to reach the detector and, as the mass of the sample decreases, the boron concentration in the sample increases. So in order to determine the concentration present in the hydrated tissue, the application of corrective coefficients is required. Evaporation mechanisms as well as various factors that could affect the process of mass variation are outlined in this work. Mass evolution for tissue samples coming from BDIX rats was registered with a semimicro analytical scale and measurements were analyzed with software developed to that end. Ambient conditions were simultaneously recorded, obtaining reproducible evaporation curves. Mathematical models found in the literature were applied for the first time to this type of samples and the best fit of the experimental data was determined. The correlation coefficients and the variability of the parameters were evaluated, pointing to Page's model as the one that best represented the evaporation curves. These studies will contribute to a more precise assessment of boron concentration in tissue samples by the Neutron Autoradiography technique.

A Limited Survey of Heavy Metal Concentrations in Fresh and Frozen Cuttlefish Ink and Mantle Used As Food.

PubMed

Conficoni, Daniele; Alberghini, Leonardo; Bissacco, Elisa; Contiero, Barbara; Giaccone, Valerio

2018-02-01

Cuttlefish ink is consumed as a delicacy worldwide. The current study is the first assessment of heavy metal concentrations in cuttlefish ink versus mantle under different storage methods. A total of 212 samples (64 of fresh mantle, 42 of frozen mantle, 64 of fresh ink, and 42 of frozen ink) were analyzed for the detection of the following heavy metals: arsenic (As), chromium (Cr), iron (Fe), lead (Pb), mercury (Hg), and cadmium (Cd). The median As concentrations were 12.9 mg/kg for fresh mantle, 8.63 mg/kg for frozen mantle, 10.8 mg/kg for frozen ink, and 0.41 mg/kg for fresh ink. The median Cr concentrations were 0.06 mg/kg for fresh mantle and frozen ink, 0.03 mg/kg for frozen mantle, and below the limit of quantification (LOQ) for fresh ink. The median Fe concentrations were 4.08 mg/kg for frozen ink, 1.51 mg/kg for fresh mantle, 0.73 mg/kg for frozen mantle, and below the LOQ for fresh ink. The median Pb concentrations of almost all samples were below the LOQ; only two frozen ink, one fresh ink, one frozen mantle, and one fresh mantle sample exceeded the limit stipulated by the European Union. The Hg concentrations were statistically similar among the four categories of samples; the median Hg concentrations were below the LOQ, and the maximum concentrations were found in frozen ink, at 1.62 mg/kg. The median Cd concentrations were 0.69 mg/kg for frozen ink and 0.11 mg/kg for frozen mantle, fresh mantle and fresh ink concentrations were below the LOQ, and in 11.3% of the tested samples, Cd concentrations were higher than the European Union limit. The probability of samples having a Cd concentration above the legal limit was 35.75 times higher in frozen than in fresh products. Fresh ink had significantly lower concentrations of As, Cr, Fe, and Cd, but the concentrations of Hg and Pb were not significantly different from those of other products. Frozen ink had significantly higher concentrations of Cd, Cr, and Fe, but concentrations of As were lower than those in fresh mantle, pointing out a possible role for the freezing process and for different fishing zones as risk factors for heavy metal contamination.

qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL

PubMed Central

2010-01-01

Background Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST. Results Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation. Conclusions As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It remains to be clarified whether an autocrine or paracrine mechanism by overexpression of receptor tyrosine kinase ligands is responsible for the tumorigenesis of wt-GIST. PMID:21171987

A multiplex real-time polymerase chain reaction assay with two internal controls for the detection of Brucella species in tissues, blood, and feces from marine mammals.

PubMed

Sidor, Inga F; Dunn, J Lawrence; Tsongalis, Gregory J; Carlson, Jolene; Frasca, Salvatore

2013-01-01

Brucellosis has emerged as a disease of concern in marine mammals in the last 2 decades. Molecular detection techniques have the potential to address limitations of other methods for detecting infection with Brucella in these species. Presented herein is a real-time polymerase chain reaction (PCR) method targeting the Brucella genus-specific bcsp31 gene. The method also includes a target to a conserved region of the eukaryotic mitochondrial 16S ribosomal RNA gene to assess suitability of extracted DNA and a plasmid-based internal control to detect failure of PCR due to inhibition. This method was optimized and validated to detect Brucella spp. in multiple sample matrices, including fresh or frozen tissue, blood, and feces. The analytical limit of detection was low, with 95% amplification at 24 fg, or an estimated 7 bacterial genomic copies. When Brucella spp. were experimentally added to tissue or fecal homogenates, the assay detected an estimated 1-5 bacteria/µl. An experiment simulating tissue autolysis showed relative persistence of bacterial DNA compared to host mitochondrial DNA. When used to screen 1,658 field-collected marine mammal tissues in comparison to microbial culture, diagnostic sensitivity and specificity were 70.4% and 98.3%, respectively. In addition to amplification in fresh and frozen tissues, Brucella spp. were detected in feces and formalin-fixed, paraffin-embedded tissues from culture-positive animals. Results indicate the utility of this real-time PCR for the detection of Brucella spp. in marine species, which may have applications in surveillance or epidemiologic investigations.

Specimen Collection and Submission Manual

DTIC Science & Technology

2016-06-01

immunoassays Specimen: tissue or bone marrow (100 mg); Whole EDTA blood or serum (0.5 ml) Nasopharyngeal or throat swab, dry or in transport medium; Sputum... Syndrome Coronavirus (MERS-CoV) – detection in clinical samples Methodology: molecular Specimen: If possible collect 3 specimen types (lower...guidelines-clinical-specimens.html) Shipping: ship cold on wet ice or ice packs. For delays exceeding 72 hours, ship frozen on dry ice. Turnaround: 1-2

Effect of different cooking methods on color, phytochemical concentration, and antioxidant capacity of raw and frozen brassica vegetables.

PubMed

Pellegrini, Nicoletta; Chiavaro, Emma; Gardana, Claudio; Mazzeo, Teresa; Contino, Daniele; Gallo, Monica; Riso, Patrizia; Fogliano, Vincenzo; Porrini, Marisa

2010-04-14

This study evaluated the effect of common cooking practices (i.e., boiling, microwaving, and basket and oven steaming) on the phytochemical content (carotenoids, chlorophylls, glucosinolates, polyphenols, and ascorbic acid), total antioxidant capacity (TAC), and color changes of three generally consumed Brassica vegetables analyzed fresh and frozen. Among cooking procedures, boiling determined an increase of fresh broccoli carotenoids and fresh Brussels sprout polyphenols, whereas a decrease of almost all other phytochemicals in fresh and frozen samples was observed. Steaming procedures determined a release of polyphenols in both fresh and frozen samples. Microwaving was the best cooking method for maintaining the color of both fresh and frozen vegetables and obtaining a good retention of glucosinolates. During all cooking procedures, ascorbic acid was lost in great amount from all vegetables. Chlorophylls were more stable in frozen samples than in fresh ones, even though steaming methods were able to better preserve these compounds in fresh samples than others cooking methods applied. The overall results of this study demonstrate that fresh Brassica vegetables retain phytochemicals and TAC better than frozen samples.

Variation in glycogen concentrations within mantle and foot tissue in Amblema plicata plicata: Implications for tissue biopsy sampling

USGS Publications Warehouse

Naimo, T.J.; Monroe, E.M.

1999-01-01

With the development of techniques to non-lethally biopsy tissue from unionids, a new method is available to measure changes in biochemical, contaminant, and genetic constituents in this imperiled faunal group. However, before its widespread application, information on the variability of biochemical components within and among tissues needs to be evaluated. We measured glycogen concentrations in foot and mantle tissue in Amblema plicata plicata (Say, 1817) to determine if glycogen was evenly distributed within and between tissues and to determine which tissue might be more responsive to the stress associated with relocating mussels. Glycogen was measured in two groups of mussels: those sampled from their native environment (undisturbed mussels) and quickly frozen for analysis and those relocated into an artificial pond (relocated mussels) for 24 months before analysis. In both undisturbed and relocated mussels, glycogen concentrations were evenly distributed within foot, but not within mantle tissue. In mantle tissue, concentrations of glycogen varied about 2-fold among sections. In addition, glycogen varied significantly between tissues in undisturbed mussels, but not in relocated mussels. Twenty-four months after relocation, glycogen concentrations had declined by 80% in mantle tissue and by 56% in foot tissue relative to the undisturbed mussels. These data indicate that representative biopsy samples can be obtained from foot tissue, but not mantle tissue. We hypothesize that mantle tissue could be more responsive to the stress of relocation due to its high metabolic activity associated with shell formation.

A comparison of sample preparation strategies for biological tissues and subsequent trace element analysis using LA-ICP-MS.

PubMed

Bonta, Maximilian; Török, Szilvia; Hegedus, Balazs; Döme, Balazs; Limbeck, Andreas

2017-03-01

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is one of the most commonly applied methods for lateral trace element distribution analysis in medical studies. Many improvements of the technique regarding quantification and achievable lateral resolution have been achieved in the last years. Nevertheless, sample preparation is also of major importance and the optimal sample preparation strategy still has not been defined. While conventional histology knows a number of sample pre-treatment strategies, little is known about the effect of these approaches on the lateral distributions of elements and/or their quantities in tissues. The technique of formalin fixation and paraffin embedding (FFPE) has emerged as the gold standard in tissue preparation. However, the potential use for elemental distribution studies is questionable due to a large number of sample preparation steps. In this work, LA-ICP-MS was used to examine the applicability of the FFPE sample preparation approach for elemental distribution studies. Qualitative elemental distributions as well as quantitative concentrations in cryo-cut tissues as well as FFPE samples were compared. Results showed that some metals (especially Na and K) are severely affected by the FFPE process, whereas others (e.g., Mn, Ni) are less influenced. Based on these results, a general recommendation can be given: FFPE samples are completely unsuitable for the analysis of alkaline metals. When analyzing transition metals, FFPE samples can give comparable results to snap-frozen tissues. Graphical abstract Sample preparation strategies for biological tissues are compared with regard to the elemental distributions and average trace element concentrations.

SPRUCE Shrub-Layer Growth Assessments in S1-Bog Plots and SPRUCE Experimental Plots beginning in 2010

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanson, P.J.; Phillips, J.R.; Brice, D.J.

This data set reports shrub layer growth assessments for the S1-Bog on the Marcell Experimental Forest in Minnesota from 2010 through 2017. Data were obtained by destructively harvesting two 0.25 m2 plots within defined plot areas of the S1-Bog or SPRUCE experimental plots. In 2015, SPRUCE plots 4, 6, 8, 10, 11, 13, 16, 17, 19 and 20 were enclosed in the SPRUCE enclosures. Prior to 2015 all data are for open ambient conditions. In early years a distinct hummock and a hollow sampling square were both collected, but in later years unsampled hollow areas became unavailable due to priormore » sampling or instrument installations. All vegetation material above the Sphagnum surface of the bog was clipped and transferred to plastic storage bags which were then frozen until the samples could be sorted. Sorting was done by species, tissue type (leaves vs. stems) and tissue age (current-year vs. older tissues).« less

A method for measuring total thiaminase activity in fish tissues

USGS Publications Warehouse

Zajicek, James L.; Tillitt, Donald E.; Honeyfield, Dale C.; Brown, Scott B.; Fitzsimons, John D.

2005-01-01

An accurate, quantitative, and rapid method for the measurement of thiaminase activity in fish samples is required to provide sufficient information to characterize the role of dietary thiaminase in the onset of thiamine deficiency in Great Lakes salmonines. A radiometric method that uses 14C-thiamine was optimized for substrate and co-substrate (nicotinic acid) concentrations, incubation time, and sample dilution. Total thiaminase activity was successfully determined in extracts of selected Great Lakes fishes and invertebrates. Samples included whole-body and selected tissues of forage fishes. Positive control material prepared from frozen alewives Alosa pseudoharengus collected in Lake Michigan enhanced the development and application of the method. The method allowed improved discrimination of thiaminolytic activity among forage fish species and their tissues. The temperature dependence of the thiaminase activity observed in crude extracts of Lake Michigan alewives followed a Q10 = 2 relationship for the 1-37??C temperature range, which is consistent with the bacterial-derived thiaminase I protein. ?? Copyright by the American Fisheries Society 2005.

European Organisation for Research and Treatment of Cancer (EORTC) Pathobiology Group standard operating procedure for the preparation of human tumour tissue extracts suited for the quantitative analysis of tissue-associated biomarkers.

PubMed

Schmitt, Manfred; Mengele, Karin; Schueren, Elisabeth; Sweep, Fred C G J; Foekens, John A; Brünner, Nils; Laabs, Juliane; Malik, Abha; Harbeck, Nadia

2007-03-01

With the new concept of 'individualized treatment and targeted therapies', tumour tissue-associated biomarkers have been given a new role in selection of cancer patients for treatment and in cancer patient management. Tumour biomarkers can give support to cancer patient stratification and risk assessment, treatment response identification, or to identifying those patients who are expected to respond to certain anticancer drugs. As the field of tumour-associated biomarkers has expanded rapidly over the last years, it has become increasingly apparent that a strong need exists to establish guidelines on how to easily disintegrate the tumour tissue for assessment of the presence of tumour tissue-associated biomarkers. Several mechanical tissue (cell) disruption techniques exist, ranging from bead mill homogenisation and freeze-fracturing through to blade or pestle-type homogenisation, to grinding and ultrasonics. Still, only a few directives have been given on how fresh-frozen tumour tissues should be processed for the extraction and determination of tumour biomarkers. The PathoBiology Group of the European Organisation for Research and Treatment of Cancer therefore has devised a standard operating procedure for the standardised preparation of human tumour tissue extracts which is designed for the quantitative analysis of tumour tissue-associated biomarkers. The easy to follow technical steps involved require 50-300 mg of deep-frozen cancer tissue placed into small size (1.2 ml) cryogenic tubes. These are placed into the shaking flask of a Mikro-Dismembrator S machine (bead mill) to pulverise the tumour tissue in the capped tubes in the deep-frozen state by use of a stainless steel ball, all within 30 s of exposure. RNA is isolated from the pulverised tissue following standard procedures. Proteins are extracted from the still frozen pulverised tissue by addition of Tris-buffered saline to obtain the cytosol fraction of the tumour or by the Tris buffer supplemented with the non-ionic detergent Triton X-100, and, after high-speed centrifugation, are found in the tissue supernatant. The resulting tissue cell debris sediment is a rich source of genomic DNA.

Significance of biological resource collection and tumor tissue bank creation.

PubMed

Yu, Ying-Yan; Zhu, Zheng-Gang

2010-01-15

Progress in the molecular oncology of gastrointestinal carcinomas depends on high quality cancer tissues for research. Recent acceleration on new technological platforms as well as the "omics" revolution increases the demands on tissues and peripheral blood for research at the DNA, mRNA and protein levels. Tissue bank creation emerges as a priority. Tumor tissue banks are facilities that are organized to collect, store and distribute samples of tumor and normal tissue for further use in basic and translational cancer research. The samples are generally obtained immediately after excision, prior to fixation, to ensure optimal preservation of proteins and nucleic acids. It is possible for surgeons or pathologists to collect fresh tissue prospectively during their routine dissection procedures. Most tissue banks are "project-driven" tumor banks, which are specialized collections of tumor samples on which their research is based. Systematic collection of all available tumor tissue is much rarer. High quality tissue banks need the collaboration of clinicians and basic scientists, but also the informed consent of patients and ethical approval. Through the standard operation procedure, snap frozen fresh tissue collection, storage and quality control for cryopreserved tissues are the pivotal factors on tissue bank construction and maintaining. The purpose of the tissue bank creation is enhancing the quality and speed on both the basic and translational research on gastrointestinal cancer. The quality assurance and quality control are handled based on reviewing HE staining slides or touch imprint cytology by pathologists.

MGMT promoter methylation in Peruvian patients with glioblastoma

PubMed Central

Belmar-Lopez, Carolina; Castaneda, Carlos A; Castillo, Miluska; García-Corrochano, Pamela; Orrego, Enrique; Meléndez, Barbara; Casavilca, Sandro; Flores, Claudio; Orrego, Enrique

2018-01-01

Purpose O6-methylguanine–DNA methyltransferase (MGMT) promoter methylation predicts the outcome and response to alkylating chemotherapy in glioblastoma. The aim of this study is to evaluate the prevalence of MGMT methylation in Peruvian glioblastoma cases. Patients and methods We evaluated retrospectively 50 cases of resected glioblastoma during the period 2008–2013 at Instituto Nacional de Enfermedades Neoplasicas in Peru. Samples consisted of paraffin embedded and frozen tumour tissue. MGMT-promoter methylation status and the expression level of MGMT gene were evaluated by methylation-specific PCR and real-time PCR, respectively. Results Unmethylated, methylated and partially methylated statuses were found in 54%, 20% and 26% of paraffin-embedded samples, respectively. Methylation status was confirmed in the Virgen de la Salud Hospital and frozen samples. There was an association between the status of MGMT-promoter methylation and the level of gene expression (p = 0.001). Methylation was associated with increased progression-free survival (p = 0.002) and overall survival (OS) (p < 0.001). Conclusion MGMT-promoter methylation frequency in Peruvian glioblastoma is similar to that reported in other populations and the detection test has been standardised. PMID:29515653

Characterization of the Tumor Secretome from Tumor Interstitial Fluid (TIF).

PubMed

Gromov, Pavel; Gromova, Irina

2016-01-01

Tumor interstitial fluid (TIF) surrounds and perfuses bodily tumorigenic tissues and cells, and can accumulate by-products of tumors and stromal cells in a relatively local space. Interstitial fluid offers several important advantages for biomarker and therapeutic target discovery, especially for cancer. Here, we describe the most currently accepted method for recovering TIF from tumor and nonmalignant tissues that was initially performed using breast cancer tissue. TIF recovery is achieved by passive extraction of fluid from small, surgically dissected tissue specimens in phosphate-buffered saline. We also present protocols for hematoxylin and eosin (H&E) staining of snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tumor sections and for proteomic profiling of TIF and matched tumor samples by high-resolution two-dimensional gel electrophoresis (2D-PAGE) to enable comparative analysis of tumor secretome and paired tumor tissue.

Detection of PAX3-FKHR and PAX7-FKHR fusion transcripts in rhabdomyosarcoma by reverse transcriptase-polymerase chain reaction using paraffin-embedded tissue.

PubMed

Chen, B F; Chen, M L; Liang, D C; Huang, Y W; Liu, H C; Chen, S H

1999-02-01

Alveolar rhabdomyosarcoma (RMS) is associated with a characteristic chromosomal translocation t(2;13)(q35;q14). The genes involved in this translocation are paired box (PAX)3 on chromosome 2 and forkhead in RMS (FKHR) on chromosome 13. An occasional variant translocation t(1;13)(p36;q14) affecting PAX7 and FKHR on chromosomes 1 and 13, respectively, has also been described. Chromosomal translocations in RMS are detected using conventional cytogenetic analysis, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) on fresh or frozen tissue samples. We describe the results of RT-PCR analysis of PAX3-FKHR and PAX7-FKHR chimeric messages in formalin-fixed, paraffin-embedded tissue samples from 17 RMS cases. RNA was extracted from formalin-fixed, paraffin-embedded RMS tissue. Oligonucleotide primers corresponding to the regions of PAX3, PAX7 and FKHR were used for the detection of PAX3-FKHR and PAX7-FKHR chimeric messages. A seminested PCR of the PCR products was used to increase the sensitivity of detection. The amplified fragments were purified and directly sequenced to confirm the specificity of the methods. The PAX3-FKHR chimeric message was detected in all three cases of alveolar RMS but not in any of the 12 embryonal and two pleomorphic RMS cases. The PAX7-FKHR fusion transcript was detected in one case of embryonal RMS. The results indicate that the RT-PCR assay is a reliable method for the detection of the PAX3-FKHR fusion transcript of alveolar RMS in formalin-fixed, paraffin-embedded tissue. This simple method enables pathologists to identify chromosomal rearrangements in RMS as a diagnostic aid in cases where fresh or frozen tissue is not available.

Establishment of a protocol for the gene expression analysis of laser microdissected rat kidney samples with affymetrix genechips

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stemmer, Kerstin; Ellinger-Ziegelbauer, Heidrun; Lotz, Kerstin

2006-11-15

Laser microdissection in conjunction with microarray technology allows selective isolation and analysis of specific cell populations, e.g., preneoplastic renal lesions. To date, only limited information is available on sample preparation and preservation techniques that result in both optimal histomorphological preservation of sections and high-quality RNA for microarray analysis. Furthermore, amplification of minute amounts of RNA from microdissected renal samples allowing analysis with genechips has only scantily been addressed to date. The objective of this study was therefore to establish a reliable and reproducible protocol for laser microdissection in conjunction with microarray technology using kidney tissue from Eker rats p.o. treatedmore » for 7 days and 6 months with 10 and 1 mg Aristolochic acid/kg bw, respectively. Kidney tissues were preserved in RNAlater or snap frozen. Cryosections were cut and stained with either H and E or cresyl violet for subsequent morphological and RNA quality assessment and laser microdissection. RNA quality was comparable in snap frozen and RNAlater-preserved samples, however, the histomorphological preservation of renal sections was much better following cryopreservation. Moreover, the different staining techniques in combination with sample processing time at room temperature can have an influence on RNA quality. Different RNA amplification protocols were shown to have an impact on gene expression profiles as demonstrated with Affymetrix Rat Genome 230{sub 2}.0 arrays. Considering all the parameters analyzed in this study, a protocol for RNA isolation from laser microdissected samples with subsequent Affymetrix chip hybridization was established that was also successfully applied to preneoplastic lesions laser microdissected from Aristolochic acid-treated rats.« less

Relative ratios of collagen composition of periarticular tissue of joints of the upper limb.

PubMed

Cheah, A; Harris, A; Le, W; Huang, Y; Yao, J

2017-07-01

We investigated the relative ratios of collagen composition of periarticular tissue of the elbow, wrist, metacarpophalangeal, proximal and distal interphalangeal joints. Periarticulat tissue, which we defined as the ligaments, palmar plate and capsule, was harvested from ten fresh-frozen cadaveric upper limbs, yielding 50 samples. The mean paired differences (95% confidence interval) of the relative ratios of collagen between the five different joints were estimated using mRNA expression of collagen in the periarticular tissue. We found that the relative collagen composition of the elbow was not significantly different to that of the proximal interphalangeal joint, nor between the proximal interphalangeal joint and distal interphalangeal joint, whereas the differences in collagen composition between all the other paired comparisons of the joints had confidence intervals that did not include zero.

Detection and Characterization of Circulating Tumour Cells from Frozen Peripheral Blood Mononuclear Cells

PubMed Central

Lu, David; Graf, Ryon P.; Harvey, Melissa; Madan, Ravi A.; Heery, Christopher; Marte, Jennifer; Beasley, Sharon; Tsang, Kwong Y.; Krupa, Rachel; Louw, Jessica; Wahl, Justin; Bales, Natalee; Landers, Mark; Marrinucci, Dena; Schlom, Jeffrey; Gulley, James L.; Dittamore, Ryan

2015-01-01

Retrospective analysis of patient tumour samples is a cornerstone of clinical research. CTC biomarker characterization offers a non-invasive method to analyse patient samples. However, current CTC technologies require prospective blood collection, thereby reducing the ability to utilize archived clinical cohorts with long-term outcome data. We sought to investigate CTC recovery from frozen, archived patient PBMC pellets. Matched samples from both mCRPC patients and mock samples, which were prepared by spiking healthy donor blood with cultured prostate cancer cell line cells, were processed “fresh” via Epic CTC Platform or from “frozen” PBMC pellets. Samples were analysed for CTC enumeration and biomarker characterization via immunofluorescent (IF) biomarkers, fluorescence in-situ hybridization (FISH) and CTC morphology. In the frozen patient PMBC samples, the median CTC recovery was 18%, compared to the freshly processed blood. However, abundance and localization of cytokeratin (CK) and androgen receptor (AR) protein, as measured by IF, were largely concordant between the fresh and frozen CTCs. Furthermore, a FISH analysis of PTEN loss showed high concordance in fresh vs. frozen. The observed data indicate that CTC biomarker characterization from frozen archival samples is feasible and representative of prospectively collected samples. PMID:28936240

Fresh vs Frozen Samples and Ambient Temperature Have Little Effect on Detection of Colorectal Cancer or Adenomas by a Fecal Immunochemical Test in a Colorectal Cancer Screening Cohort in Germany.

PubMed

Chen, Hongda; Werner, Simone; Brenner, Hermann

2017-10-01

Fecal immunochemical tests (FITs) are used in colorectal cancer (CRC) screening. We compared detection of CRCs and colorectal neoplasms by FITs using fresh samples (collected into buffer-filled tubes) vs frozen samples, and we assessed the effects of seasonal variations in ambient temperature on test performance. We performed a prospective study of 3466 individuals (50% male; mean age, 62 years) undergoing screening colonoscopies at 20 gastroenterology practices in southern Germany from November 2008 through September 2014. Frozen stool samples (collected and frozen by patients through February 2012, n = 1644) and fresh stool samples (collected by patients into buffer-filled tubes after February 2012, n = 1822) were obtained; hemoglobin (Hgb) concentrations were measured by using a commercial, quantitative FIT (cutoff value for positive result, 17 μg Hgb/g feces). Colonoscopy results were used as the gold standard, with results categorized as CRC, advanced adenoma, non-advanced adenoma, or no colorectal neoplasm. Differences in detection of colorectal neoplasms with fresh vs frozen samples were compared by using Wilcoxon rank sum test (continuous variables) and Fisher exact test (categorical variables). We also compared test performance when samples were collected during different seasons (based on outdoor temperature less than 8°, 8°-15°, or more than 15°). Of the samples analyzed by FIT, 12.8% of frozen stool samples (95% confidence interval [CI], 11.3%-14.5%) and 8.7% of fresh stool samples (95% CI, 7.5%-10.1%) had positive results (P value for difference < .001). When adjusting the Hgb cutoff value to produce the same percentage of positive results for fresh and frozen samples (10% and 5%), FIT with frozen vs fresh samples detected colorectal neoplasms with similar levels of sensitivity and specificity. For example, at cutoff values that produced 5% positive results for each sample type, FIT detected advanced neoplasms with 27.8% sensitivity when frozen samples were used (95% CI, 21.4%-35.1%) and 25.6% sensitivity when fresh samples were used (95% CI, 19.8%-32.1%). Specificity values were 97.7% when frozen samples were used (95% CI, 96.8%-98.4%) and 97.6% when fresh samples were used (95% CI, 96.7%-98.3%). We did not observe any differences in detection of neoplasms during different seasons that were based on outdoor temperature. In a prospective study of 3466 individuals who underwent screening colonoscopies and received FITs, we found that use of fresh vs frozen samples slightly affected positivity rates and the proportions of CRCs or adenomas detected at the recommended Hgb cutoff value. However, after we adjusted Hgb cutoff values to produce equal proportions of positive results for fresh vs frozen samples, the performance of the FIT was similar with each sample type. Season of sample collection (based on outdoor temperature) did not affect detection of CRC using either sample type in this study from Middle Europe. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Chapter 15 Development of a Nationwide Network for Ovarian Tissue Cryopreservation.

PubMed

Liebenthron, Jana; Montag, Markus

2017-01-01

Ovarian tissue cryopreservation is gaining much interest since the publication of the first live birth after retransplantation of frozen-thawed tissue in 2004 (Donnez et al., Lancet 364:1405-1410, 2004). In contrast to cryopreservation of gametes and embryos, ovarian tissue freezing is a complex requiring a proper approach in order to make this a viable option for fertility preservation of cancer patients. Due to the need in terms of laboratory space, equipment, personnel, and adequate logistics, an ovarian tissue cryobank is most economic if managed as a centralized service unit that interacts with numerous clinics covering the surgical part. Transportation of ovarian tissue under appropriate conditions from the surgical unit to the cryobank for subsequent preparation and freezing has been shown to have no impact on cryo-survival (Schmidt et al., Hum Reprod 18:2654-2659, 2003; Isachenko et al., Fertil Steril 91:1556-1559, 2009). Several children have been born after retransplantation of such tissue that was derived from the cryobank in Bonn, Germany (Homepage FertiPROTEKT. http://www.fertiprotekt.de ). This cryobank is one of the largest in the world with more than 1300 tissue samples that were frozen from 2003 until today. It is integrated in the network FertiPROTEKT (Homepage FertiPROTEKT. http://www.fertiprotekt.de ) and is served by 108 surgical centers that are located all over Germany. The concept of this cryobank is a blueprint for success and has recently been used for another regionally centralized cryobank in Beijing, China. In this chapter the most important topics that need to be considered while creating a centralized cryobank within a national or regional network are highlighted.

Histochemical detection of lead and zinc in plant tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tung, G.; Temple, P.J.

1975-01-01

Histochemical studies on uptake and localization of lead and zinc in plant tissues were carried out. A histochemical stain technique was developed to differentiate zinc from lead. Lead was detected in plant tissues by soaking fresh plant materials in freshly prepared sodium rhodizonate stain (0.2% Na rhodizonate acidified to pH3 with glacial acetic acid). Samples were evacuated 5 min and soaked for 30 min before embedding in the congealed stain, then sectioned with a cryostat and examined under a light microscope. Lead particles in plant tissues were stained scarlet-red. Gelatinous, proteinaceous or saccharic embedding materials normally used to prepare plantmore » sampled for sectioning in the cryostat interfered with the color reaction. Sectioning plant samples without staining whole tissues resulted in a weakened response to the stain. Color of stained sample materials were retained for several months if stored in a frozen condition. This technique was used to detect lead both inside and on the surface of plant samples collected in the vicinity of highway and industrial lead sources and to trace the pathways of lead uptake from the air or from contaminated soils. A sodium rhodizonate technique was also developed to be specific for zinc in plant tissues. Plant samples were soaked in a neutral Na-rhodizonate in phosphate buffer at pH 7.5 for observation. The color of zinc developed to produce a purplish or reddish-brown color.« less

Measurement of stable isotopic enrichment and concentration of long-chain fatty acyl-carnitines in tissue by HPLC-MS.

PubMed

Sun, Dayong; Cree, Melanie G; Zhang, Xiao-Jun; Bøersheim, Elisabet; Wolfe, Robert R

2006-02-01

We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.

The use of frozen plasma samples in thromboelastometry.

PubMed

Schoergenhofer, Christian; Buchtele, Nina; Schwameis, Michael; Bartko, Johann; Jilma, Bernd; Jilma-Stohlawetz, Petra

2017-11-01

Thromboelastometry is increasingly used in the clinical and scientific setting. The use of frozen plasma samples may be useful in overcoming certain limitations such as local and timely availability. Whole blood (WB) samples of 20 healthy volunteers were obtained, and plasma was generated. NATEM (n = 20), EXTEM (n = 20) and INTEM (n = 8) analyses were performed in WB, fresh plasma and frozen and thawed plasma. Dabigatran (500, 1000 ng/ml), rivaroxaban (100, 200 ng/ml) or alteplase (333 ng/ml) were added ex vivo to WB, and thromboelastometry was performed in WB and in frozen and thawed plasma samples. Clot formation time, mean clot firmness and the area under the curve were significantly altered in plasma compared to WB. In INTEM and EXTEM analysis, clotting time (CT) was comparable between WB (100%) and fresh (INTEM 114% and EXTEM 93%, ratio of the means) and frozen plasma samples (85 and 99%), whereas in NATEM analysis, the CT increased in fresh (193%) and frozen plasma samples (130%). Dabigatran dose-dependently increased the CT approximately 5- and 9-fold in WB and even more pronounced 10- and 26-fold in plasma. Accordingly, rivaroxaban dose-dependently increased the CT 2- and 2.7-fold in WB, and 3.5- and 4-fold in plasma samples. Hyperfibrinolysis was achieved by addition of alteplase in all WB samples and was reproducible in plasma samples. In conclusion, thromboelastometry, especially INTEM and EXTEM analyses, is possible using frozen and stored plasma samples with comparable results to the corresponding whole blood samples.

PBRM1 and VHL expression correlate in human clear cell renal cell carcinoma with differential association with patient's overall survival.

PubMed

Högner, Anica; Krause, Hans; Jandrig, Burkhard; Kasim, Mumtaz; Fuller, Tom Florian; Schostak, Martin; Erbersdobler, Andreas; Patzak, Andreas; Kilic, Ergin

2018-03-01

To identify the clinicopathological association of PBRM1 (Polybromo-1 gene) and VHL (von Hippel-Lindau gene) expression at mRNA and protein levels in clear cell renal cell carcinoma (ccRCC) and its role in tumor progression. Immunohistochemical analysis, Western blotting and qPCR analysis of PBRM1 and VHL were performed on fresh-frozen ccRCC and adjacent normal tissue obtained from 70 patients who underwent radical nephrectomy. In addition, a tissue microarray (TMA) from specimens of 326 ccRCC patients was used to evaluate the effect of loss of PBRM1 and VHL immunohistological expression on clinicopathological features as well as patient survival. In frozen tissue, PBRM1 and VHL mRNA were significantly down-regulated in most ccRCC tumors (77.6%/80.6%). Simultaneous weak PBRM1 and VHL protein expression was observed in 21.4% of frozen tumors. In the TMA samples, weak PBRM1 and VHL immunohistochemical staining was observed in 60.4% of the cases and was correlated (P<0.001). The association of PBRM1 and VHL immunohistochemical expression with clinicopathological parameters depicts a variable picture: predominantly weak PBRM1 and VHL expression were significantly associated with higher Fuhrman grade (P = 0.012 and 0.024, respectively) but only weak VHL expression was associated with a higher pT stage (P = 0.023). PBRM1 expression did not affect the overall survival, whereas weak VHL expression was associated with decreased patient overall survival (P = 0.013). Our data suggest that reduced expression of PBRM1 and VHL is correlated with an increased tumor aggressiveness. Low VHL expression was identified as a risk factor for worse patient overall survival, independently from PBRM1 expression pattern. Copyright © 2018 Elsevier Inc. All rights reserved.

Feline spermatozoa from fresh and cryopreserved testicular tissues have comparable ability to fertilize matured oocytes and sustain the embryo development after intracytoplasmic sperm injection.

PubMed

Buarpung, S; Tharasanit, T; Comizzoli, P; Techakumphu, M

2013-01-01

Cryopreservation of testicular tissue associated with intracytoplasmic sperm injection (ICSI) is a critical tool that still needs to be explored for preserving the fertility of endangered species. Using the domestic cat as a model for wild felids, the study aimed at determining the effect of different cryoprotectants and freezing techniques (two-step freezing vs. controlled slow freezing) on the sperm quality (membrane and DNA integrity). Then, spermatozoa were extracted from frozen-thawed testicular tissues and used for ICSI to assess early gamete activation or developmental competence in vitro. The percentage of spermatozoa with intact plasma membrane was not different (P > 0.05) among nonfrozen control, glycerol-, and ethylene glycol-frozen tissues (63.2 ± 2%, 58.2 ± 2.6%, 53.3 ± 2.3%, respectively). However, these percentages were significantly lower (P < 0.05) in groups of dimethyl sulfoxide (46.3 ± 3.3%) and 1,2 propanediol (44.3 ± 2.9%) when compared with control. Conventional freezing combined with 5% (vol/vol) glycerol best preserved sperm membrane integrity (55.0 ± 2.7%) when compared with other freezing techniques. The incidence of DNA fragmentation was found to be low (0.2%-1.1%) in all freezing protocols. After ICSI with frozen testicular spermatozoa, male and female gametes were asynchronously activated and the percentages of normal fertilization at 6, 12, and 18 hours were 11.2%, 20.6%, and 22.1%, respectively. Metaphase II-arrested oocytes containing or not a decondensed sperm head were predominantly found after ICSI with frozen testicular spermatozoa. Although two-pronucleus formation could be observed as soon as 6 hours post ICSI (10%), the rate increased dramatically after 12 and 18 hours post ICSI (17.2% and 19.5%, respectively). ICSI using frozen-thawed testicular spermatozoa yielded cleavage (32.7%), morula (6.5%), and blastocyst (4.4%) percentages similar to nonfrozen control (P > 0.05). It is concluded that conventional freezing technique with glycerol as a principle cryoprotectant is simplified and applicable for cat testicular tissue cryopreservation. We also demonstrate for the first time that feline spermatozoa derived from frozen-thawed testicular tissues retain their fertilizing ability and can be used to produce ICSI-derived embryos. Copyright © 2013 Elsevier Inc. All rights reserved.

Contraction-free, fume-fixed longitudinal sections of fresh frozen muscle

NASA Technical Reports Server (NTRS)

Riley, Danny A.; Slocum, Glenn R.

1988-01-01

Contraction damage occurring when longitudinal frozen sections of fresh unfixed muscles are thawed on microscope slides has limited histological examination of this tissue mainly to cross sections. Longitudinally oriented sections are advantageous for investigating properties that vary along the length of the muscle fibers. A fume fixation technique has been developed for preventing contraction of thick longitudinal frozen sections. The technique is compatible with histochemical staining of enzymes.

Intercalary frozen autograft for reconstruction of malignant bone and soft tissue tumours.

PubMed

Zekry, Karem M; Yamamoto, Norio; Hayashi, Katsuhiro; Takeuchi, Akihiko; Higuchi, Takashi; Abe, Kensaku; Taniguchi, Yuta; Alkhooly, Ali Zein A A; Abd-Elfattah, Ahmed Saleh; Fouly, Ezzat H; Ahmed, Adel Refaat; Tsuchiya, Hiroyuki

2017-07-01

In 1999, we developed a technique using frozen autografts-tumour-containing bone treated with liquid nitrogen-for the reconstruction of malignant bone tumours. The purpose of this study was to evaluate the functional and oncological outcomes of frozen autografts for intercalary reconstruction of malignant bones and soft tissue tumours. This retrospective study was designed to assess 34 patients of mean age 35 (range, 6-79) years. The mean follow-up period was 62 (24-214) months. The median length of the frozen autografts was 138.4 ± 60.39 (50-290) mm. Postsurgically, 20 patients remained disease-free, seven patients survived with no evidence of disease, five patients were alive with disease, and two patients died of disease. The five- and ten-year survival rates of the frozen autografts were 91.2% and the mean International Society of Limb Salvage score was 90%. Complete bony union was achieved in 97% of the patients. There were five cases of nonunion, six cases of fracture, two cases of deep infection and four cases of local recurrence. Utilizing intercalary frozen autografts for patients with a nonosteolytic primary or secondary bone tumour without involvement of the subchondral bone is a good alternative treatment, because it is a straightforward biological technique and can provide excellent limb function.

High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq

PubMed Central

Kondrashova, Olga; Love, Clare J.; Lunke, Sebastian; Hsu, Arthur L.; Waring, Paul M.; Taylor, Graham R.

2015-01-01

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity. PMID:26569395

The Christchurch Tissue Bank to support cancer research.

PubMed

Morrin, Helen; Gunningham, Sarah; Currie, Margaret; Dachs, Gabi; Fox, Stephen; Robinson, Bridget

2005-11-11

To report on the development of a central resource of consented cancer tissues for researchers to use for ethically approved projects, and to describe the banking process. The development of tissue banking in Christchurch, New Zealand is described, including the number and main types of samples collected. The consent forms have evolved with several new donor options added between 1996 and 2004. Since June 2004, disposal of tissues by a karakia (blessing) has been offered. Characteristics of each tissue including amount, location in the bank, and relevant clinicopathological data have been recorded prospectively in a detailed secure relational database. The changes in the consent form and donor options are described. Most donors (99.6%) consented to allow access to medical records (since May 2002); 98.3% to tissue being sent overseas (since May 2003), 97.4% to commercial research (since May 2003), and 35.6% requested disposal with a karakia. Since May 2003, 87% of donors have been Caucasian, 5.1% Maori, and the remainder composed of other categories as stated on the 2001 New Zealand census format. By March 2005, samples have been banked from more than 2000 donors. For each of the last 4 years, samples have been collected from more than 300 donors, including fresh-frozen tissue, DNA preparations, serum, plasma, and paraffin blocks. The predominant tissues are from donors with cancers of the breast, colon, urological, and gynaecological sites. The Christchurch Tissue Bank is a successful model for potential New Zealand-wide application, providing quality tissue samples for cancer research whilst appropriately addressing ethical, legal, and cultural aspects of their collection.

Effect of gamma-irradiation on frozen shrimps for decontamination of pathogenic bacteria

NASA Astrophysics Data System (ADS)

Ito, Hitoshi; Rashid, Harun Or; Sangthong, Naruemon; Adulyatham, Pitaya; Rattagool, Pongpen; Ishigaki, Isao

1993-07-01

Twelve samples of imported frozen shrimps were used in this study. The total aerobic bacteria were at 2 × 10 4 to 6 × 10 6 per gram. A few of Vibrio parahaemolyticus, V. mimicus, V. alginolyticus, V. vulnificus, V. fluvialis and Listeria monocytogenes were isolated from many samples. However, Salmonella was not detected in any of the samples. After exposure to 4-5 kGy of gamma-rays, the total aerobic bacteria in frozen shrimps were reduced by approximately 2-3 log cycles. The dose necessary to reduce the vibrio isolates and Aeromonas hydrophila at a level of below 10 -4 per gram was about 3 kGy in frozen shrimps, whereas about 3.5 kGy was required for L. monocytogenes and Salmonella typhimurium. In this study, unpleasant off-odor was clearly detected in the non-frozen shrimps irradiated at 2.5 kGy. On the other hand, off-odor was negligible in the frozen product below 5 kGy irradiation. No remarkable changes of peroxide values were also obtained up to 9 kGy of irradiation in the frozen shrimps. However peroxide values of non-frozen shrimps were clearly increased even irradiated at 4 kGy. Trimethylamine content was not changed at doses below 10 kGy in both of frozen and non-frozen shrimps. Shelf-life of defrosted shrimps were extended ca. 2 times under non-frozen market conditions.

The PAXgene® Tissue System Preserves Phosphoproteins in Human Tissue Specimens and Enables Comprehensive Protein Biomarker Research

PubMed Central

Gündisch, Sibylle; Schott, Christina; Wolff, Claudia; Tran, Kai; Beese, Christian; Viertler, Christian; Zatloukal, Kurt; Becker, Karl-Friedrich

2013-01-01

Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE) tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assay (ELISA) to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology. PMID:23555997

Frozen chips: an unusual cause of severe frostbite injury

PubMed Central

Graham, C.; Stevenson, J.

2000-01-01

A case of severe frostbite injury to the right foot is presented. This was caused by the inappropriate application of a bag of frozen chips to the foot in an attempt to ease non-specific pain. No specific acute traumatic injury was identified. As the patient was a teacher of physical education, the pain had initially been assumed to originate from a minor musculoskeletal injury. Full recovery ensued after surgical excision of necrotic tissue and split skin grafting. The danger of inappropriate overenthusiastic use of ice packs or other frozen material to treat soft tissue injuries is emphasised. The need for education to prevent similar future injuries is discussed. Key Words: cold injury; frostbite; ice pack; skin; necrosis PMID:11049150

The effects of soy on freezable bread dough: a magnetic resonance study.

PubMed

Simmons, Amber L; Vodovotz, Yael

2012-11-15

Hygroscopic soy ingredients were hypothesised to slow the rate of water migration in unleavened bread dough during frozen storage. Thawed soy (18% dry weight) and wheat dough samples were assessed using non-destructive nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) for up to 8 wks frozen storage time. MRI suggested a spatially homogeneous, net increase in proton mobility with frozen storage and, with solution state proton NMR, distinct "free" and "bound" states were discerned. T(2) relaxation times of the majority proton population suggested increased mobility with frozen storage time, and statistical difference from the fresh sample was seen later for the soy samples than the wheat samples. As seen by (13)C-solid state NMR, the crystallinity of the starch was not affected by either soy addition or frozen storage. In conclusion, addition of soy to bakery products led to slightly enhanced preservation of "fresh" characteristics of the dough during frozen storage. Copyright © 2012 Elsevier Ltd. All rights reserved.

Autofluorescence of bovine ligamentum nuchae, cartilage, heart valve and lung measured by microscopy and fibre optics.

PubMed

Swatland, H J

1988-09-01

The fluorescence of bovine tissues was measured post mortem by microscopy of frozen sections and by using optical fibres to excite fluorescence and to measure fluorescence emission spectra. Mechanical disruption of the tissue (by comminution or sectioning) did not appreciably change tissue fluorescence spectra. Ligamentum nuchae had the strongest fluorescence and lung tissue had the weakest. In samples measured with a minimum prior exposure to ultraviolet light, the peak fluorescence emission was at 410 or 420 nm (with excitation at 365 nm). Exposure to ultraviolet light for about 1 minute shifted the fluorescence peak to 450 to 470 nm. Further exposure (about 30 minutes) caused a loss of the 450 to 470 nm fluorescence peak, while emissions above 530 nm were maintained or strengthened. Microscopy showed that the fluorescence that was measured by fibre optics from intact connective tissues originated mostly from collagen and elastin fibres.

Effect of sugar and acid on the acceptability of frozen yogurt to a student population.

PubMed

Guinard, J X; Little, C; Marty, C; Palchak, T R

1994-05-01

One hundred and forty-one college students tasted and rated on a nine-point hedonic scale their degree of liking for nine samples of vanilla frozen yogurt varying in sugar and lactic acid. Subjects were also asked to complete a questionnaire about consumption of frozen yogurt and other dairy products. Degree of liking differed significantly among samples, and the samples best liked were those with the lowest acidity, .23 to .29%, independent of sugar concentration. Degree of liking of frozen yogurt failed to correlate with dairy product consumption or hunger at the time of testing. No significant difference existed between male and female students for overall degree of liking of frozen yogurt or overall dairy product intake, yet the questionnaire revealed a significantly higher consumption of frozen yogurt among female students. The results of this study suggest that, for the student population tested, frozen yogurt should combine the sensory properties of ice cream (low acidity) with the nutritional properties of yogurt (low fat, active enzyme culture).

A comparative study of vascular injection fluids in fresh-frozen and embalmed human cadaver forearms.

PubMed

Doomernik, D E; Kruse, R R; Reijnen, M M P J; Kozicz, T L; Kooloos, J G M

2016-10-01

Over the years, various vascular injection products have been developed to facilitate anatomical dissections. This study aimed to compare the most commonly used vascular injection products in fresh-frozen and formalin-embalmed cadaver specimens. An overview of the properties, advantages and limitations of each substance was given, and a comparison of vascular infusion procedures in both preservation methods was made. A literature search was performed in order to identify the most commonly used vascular injection products. Acrylic paint, latex, gelatin, silicone, Araldite F and Batson's No. 17 were selected for the study. One fresh-frozen and one embalmed cadaver forearm were infused with each injection product according to a uniform protocol. The curing time, skin- and subcutaneous tissue penetration, degree of filling of the arterial tree, extravasations, consistency of the injected vessels during dissection, and the costs of each injection fluid were noted. There was a large variation between the injection fluids in processing- and curing time, colour intensity, flexibility, fragility, elasticity, strength, toxicity and costs. All fluids were suitable for infusion. The penetration of injection fluid into the skin and subcutaneous tissue was significantly better in fresh-frozen specimens (P = 0.002 and P = 0.009, respectively), with significantly smaller branches casted (P = 0.004). Vascular infusion of fresh-frozen cadaver specimens results in a significantly better filled coloured arterial tree, enabling more detail to be achieved and smaller branches casted. The biomechanical properties of fresh-frozen soft tissues are less affected compared with formalin fixation. All the injection fluids studied are suitable for vascular infusion, but their different properties ensure that certain products and procedures are more suitable for specific study purposes. © 2016 Anatomical Society.

Correlations of magnetic resonance imaging findings with clinical symptom severity and prognosis of frozen shoulder.

PubMed

Yoon, Jong Pil; Chung, Seok Won; Lee, Byung Joo; Kim, Hyung Sup; Yi, Jae Hyuck; Lee, Hyun-Joo; Jeong, Won-Ju; Moon, Sung Gyu; Oh, Kyung-Soo; Yoon, Seok Tae

2017-10-01

To evaluate the correlation between indirect magnetic resonance (MR) arthrographic imaging findings and the clinical symptoms and prognosis of patients with frozen shoulder. Indirect MR arthrography was performed for 52 patients with primary frozen shoulder (mean age 55.1 ± 9.0 years) and 52 individuals without frozen shoulder (mean age 53.1 ± 10.7 years); capsular thickening and enhancement of the axillary recess as well as soft tissue thickening of the rotator interval were evaluated. Clinical symptom severity was assessed using the Visual Analogue Scale for Pain (VAS Pain), simple shoulder test (SST), Constant score, American Shoulder and Elbow Surgeons (ASES) score, and range of motion (ROM). At 6-month follow-up, we evaluated whether MR arthrography findings correlated with the clinical symptoms and prognosis. Capsular thickening and enhancement of the axillary recess as well as soft tissue thickening of the rotator interval were significantly greater in the patient group than in the controls (p < 0.001). Capsular thickening of the axillary recess did not correlate with clinical symptoms or ROM (n.s.); however, capsular enhancement correlated with clinical symptom severity according to VAS Pain (p = 0.005), SST (p = 0.046), and ASES scores (p = 0.009). Soft tissue thickening of the rotator interval did not correlate with clinical symptom severity, but was associated with external rotation limitation (p = 0.002). However, none of the parameters correlated with clinical symptoms at 6-month follow-up. Indirect MR arthrography provided ancillary findings, especially with capsular enhancement, for evaluating clinical symptom severity of frozen shoulder, but did not reflect the prognosis. MR findings in frozen shoulder should not replace clinical judgments regarding further prognosis and treatment decisions. IV.

Comparative biomarker expression and RNA integrity in biospecimens derived from radical retropubic and robot-assisted laparoscopic prostatectomies.

PubMed

Ricciardelli, Carmela; Bianco-Miotto, Tina; Jindal, Shalini; Dodd, Thomas J; Cohen, Penelope A; Marshall, Villis R; Sutherland, Peter D; Samaratunga, Hemamali; Kench, James G; Dong, Ying; Wang, Hong; Clements, Judith A; Risbridger, Gail P; Sutherland, Robert L; Tilley, Wayne D; Horsfall, David J

2010-07-01

Knowledge of preanalytic conditions that biospecimens are subjected to is critically important because novel surgical procedures, tissue sampling, handling, and storage might affect biomarker expression or invalidate tissue samples as analytes for some technologies. We investigated differences in RNA quality, gene expression by quantitative real-time PCR, and immunoreactive protein expression of selected prostate cancer biomarkers between tissues from retropubic radical prostatectomy (RRP) and robot-assisted laparoscopic prostatectomy (RALP). Sections of tissue microarray of 23 RALP and 22 RRP samples were stained with antibodies to androgen receptor (AR) and prostate-specific antigen (PSA) as intersite controls, and 14 other candidate biomarkers of research interest to three laboratories within the Australian Prostate Cancer BioResource tissue banking network. Quantitative real-time PCR was done for AR, PSA (KLK3), KLK2, KLK4, and HIF1A on RNA extracted from five RALP and five RRP frozen tissue cores. No histologic differences were observed between RALP and RRP tissue. Biomarker staining grouped these samples into those with increased (PSA, CK8/18, CKHMW, KLK4), decreased (KLK2, KLK14), or no change in expression (AR, ghrelin, Ki67, PCNA, VEGF-C, PAR2, YB1, p63, versican, and chondroitin 0-sulfate) in RALP compared with RRP tissue. No difference in RNA quality or gene expression was detected between RALP and RRP tissue. Changes in biomarker expression between RALP and RRP tissue exist at the immunoreactive protein level, but the etiology is unclear. Future studies should account for changes in biomarker expression when using RALP tissues, and mixed cohorts of RALP and RRP tissue should be avoided.

Scanning electron microscopy of high-pressure-frozen sea urchin embryos.

PubMed

Walther, P; Chen, Y; Malecki, M; Zoran, S L; Schatten, G P; Pawley, J B

1993-12-01

High-pressure-freezing permits direct cryo-fixation of sea urchin embryos having a defined developmental state without the formation of large ice crystals. We have investigated preparation protocols for observing high-pressure-frozen and freeze-fractured samples in the scanning electron microscope. High-pressure-freezing was superior to other freezing protocols, because the whole bulk sample was reasonably well frozen and the overall three-dimensional shape of the embryos was well preserved. The samples were either dehydrated by freeze-substitution and critical-point-drying, or imaged in the partially hydrated state, using a cold stage in the SEM. During freeze-substitution the samples were stabilized by fixatives. The disadvantage of this method was that shrinking and extraction effects, caused by the removal of the water, could not be avoided. These disadvantages were avoided when the sample was imaged in the frozen-hydrated state using a cold-stage in the SEM. This would be the method of choice for morphometric studies. Frozen-hydrated samples, however, were very beam sensitive and many structures remained covered by the ice and were not visible. Frozen-hydrated samples were partially freeze-dried to make visible additional structures that had been covered by ice. However, this method also caused drying artifacts when too much water was removed.

Validation of the Complexity INdex in SARComas prognostic signature on formalin-fixed, paraffin-embedded, soft tissue sarcomas.

PubMed

Le Guellec, S; Lesluyes, T; Sarot, E; Valle, C; Filleron, T; Rochaix, P; Valentin, T; Pérot, G; Coindre, J-M; Chibon, F

2018-05-31

Prediction of metastatic outcome in sarcomas is challenging for clinical management since they are aggressive and carry a high metastatic risk. A 67-gene expression signature, the Complexity INdex in SARComas (CINSARC), has been identified as a better prognostic factor than the reference pathological grade. Since it cannot be applied easily in standard laboratory practice, we assessed its prognostic value using nanoString on formalin-fixed, paraffin-embedded (FFPE) blocks to evaluate its potential in clinical routine practice and guided therapeutic management. A code set consisting of 67 probes derived from the 67 genes of the CINSARC signature was built and named NanoCind®. To compare the performance of RNA-seq and nanoString (NanoCind®), we used expressions of various sarcomas (n=124, frozen samples) using both techniques and compared predictive values based on CINSARC risk groups and clinical annotations. We also used nanoString on FFPE blocks (n=67) and matching frozen and FFPE samples (n=45) to compare their level of agreement. Metastasis-free survival and agreement values in classification groups were evaluated. CINSARC strongly predicted metastatic outcome using nanoString on frozen samples (HR = 2.9, 95% CI 1.23-6.82) with similar risk-group classifications (86%). While more than 50% of FFPE blocks were not analyzable by RNA-seq owing to poor RNA quality, all samples were analyzable with nanoString. When similar (risk-group) classifications were measured with frozen tumors (RNA-seq) compared to FFPE blocks (84% agreement), the CINSARC signature was still a predictive factor of metastatic outcome with nanoString on FFPE samples (HR = 4.43, 95% CI 1.25-15.72). CINSARC is a material-independent prognostic signature for metastatic outcome in sarcomas and outperforms histological grade. Unlike RNA-seq, nanoString is not influenced by the poor quality of RNA extracted from FFPE blocks. The CINSARC signature can potentially be used in combination with nanoString (NanoCind®) in routine clinical practice on FFPE blocks to predict metastatic outcome.

Banking for the future: an Australian experience in brain banking.

PubMed

Sarris, M; Garrick, T M; Sheedy, D; Harper, C G

2002-06-01

The New South Wales (NSW) Tissue Resource Centre (TRC) has been set up to provide Australian and international researchers with fixed and frozen brain tissue from cases that are well characterised, both clinically and pathologically, for projects related to neuropsychiatric and alcohol-related disorders. A daily review of the Department of Forensic Medicine provides initial information regarding a potential collection. If the case adheres to the strict inclusion criteria, the pathologist performing the postmortem examination is approached regarding retention of the brain tissue. The next of kin of the deceased is then contacted requesting permission to retain the brain for medical research. Cases are also obtained through donor programmes, where donors are assessed and consent to donate their brain during life. Once the brain is removed at autopsy, the brain is photographed, weighed and the volume determined, the brainstem and cerebellum are removed. The two hemispheres are divided, one hemisphere is fresh frozen and one fixed (randomised). Prior to freezing, the hemisphere is sliced into 1-cm coronal slices and a set of critical area blocks is taken. All frozen tissues are kept bagged at -80 degrees C. The other hemisphere is fixed in 15% buffered formalin for 2 weeks, embedded in agar and sliced at 3-mm intervals in the coronal plane. Tissue blocks from these slices are used for neuropathological analysis to exclude any other pathology. The TRC currently has 230 cases of both fixed and frozen material that has proven useful in a range of techniques in many research projects. These techniques include quantitative analyses of brain regions using neuropathological, neurochemical, neuropharmacological and gene expression assays.

RNA Extraction from Animal and Human's Cancerous Tissues: Does Tissue Matter?

PubMed

Samadani, Ali Akbar; Nikbakhsh, Novin; Fattahi, Sadegh; Pourbagher, Roghayeh; Aghajanpour Mir, Seyyed Mohsen; Mousavi Kani, Narges; Abedian, Zeinab; Akhavan-Niaki, Haleh

2015-01-01

The reliability of gene expression profiling, based technologies and methods to find transcriptional differences representative of the original samples is influenced by the quality of the extracted RNA. Hence, RNA extraction is the first step to investigate the gene expression and its function. Consequently, the quality of extracted RNA is really significant. Correspondingly, this research was accomplished to optimize the RNA extraction methods and compare the amounts of tissue or quality of tissue. Relatively, the cancerous tissue of human stomach in fresh and frozen conditions and also the mouse fresh tissue were studied. Some factors like the amount of samples, efficacy differences of diverse extraction buffers (TriPure, Trizol) and also the efficacy of b-mercaptoethanol were compared and investigated. The results indicated that the less amount (1-2 mg) compared to other amounts (2-5 mg, 5-15 mg) yielded the best quality and the RNA bands (5S, 18S, 28S) were observed perfectly. Relatively, comparing and measuring some kinds of buffers (Trizol, TriPure) indicated no difference in RNA extraction quality. The last investigated factor was the effect of b- mercaptoethanol which was used along with TriPure to remove the RNAse. Conclusively, no effective impression was observed.

Tissue recommendations for precision cancer therapy using next generation sequencing: a comprehensive single cancer center’s experiences

PubMed Central

Hong, Mineui; Bang, Heejin; Van Vrancken, Michael; Kim, Seungtae; Lee, Jeeyun; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Sun, Jong-Mu; Lee, Se Hoon; Ahn, Myung-Ju; Park, Keunchil; Kim, Duk Hwan; Lee, Seunggwan; Park, Woongyang; Kim, Kyoung-Mee

2017-01-01

To generate accurate next-generation sequencing (NGS) data, the amount and quality of DNA extracted is critical. We analyzed 1564 tissue samples from patients with metastatic or recurrent solid tumor submitted for NGS according to their sample size, acquisition method, organ, and fixation to propose appropriate tissue requirements. Of the 1564 tissue samples, 481 (30.8%) consisted of fresh-frozen (FF) tissue, and 1,083 (69.2%) consisted of formalin-fixed paraffin-embedded (FFPE) tissue. We obtained successful NGS results in 95.9% of cases. Out of 481 FF biopsies, 262 tissue samples were from lung, and the mean fragment size was 2.4 mm. Compared to lung, GI tract tumor fragments showed a significantly lower DNA extraction failure rate (2.1 % versus 6.1%, p = 0.04). For FFPE biopsy samples, the size of biopsy tissue was similar regardless of tumor type with a mean of 0.8 × 0.3 cm, and the mean DNA yield per one unstained slide was 114 ng. We obtained highest amount of DNA from the colorectum (2353 ng) and the lowest amount from the hepatobiliary tract (760.3 ng) likely due to a relatively smaller biopsy size, extensive hemorrhage and necrosis, and lower tumor volume. On one unstained slide from FFPE operation specimens, the mean size of the specimen was 2.0 × 1.0 cm, and the mean DNA yield per one unstained slide was 1800 ng. In conclusions, we present our experiences on tissue requirements for appropriate NGS workflow: > 1 mm2 for FF biopsy, > 5 unstained slides for FFPE biopsy, and > 1 unstained slide for FFPE operation specimens for successful test results in 95.9% of cases. PMID:28477007

Increasing the speed of tumour diagnosis during surgery with selective scanning Raman microscopy

NASA Astrophysics Data System (ADS)

Kong, Kenny; Rowlands, Christopher J.; Varma, Sandeep; Perkins, William; Leach, Iain H.; Koloydenko, Alexey A.; Pitiot, Alain; Williams, Hywel C.; Notingher, Ioan

2014-09-01

One of the main challenges in cancer surgery is ensuring that all tumour cells are removed during surgery, while sparing as much healthy tissue as possible. Histopathology, the gold-standard technique for cancer diagnosis, is often impractical for intra-operative use because of the time-consuming tissue preparation procedures (sectioning and staining). Raman micro-spectroscopy is a powerful technique that can discriminate between tumours and healthy tissues with high accuracy, based entirely on intrinsic chemical differences. However, raster-scanning Raman micro-spectroscopy is a slow imaging technique that typically requires data acquisition times as long as several days for typical tissue samples obtained during surgery (1 × 1 cm2) - in particular when high signal-to-noise ratio spectra are required to ensure accurate diagnosis. In this paper we present two techniques based on selective sampling Raman micro-spectroscopy that can overcome these limitations. In selective sampling, information regarding the spatial features of the tissue, either measured by an alternative optical technique or estimated in real-time from the Raman spectra, can be used to drastically reduce the number of Raman spectra required for diagnosis. These sampling strategies allowed diagnosis of basal cell carcinoma in skin tissue samples excised during Mohs micrographic surgery faster than frozen section histopathology, and two orders of magnitude faster than previous techniques based on raster-scanning Raman microscopy. Further development of these techniques may help during cancer surgery by providing a fast and objective way for surgeons to ensure the complete removal of tumour cells while sparing as much healthy tissue as possible.

Fresh-frozen Complete Extensor Mechanism Allograft versus Autograft Reconstruction in Rabbits

PubMed Central

Chen, Guanyin; Zhang, Hongtao; Ma, Qiong; Zhao, Jian; Zhang, Yinglong; Fan, Qingyu; Ma, Baoan

2016-01-01

Different clinical results have been reported in the repair of extensor mechanism disruption using fresh-frozen complete extensor mechanism (CEM) allograft, creating a need for a better understanding of fresh-frozen CME allograft reconstruction. Here, we perform histological and biomechanical analyses of fresh-frozen CEM allograft or autograft reconstruction in an in vivo rabbit model. Our histological results show complete incorporation of the quadriceps tendon into the host tissues, patellar survival and total integration of the allograft tibia, with relatively fewer osteocytes, into the host tibia. Vascularity and cellularity are reduced and delayed in the allograft but exhibit similar distributions to those in the autograft. The infrapatellar fat pad provides the main blood supply, and the lowest cellularity is observed in the patellar tendon close to the tibia in both the allograft and autograft. The biomechanical properties of the junction of quadriceps tendon and host tissues and those of the allograft patellar tendon are completely and considerably restored, respectively. Therefore, fresh-frozen CEM allograft reconstruction is viable, but the distal patellar tendon and the tibial block may be the weak links of the reconstruction. These findings provide new insight into the use of allograft in repairing disruption of the extensor mechanism. PMID:26911538

Fresh-frozen Complete Extensor Mechanism Allograft versus Autograft Reconstruction in Rabbits.

PubMed

Chen, Guanyin; Zhang, Hongtao; Ma, Qiong; Zhao, Jian; Zhang, Yinglong; Fan, Qingyu; Ma, Baoan

2016-02-25

Different clinical results have been reported in the repair of extensor mechanism disruption using fresh-frozen complete extensor mechanism (CEM) allograft, creating a need for a better understanding of fresh-frozen CME allograft reconstruction. Here, we perform histological and biomechanical analyses of fresh-frozen CEM allograft or autograft reconstruction in an in vivo rabbit model. Our histological results show complete incorporation of the quadriceps tendon into the host tissues, patellar survival and total integration of the allograft tibia, with relatively fewer osteocytes, into the host tibia. Vascularity and cellularity are reduced and delayed in the allograft but exhibit similar distributions to those in the autograft. The infrapatellar fat pad provides the main blood supply, and the lowest cellularity is observed in the patellar tendon close to the tibia in both the allograft and autograft. The biomechanical properties of the junction of quadriceps tendon and host tissues and those of the allograft patellar tendon are completely and considerably restored, respectively. Therefore, fresh-frozen CEM allograft reconstruction is viable, but the distal patellar tendon and the tibial block may be the weak links of the reconstruction. These findings provide new insight into the use of allograft in repairing disruption of the extensor mechanism.

Freezing does not alter multiscale tendon mechanics and damage mechanisms in tension.

PubMed

Lee, Andrea H; Elliott, Dawn M

2017-12-01

It is common in biomechanics to use previously frozen tissues, where it is assumed that the freeze-thaw process does not cause consequential mechanical or structural changes. We have recently quantified multiscale tendon mechanics and damage mechanisms using previously frozen tissue, where damage was defined as an irreversible change in the microstructure that alters the macroscopic mechanical parameters. Because freezing has been shown to alter tendon microstructures, the objective of this study was to determine if freezing alters tendon multiscale mechanics and damage mechanisms. Multiscale testing using a protocol that was designed to evaluate tendon damage (tensile stress-relaxation followed by unloaded recovery) was performed on fresh and previously frozen rat tail tendon fascicles. At both the fascicle and fibril levels, there was no difference between the fresh and frozen groups for any of the parameters, suggesting that there is no effect of freezing on tendon mechanics. After unloading, the microscale fibril strain fully recovered, and interfibrillar sliding only partially recovered, suggesting that the tendon damage is localized to the interfibrillar structures and that mechanisms of damage are the same in both fresh and previously frozen tendons. © 2017 New York Academy of Sciences.

Use of FTA filter paper for the molecular detection of Newcastle disease virus.

PubMed

Perozo, Francisco; Villegas, Pedro; Estevez, Carlos; Alvarado, Iván; Purvis, Linda B

2006-04-01

The feasibility of using Flinders Technology Associates filter papers (FTA cards) to collect allantoic fluid and chicken tissue samples for Newcastle disease virus (NDV) molecular detection was evaluated. Trizol RNA extraction and one-step reverse transcriptase-polymerase chain reaction (RT-PCR) were used. FTA cards allowed NDV identification from allantoic fluid with a titre of 10(5.8) median embryo lethal doses/ml. The inactivated virus remained stable on the cards for 15 days. NDV was detected from FTA imprints of the trachea, lung, caecal tonsil and cloacal faeces of experimentally infected birds. RT-PCR detection from FTA cards was confirmed by homologous frozen-tissue RT-PCR and virus isolation. Direct nucleotide sequence of the amplified F gene allowed prediction of NDV virulence. No virus isolation was possible from the FTA inactivated samples, indicating viral inactivation upon contact. The FTA cards are suitable for collecting and transporting NDV-positive samples, providing a reliable source of RNA for molecular characterization and a hazard-free sample.

Effect of blanching treatments on antioxidant activity of frozen green capsicum (Capsicum annuum L. var bell pepper) using radical scavenging activity (DPPH) assay

NASA Astrophysics Data System (ADS)

Azizzuddin, Norafida; Abdullah, Aminah

2016-11-01

Blanching treatments are needed to deactivate enzymes in frozen vegetables. Antioxidant activity using DPPH radical scavenging activity assay were evaluated in steaming, boiling water, and microwave blanching at different temperature, time and microwave power level on frozen green capsicum. Green capsicum was chosen for frozen treatment compared to other capsicum with different maturity index because of the firm texture. The objective of this study was to compare the antioxidant activity of frozen green capsicum between conventional and Oxi Count Kit® assay for DPPH radical scavenging activity. Results showed frozen green capsicum blanched using microwave at high level/90 seconds (sample J) contained higher level of DPPH in both conventional method and Oxi Count Kit® compared to other treatments. However, there were no significant differences between sample J and fresh sample (sample A). Overall, the sequences from highest to lowest in blanching treatments for both DPPH conventional method, and DPPH Oxi Count Kit® were J (microwave high level/90 seconds) > A (Fresh) > H (Microwave Medium Level/120 seconds) > D (Boiling Water 80°C/150 seconds) > K (Microwave High Level/120 seconds) > I (Microwave Medium Level/150 seconds) > F (Microwave Low Level/150 seconds)> B (Steam 100°C/150 seconds) > E (Boiling Water 100°C /120 seconds) > G (Microwave Low Level /180 seconds)> C (Steam 100°C/180 seconds). Almost all frozen green capsicum samples showed no significant differences for comparison between test using DPPH conventional method and Oxi Count Kit®. Frozen storage for 0, and 3rd months showed no significant differences which indicate no changes on antioxidant activity during frozen storage at -18°C.

Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy

PubMed Central

Zhao, Yongxin; Bucur, Octavian; Irshad, Humayun; Chen, Fei; Weins, Astrid; Stancu, Andreea L.; Oh, Eun-Young; DiStasio, Marcello; Torous, Vanda; Glass, Benjamin; Stillman, Isaac E.; Schnitt, Stuart J.; Beck, Andrew H.; Boyden, Edward S.

2017-01-01

Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding the specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin (H&E), and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ~70 nm resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes, and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, which previously required electron microscopy (EM), and demonstrate high-fidelity computational discrimination between early breast neoplastic lesions that to date have challenged human judgment. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research. PMID:28714966

Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.

PubMed

Zhao, Yongxin; Bucur, Octavian; Irshad, Humayun; Chen, Fei; Weins, Astrid; Stancu, Andreea L; Oh, Eun-Young; DiStasio, Marcello; Torous, Vanda; Glass, Benjamin; Stillman, Isaac E; Schnitt, Stuart J; Beck, Andrew H; Boyden, Edward S

2017-08-01

Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding a specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin, and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ∼70-nm-resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, a process that previously required electron microscopy, and we demonstrate high-fidelity computational discrimination between early breast neoplastic lesions for which pathologists often disagree in classification. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research.

Survey of Salmonella and Campylobacter contamination of whole, raw poultry on retail sale in Wales in 2003.

PubMed

Meldrum, R J; Tucker, I D; Smith, R M M; Edwards, C

2005-07-01

A survey of the Salmonella and Campylobacter contamination of raw, whole chickens available to consumers in Wales was performed between March and December 2003. In total, 736 samples were taken, and overall contamination rates of 73.1% for Campylobacter and 5.7% for Salmonella were found. This survey follows a survey performed during 2001 to 2002 by Welsh local authorities and the National Public Health Service for Wales that established updated baseline rates for both pathogens in raw, whole chicken available to consumers in Wales. This survey indicated no difference in Campylobacter rates between fresh and frozen samples or between samples taken from retailers and local butchers, but significant differences existed in Salmonella rates between fresh and frozen samples and between those sampled from retailers and butchers, with frozen chickens and samples taken from retailers having significantly higher rates. However, the difference in Salmonella isolation rate between retailers and butchers was found to be due to the differences in the proportions of fresh and frozen chickens sampled from these locations, with a significantly higher number of frozen chickens (with a higher Salmonella rate) being sampled from retailers.

Novel Polymerase Spiral Reaction (PSR) for rapid visual detection of Bovine Herpesvirus 1 genomic DNA from aborted bovine fetus and semen.

PubMed

Malla, Javed Ahmed; Chakravarti, Soumendu; Gupta, Vikas; Chander, Vishal; Sharma, Gaurav Kumar; Qureshi, Salauddin; Mishra, Adhiraj; Gupta, Vivek Kumar; Nandi, Sukdeb

2018-02-20

Bovine herpesvirus-1 (BHV-1) is a major viral pathogen affecting bovines leading to various clinical manifestations and causes significant economic impediment in modern livestock production system. Rapid, accurate and sensitive detection of BHV-1 infection at frozen semen stations or at dairy herds remains a priority for control of BHV-1 spread to susceptible population. Polymerase Spiral Reaction (PSR), a novel addition in the gamut of isothermal techniques, has been successfully implemented in initial optimization for detection of BHV-1 genomic DNA and further validated in clinical samples. The developed PSR assay has been validated for detection of BHV-1 from bovine semen (n=99), a major source of transmission of BHV-1 from breeding bulls to susceptible dams in artificial insemination programs. The technique has also been used for screening of BHV-1 DNA from suspected aborted fetal tissues (n=25). The developed PSR technique is 100 fold more sensitive than conventional PCR and comparable to real-time PCR. The PSR technique has been successful in detecting 13 samples positive for BHV-1 DNA in bovine semen, 4 samples more than conventional PCR. The aborted fetal tissues were negative for presence of BHV-1 DNA. The presence of BHV-1 in bovine semen samples raises a pertinent concern for extensively screening of semen from breeding bulls before been used for artificial insemination process. PSR has all the attributes for becoming a method of choice for rapid, accurate and sensitive detection of BHV-1 DNA at frozen semen stations or at dairy herds in resource constrained settings. Copyright © 2017 Elsevier B.V. All rights reserved.

Three-dimensional reconstruction of frozen and thawed plant tissues from microscopic images

USDA-ARS?s Scientific Manuscript database

Histological analysis of frozen and thawed plants has been conducted for many years but the observation of individual sections only provides a 2 dimensional representation of a 3 dimensional phenomenon. Most techniques for viewing internal plant structure in 3 dimensions is either low in resolution...

Evaluation of digestion methods for analysis of trace metals in mammalian tissues and NIST 1577c.

PubMed

Binder, Grace A; Metcalf, Rainer; Atlas, Zachary; Daniel, Kenyon G

2018-02-15

Digestion techniques for ICP analysis have been poorly studied for biological samples. This report describes an optimized method for analysis of trace metals that can be used across a variety of sample types. Digestion methods were tested and optimized with the analysis of trace metals in cancerous as compared to normal tissue as the end goal. Anthropological, forensic, oncological and environmental research groups can employ this method reasonably cheaply and safely whilst still being able to compare between laboratories. We examined combined HNO 3 and H 2 O 2 digestion at 170 °C for human, porcine and bovine samples whether they are frozen, fresh or lyophilized powder. Little discrepancy is found between microwave digestion and PFA Teflon pressure vessels. The elements of interest (Cu, Zn, Fe and Ni) yielded consistently higher and more accurate values on standard reference material than samples heated to 75 °C or samples that utilized HNO 3 alone. Use of H 2 SO 4 does not improve homogeneity of the sample and lowers precision during ICP analysis. High temperature digestions (>165 °C) using a combination of HNO 3 and H 2 O 2 as outlined are proposed as a standard technique for all mammalian tissues, specifically, human tissues and yield greater than 300% higher values than samples digested at 75 °C regardless of the acid or acid combinations used. The proposed standardized technique is designed to accurately quantify potential discrepancies in metal loads between cancerous and healthy tissues and applies to numerous tissue studies requiring quick, effective and safe digestions. Copyright © 2017 Elsevier Inc. All rights reserved.

Rheological properties and bread quality of frozen yeast-dough with added wheat fiber.

PubMed

Adams, Vivian; Ragaee, Sanaa M; Abdel-Aal, El-Sayed M

2017-01-01

The rheological characteristics of frozen dough are of great importance in bread-making quality. The effect of addition of commercial wheat aleurone and bran on rheological properties and final bread quality of frozen dough was studied. Wheat aleurone (A) and bran (B) containing 240 g kg -1 and 200 g kg -1 arabinoxylan (AX), respectively, were incorporated into refined wheat flour at 150 g kg -1 substitution level (composite A and B, respectively). Dough samples of composite A and B in addition to two reference dough samples, refined flour (ref A) and whole wheat flour (ref B) were stored at -18°C for 9 weeks. Frozen stored composite dough samples contained higher amounts of bound water, less freezable water and exhibited fewer modifications in gluten network during frozen storage based on data from differential scanning calorimetry and nuclear magnetic resonance spectroscopy. Bread made from composite frozen dough had higher loaf volume compared to ref A or ref B throughout the storage period. The incorporation of wheat fiber into refined wheat flour produced dough with minimum alterations in its rheological properties during 9 weeks of frozen storage compared to refined and 100% wheat flour dough samples. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

In situ hybridisation of EBV DNA-DNA hybrids using wet heat in polypropylene containers.

PubMed Central

Labrecque, L G

1992-01-01

AIMS: To explore procedures designed to optimise DNA-DNA in situ hybridisation, using cells infected with Epstein-Barr virus (EBV) and tissues and subfragments of the EBV DNA as probes. METHODS: The denaturation step occurred in a polypropylene container, using wet heat generated by a hot water container, the pressure cooker, or the microwave oven, without coverslips, reaching a temperature of 121 degrees C or more in these two last systems. Two different visualisation systems were used. RESULTS: Fixed cells and tumours harbouring a high and medium to low copy number (a few hundreds to 33 copies per cell), were clearly labelled, using a simple reiterated subfragment (BamW) of the EBV DNA, and fresh frozen cells, harbouring a very low copy number (one to two on average) labelled using BamW as well as BamH (single non-reiterated 6 kilobase subfragment). CONCLUSION: This is a valuable alternative technique for DNA-DNA ISH that can be used in fresh frozen samples as well as fixed samples. Images PMID:1336018

Microfluidics for rapid cytokeratin immunohistochemical staining in frozen sections.

PubMed

Brajkovic, Saska; Dupouy, Diego G; de Leval, Laurence; Gijs, Martin Am

2017-08-01

Frozen sections (FS) of tumor samples represent a cornerstone of pathological intraoperative consultation and have an important role in the microscopic analysis of specimens during surgery. So far, immunohistochemical (IHC) stainings on FS have been demonstrated for a few markers using manual methods. Microfluidic technologies have proven to bring substantial improvement in many fields of diagnostics, though only a few microfluidic devices have been designed to improve the performance of IHC assays. In this work, we show optimization of a complete pan-cytokeratin chromogenic immunostaining protocol on FS using a microfluidic tissue processor into a protocol taking <12 min. Our results showed specificity and low levels of background. The dimensions of the microfluidic prototype device are compatible with the space constraints of an intraoperative pathology laboratory. We therefore anticipate that the adoption of microfluidic technologies in the field of surgical pathology can significantly improve the way FSs influence surgical procedures.

Microfluidics for rapid cytokeratin immunohistochemical staining in frozen sections

PubMed Central

Brajkovic, Saska; Dupouy, Diego G.; de Leval, Laurence; Gijs, Martin A. M.

2017-01-01

Frozen sections (FS) of tumor samples represent a cornerstone of pathological intraoperative consultation and play an important role in the microscopic analysis of specimens during surgery. So far, immunohistochemical (IHC) stainings on FS have been demonstrated for a few markers using manual methods. Microfluidic technologies have proven to bring substantial improvement in many fields of diagnostics, though only a few microfluidic devices have been designed to improve the performance of IHC assays. In this work, we show optimization of a complete pan-cytokeratin chromogenic immunostaining protocol on FS using a microfluidic tissue processor, into a protocol taking less than 12 minutes. Our results showed specificity and low levels of background. The dimensions of the microfluidic prototype device are compatible with the space constraints of an intraoperative pathology laboratory. We therefore anticipate that the adoption of microfluidic technologies in the field of surgical pathology can significantly improve the way FSs influence surgical procedures. PMID:28553936

Establishment and Characterization of Primary Glioblastoma Cell Lines from Fresh and Frozen Material: A Detailed Comparison

PubMed Central

Mullins, Christina Susanne; Schneider, Björn; Stockhammer, Florian; Krohn, Mathias; Classen, Carl Friedrich; Linnebacher, Michael

2013-01-01

Background Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. High molecular heterogeneity of GBM tumors is well recognized, forming the rationale for molecular tests required before administration of several of the novel therapeutics rapidly entering the clinics. One model that has gained wide acceptance is the primary cell culture model. The laborious and time consuming process is rewarded with a relative high success rate (about 60%). We here describe and evaluate a very simple cryopreservation procedure for GBM tissue prior to model establishment that will considerably reduce the logistic complexity. Methods Twenty-seven GBM samples collected ad hoc were prepared for primary cell culture freshly from surgery (#1) and after cryopreservation (#2). Results Take rates after cryopreservation (59%) were as satisfactory as from fresh tissue (63%; p = 1.000). We did not observe any relevant molecular or phenotypic differences between cell lines established from fresh or vitally frozen tissue. Further, sensitivity both towards standard chemotherapeutic agents (Temozolomide, BCNU and Vincristine) and novel agents like the receptor tyrosine kinase inhibitor Imatinib did not differ. Conclusions Our simple cryopreservation procedure facilitates collection, long-time storage and propagation (modeling) of clinical GBM specimens (potentially also from distant centers) for basic research, (pre-) clinical studies of novel therapies and individual response prediction. PMID:23951083

The BUME method: a new rapid and simple chloroform-free method for total lipid extraction of animal tissue

NASA Astrophysics Data System (ADS)

Löfgren, Lars; Forsberg, Gun-Britt; Ståhlman, Marcus

2016-06-01

In this study we present a simple and rapid method for tissue lipid extraction. Snap-frozen tissue (15-150 mg) is collected in 2 ml homogenization tubes. 500 μl BUME mixture (butanol:methanol [3:1]) is added and automated homogenization of up to 24 frozen samples at a time in less than 60 seconds is performed, followed by a 5-minute single-phase extraction. After the addition of 500 μl heptane:ethyl acetate (3:1) and 500 μl 1% acetic acid a 5-minute two-phase extraction is performed. Lipids are recovered from the upper phase by automated liquid handling using a standard 96-tip robot. A second two-phase extraction is performed using 500 μl heptane:ethyl acetate (3:1). Validation of the method showed that the extraction recoveries for the investigated lipids, which included sterols, glycerolipids, glycerophospholipids and sphingolipids were similar or better than for the Folch method. We also applied the method for lipid extraction of liver and heart and compared the lipid species profiles with profiles generated after Folch and MTBE extraction. We conclude that the BUME method is superior to the Folch method in terms of simplicity, through-put, automation, solvent consumption, economy, health and environment yet delivering lipid recoveries fully comparable to or better than the Folch method.

Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

PubMed

Metildi, Cristina A; Kaushal, Sharmeela; Luiken, George A; Talamini, Mark A; Hoffman, Robert M; Bouvet, Michael

2014-04-01

The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model. © 2013 Wiley Periodicals, Inc.

Histology of a Woolly Mammoth (Mammuthus primigenius) Preserved in Permafrost, Yamal Peninsula, Northwest Siberia.

PubMed

Papageorgopoulou, Christina; Link, Karl; Rühli, Frank J

2015-06-01

In 2007, the baby woolly mammoth (Mammuthus primigenius) named Lyuba was found frozen in the Siberian tundra permafrost along the Yuribey River. She was proclaimed the best-preserved mammoth discovery. As part of the endoscopic examination of Lyuba, tissue samples of hair, muscle, and internal organs were taken. The sectioned biopsies were stained using standard and special histological stains. In general, the microscopic preservation of the tissue was good although no clearly identifiable cell nuclei were found by standard staining methods. Only a few cell nuclei could be identified in some samples when fluorescence stained with DAPI. The best-preserved structures were collagen fibers and muscle tissue, which gave some structural resemblance to the organs. In the hairs, evidence of pigmentation, a scaly surface, diagonal intra-hair structures, and a medulla were seen. Fat droplets could be identified with Sudan Red in the subcutaneous fat sample and in several organs. Bacteria were seen on the lumen side of the small intestine and caecum, and in the liver and lung tissue. In addition, fungi and pollen were seen in the lung sample. In the wall of the caecum and small intestine, blood vessels and nerves were visualized. Iron was identified in the vivianite sample. Some biopsies compared well structurally with the African elephant tissue sections. The histological findings support the theory that Lyuba drowned in muddy water. The microscopic tissue preservation and cell nuclei destruction indicate that Lyuba's body underwent at least one freeze-thaw cycle. © 2015 Wiley Periodicals, Inc.

Muscle Fiber Orientation Angle Dependence of the Tensile Fracture Behavior of Frozen Fish Muscle

NASA Astrophysics Data System (ADS)

Hagura, Yoshio; Okamoto, Kiyoshi; Suzuki, Kanichi; Kubota, Kiyoshi

We have proposed a new cutting method for frozen fish named "cryo-cutting". This method applied tensile fracture force or bending fracture force to the frozen fish at appropriate low temperatures. In this paper, to clarify cryo-cutting mechanism, we analyzed tensile fracture behavior of the frozen fish muscle. In the analysis, the frozen fish muscle was considered unidirectionally fiber-reinforced composite material which consisted of fiber (muscle fiber) and matrix (connective tissue). Fracture criteria (maximum stress criterion, Tsai-Hill criterion) for the unidirectionally fiber-reinforced composite material were used. The following results were obtained: (1) By using Tsai-Hill criterion, muscle fiber orientation angle dependence of the tensile fracture stress could be calculated. (2) By using the maximum stress theory jointly with Tsai-Hill criterion, muscle fiber orientation angle dependence of the fracture mode of the frozen fish muscle could be estimated.

Evaluation of formalin-fixed paraffin-embedded tissues obtained from vaccine site-associated sarcomas of cats for DNA of feline immunodeficiency virus.

PubMed

Kidney, B A; Ellis, J A; Haines, D M; Jackson, M L

2000-09-01

To evaluate the use of a polymerase chain reaction (PCR) method for detection of feline immunodeficiency virus (FIV) DNA, using formalin-fixed paraffin-embedded (FFPE) tissues, and to use this method to evaluate tissues obtained from vaccine site-associated sarcomas (VSS) of cats for FIV DNA. 50 FFPE tissue blocks from VSS of cats and 50 FFPE tissue blocks from cutaneous non-vaccine site-associated fibrosarcomas (non-VSS) of cats. DNA was extracted from FFPE sections of each tumor and regions of the gag gene of FIV were amplified by a PCR, using 3 sets of primers. Sensitivity of the method was compared between frozen and FFPE tissues, using splenic tissue obtained from a cat that had been experimentally infected with FIV. We did not detect FIV DNA in VSS or non-VSS tissues. Sensitivity of the PCR method was identical for frozen or FFPE tissues. It is possible to detect FIV DNA in FFPE tissues by use of a PCR. We did not find evidence to support direct FIV involvement in the pathogenesis of VSS in cats.

Mass Spectrometry Imaging of Drug Related Crystal-Like Structures in Formalin-Fixed Frozen and Paraffin-Embedded Rabbit Kidney Tissue Sections

NASA Astrophysics Data System (ADS)

Bruinen, Anne L.; van Oevelen, Cateau; Eijkel, Gert B.; Van Heerden, Marjolein; Cuyckens, Filip; Heeren, Ron M. A.

2016-01-01

A multimodal mass spectrometry imaging (MSI) based approach was used to characterize the molecular content of crystal-like structures in a frozen and paraffin embedded piece of a formalin-fixed rabbit kidney. Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) imaging and desorption electrospray ionization (DESI) mass spectrometry imaging were combined to analyze the frozen and paraffin embedded sample without further preparation steps to remove the paraffin. The investigated rabbit kidney was part of a study on a drug compound in development, in which severe renal toxicity was observed in dosed rabbits. Histological examination of the kidney showed tubular degeneration with precipitation of crystal-like structures in the cortex, which were assumed to cause the renal toxicity. The MS imaging approach was used to find out whether the crystal-like structures were composed of the drug compound, metabolites, or an endogenous compound as a reaction to the drug administration. The generated MALDI-MSI data were analyzed using principal component analysis. In combination with the MS/MS results, this way of data processing demonstrates that the crystal structures were mainly composed of metabolites and relatively little parent drug.

Effective melanin depigmentation of human and murine ocular tissues: an improved method for paraffin and frozen sections.

PubMed

Manicam, Caroline; Pitz, Susanne; Brochhausen, Christoph; Grus, Franz H; Pfeiffer, Norbert; Gericke, Adrian

2014-01-01

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. 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. 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. Warm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues.

Heating or freezing bone. Effects on angiogenesis induction and growth potential in mice.

PubMed

Leunig, M; Yuan, F; Berk, D A; Gerweck, L E; Jain, R K

1996-08-01

We have characterized the effect of bone graft treatment by heating or freezing (with or without dimethyl sulfoxide (DMSO)). Tissue culture and dorsal skin-fold chambers in mice were used as sites to quantify the effect on angiogenesis, growth and calcification of neonatal femora. Fresh femora increased in both length and cartilage diameter (calcification in vivo only), but cryopreservation or heating abolished the increase in femoral dimensions. In vivo, femora of all experimental groups elicited an angiogenic response from the host tissue, which was most pronounced for fresh femora, weaker for DMSO-preserved frozen bone and poor for unprotected frozen bone and boiled femora. Freezing in the presence of a cryopreservative (DMSO) was found to preserve the angiogenic potential of frozen bone, whereas unprotected heating or freezing significantly impaired angiogenesis induction and growth potential.

Frozen Section Evaluation of Margin Status in Primary Squamous Cell Carcinomas of the Head and Neck: A Correlation Study of Frozen Section and Final Diagnoses.

PubMed

Layfield, Eleanor M; Schmidt, Robert L; Esebua, Magda; Layfield, Lester J

2018-06-01

Frozen section is routinely used for intraoperative margin evaluation in carcinomas of the head and neck. We studied a series of frozen sections performed for margin status of head and neck tumors to determine diagnostic accuracy. All frozen sections for margin control of squamous carcinomas of the head and neck were studied from a 66 month period. Frozen and permanent section diagnoses were classified as negative or malignant. Correlation of diagnoses was performed to determine accuracy. One thousand seven hundred and ninety-six pairs of frozen section and corresponding permanent section diagnoses were obtained. Discordances were found in 55 (3.1%) pairs. In 35 pairs (1.9%), frozen section was reported as benign, but permanent sections disclosed carcinoma. In 21 cases, the discrepancy was due to sampling and in the remaining cases it was an interpretive error. In 20 cases (1.1%), frozen section was malignant, but the permanent section was interpreted as negative. Frozen section is an accurate method for evaluation of operative margins for head and neck carcinomas with concordance between frozen and permanent results of 97%. Most errors are false negative results with the majority of these being due to sampling issues.

K-Ras mutation detection in liquid biopsy and tumor tissue as prognostic biomarker in patients with pancreatic cancer: a systematic review with meta-analysis.

PubMed

Li, Tao; Zheng, Yuanting; Sun, Hong; Zhuang, Rongyuan; Liu, Jing; Liu, Tianshu; Cai, Weimin

2016-07-01

K-Ras gene mutations have been found in most pancreatic cancers; however, conflicting data on the prognostic value of K-Ras mutations in pancreatic cancer have been published. We conducted a meta-analysis to assess its prognostic significance. Literature searches of PubMed, EMBASE, Cochrane Library, Web of Science and Google Scholar were performed through December 2015 to identify publications exploring the association of K-Ras mutation with overall survival. Forty eligible studies involving 3427 patients with pancreatic cancer were included in the present meta-analysis. Our analysis showed a hazard ratio (HR) of negative association with survival of 1.61 [95 % confidence interval (CI) 1.36-1.90; p < 0.01] in K-Ras mutant pancreatic cancer patients. In subgroup analyses, K-Ras mutations detected in tumor tissues and in liquid biopsies had HRs of 1.37 (95 % CI 1.20-1.57; p < 0.01) and 3.16 (95 % CI 2.1-4.71; p < 0.01), respectively. In addition, the HR was higher when K-Ras mutations were detected in fresh frozen samples (HR = 2.01, 95 % CI 1.28-3.16, p = 0.002) than in formalin-fixed, paraffin-embedded (FFPE) samples (HR = 1.29, 95 % CI 1.12-1.49, p < 0.01). Though K-Ras alterations are more frequent among non-East Asian individuals than East Asian individuals, there were no significant differences in HRs of survival between the two ethnic subgroups. In conclusion, this meta-analysis suggests that K-Ras mutations are associated with a worse overall survival in pancreatic cancer patients, especially when mutations are detected in liquid biopsies or fresh frozen tumor tissue samples.

Toxoplasma gondii transmission by artificial insemination in sheep with experimentally contaminated frozen semen.

PubMed

Consalter, Angélica; Silva, Andressa F; Frazão-Teixeira, Edwards; Matos, Luis F; de Oliveira, Francisco C R; Leite, Juliana S; Silva, Franciele B F; Ferreira, Ana M R

2017-03-01

Toxoplasma gondii is a parasite considered one of the major causes of reproductive problems in sheep. Furthermore, the presence of the agent in ram semen urges the possibility of sexual transmission in this species. The aim of this study was to evaluate if ram's frozen semen spiked with T. gondii tachyzoites would be able to cause infection in sheep by laparoscopic artificial insemination (AI). Nine ewes tested seronegative to anti-T. gondii antibodies by the modified agglutination test (MAT) were superovulated and inseminated to collect embryos. Animals were divided into two groups: G1 (n = 5), ewes inseminated with semen containing 4 × 10 7 tachyzoites; and G2 (n = 4), ewes inseminated with tachyzoite-free semen (control group). To confirm infection, ewe's blood samples were collected on days -14, -7, 0, 7, 14, 21, 28, 35, 49 and 57 after AI for analysis by MAT and PCR. Tissue samples of these ewes were also collected for histopathology and immunohistochemistry (IHC). Seven days after AI, all ewes of group G1 had specific antibodies to T. gondii, while those of G2 were negative. Toxoplasma gondii DNA was detected in the blood of one ewe and parasites were observed in tissues of all five animals inseminated with contaminated semen, indicating that semen freezing protocol does not affect T. gondii transmission by artificial insemination in sheep. Copyright © 2016 Elsevier Inc. All rights reserved.

On-the-spot lung cancer differential diagnosis by label-free, molecular vibrational imaging and knowledge-based classification

NASA Astrophysics Data System (ADS)

Gao, Liang; Li, Fuhai; Thrall, Michael J.; Yang, Yaliang; Xing, Jiong; Hammoudi, Ahmad A.; Zhao, Hong; Massoud, Yehia; Cagle, Philip T.; Fan, Yubo; Wong, Kelvin K.; Wang, Zhiyong; Wong, Stephen T. C.

2011-09-01

We report the development and application of a knowledge-based coherent anti-Stokes Raman scattering (CARS) microscopy system for label-free imaging, pattern recognition, and classification of cells and tissue structures for differentiating lung cancer from non-neoplastic lung tissues and identifying lung cancer subtypes. A total of 1014 CARS images were acquired from 92 fresh frozen lung tissue samples. The established pathological workup and diagnostic cellular were used as prior knowledge for establishment of a knowledge-based CARS system using a machine learning approach. This system functions to separate normal, non-neoplastic, and subtypes of lung cancer tissues based on extracted quantitative features describing fibrils and cell morphology. The knowledge-based CARS system showed the ability to distinguish lung cancer from normal and non-neoplastic lung tissue with 91% sensitivity and 92% specificity. Small cell carcinomas were distinguished from nonsmall cell carcinomas with 100% sensitivity and specificity. As an adjunct to submitting tissue samples to routine pathology, our novel system recognizes the patterns of fibril and cell morphology, enabling medical practitioners to perform differential diagnosis of lung lesions in mere minutes. The demonstration of the strategy is also a necessary step toward in vivo point-of-care diagnosis of precancerous and cancerous lung lesions with a fiber-based CARS microendoscope.

NASA's Rodent Research Project: Validation of Flight Hardware, Operations and Science Capabilities for Conducting Long Duration Experiments in Space

NASA Technical Reports Server (NTRS)

Choi, S. Y.; Beegle, J. E.; Wigley, C. L.; Pletcher, D.; Globus, R. K.

2015-01-01

Research using rodents is an essential tool for advancing biomedical research on Earth and in space. Rodent Research (RR)-1 was conducted to validate flight hardware, operations, and science capabilities that were developed at the NASA Ames Research Center. Twenty C57BL/6J adult female mice were launched on Sept 21, 2014 in a Dragon Capsule (SpaceX-4), then transferred to the ISS for a total time of 21-22 days (10 commercial mice) or 37 (10 validation mice). Tissues collected on-orbit were either rapidly frozen or preserved in RNA later at less than or equal to -80 C (n=2/group) until their return to Earth. Remaining carcasses were rapidly frozen for dissection post-flight. The three controls groups at Kennedy Space Center consisted of: Basal mice euthanized at the time of launch, Vivarium controls, housed in standard cages, and Ground Controls (GC), housed in flight hardware within an environmental chamber. FLT mice appeared more physically active on-orbit than GC, and behavior analysis are in progress. Upon return to Earth, there were no differences in body weights between FLT and GC at the end of the 37 days in space. RNA was of high quality (RIN greater than 8.5). Liver enzyme activity levels of FLT mice and all control mice were similar in magnitude to those of the samples that were optimally processed in the laboratory. Liver samples collected from the intact frozen FLT carcasses had RNA RIN of 7.27 +/- 0.52, which was lower than that of the samples processed on-orbit, but similar to those obtained from the control group intact carcasses. Nonetheless, the RNA samples from the intact carcasses were acceptable for the most demanding transcriptomic analyses. Adrenal glands, thymus and spleen (organs associated with stress response) showed no significant difference in weights between FLT and GC. Enzymatic activity was also not significantly different. Over 3,000 tissues collected from the four groups of mice have become available for the Biospecimen Sharing Program. Together, these validation flight findings demonstrate the capability to support long-duration RR on the ISS to achieve both basic science and biomedical objectives.

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.

PubMed

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 measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples.

Assessment of the influence of different sample processing and cold storage duration on plant free proline content analyses.

PubMed

Teklić, Tihana; Spoljarević, Marija; Stanisavljević, Aleksandar; Lisjak, Miroslav; Vinković, Tomislav; Parađiković, Nada; Andrić, Luka; Hancock, John T

2010-01-01

A method which is widely accepted for the analysis of free proline content in plant tissues is based on the use of 3% sulfosalicylic acid as an extractant, followed by spectrophotometric quantification of a proline-ninhydrin complex in toluene. However, sample preparation and storage may influence the proline actually measured. This may give misleading or difficult to compare data. To evaluate free proline levels fresh and frozen strawberry (Fragaria × ananassa Duch.) leaves and soybean [Glycine max (L.) Merr.] hypocotyl tissues were used. These were ground with or without liquid nitrogen and proline extracted with sulfosalicylic acid. A particular focus was the influence of plant sample cold storage duration (1, 4 and 12 weeks at -20°C) on tissue proline levels measured. The free proline content analyses, carried out in leaves of Fragaria × ananassa Duch. as well as in hypocotyls of Glycine max (L.) Merr., showed a significant influence of the sample preparation method and cold storage period. Long-term storage of up to 12 weeks at -20°C led to a significant increase in the measured proline in all samples analysed. The observed changes in proline content in plant tissue samples stored at -20°C indicate the likelihood of the over-estimation of the proline content if the proline analyses are delayed. Plant sample processing and cold storage duration seem to have an important influence on results of proline analyses. Therefore it is recommended that samples should be ground fresh and analysed immediately. Copyright © 2010 John Wiley & Sons, Ltd.

Effects of various cryoprotectants on the quality of frozen-thawed immature bovine (Qinchuan cattle) calf testicular tissue.

PubMed

Zhang, X-G; Li, H; Hu, J-H

2017-11-01

To investigate the effects of different concentrations of various cryoprotectants (CPs) on the cell viability as well as expression of spermatogenesis-related genes, such as CREM, Stra8 and HSP70-2 in frozen-thawed bovine calf testicular tissue, immature bovine (Qinchuan cattle) calf testicular tissue was collected and cryopreserved in the cryomedia containing different concentrations (5%, 10%, 15% and 20%) of the following three CPs: glycerol, ethylene glycol (EG) and dimethyl sulphoxide (DMSO) respectively. After 1 month cryopreservation in liquid nitrogen, cell viability was evaluated using Trypan blue exclusion under a bright-field microscope. The mRNA expression of the three genes was also evaluated using qRT-PCR. The results indicated that different concentrations of glycerol, EG and DMSO in cryomedia during cryopreservation could protect bovine calf testicular tissue in various ways to avoid freezing or cryopreservation-induced expression changes in spermatogenesis-related genes. The highest cell viability and the three spermatogenesis-related genes (CREM, Stra8 and HSP70-2) expression level came from the cryomedia containing glycerol, EG and DMSO at 10% concentration respectively (p < .05). Meanwhile, compared with the other CPs, the frozen-thawed bovine calf testicular tissue treated with 10% DMSO exhibited the highest cell viability and mRNA expression level of the spermatogenesis-related genes (CREM, Stra8 and HSP70-2). © 2017 Blackwell Verlag GmbH.

Ambient Mass Spectrometry in Cancer Research.

PubMed

Takats, Z; Strittmatter, N; McKenzie, J S

2017-01-01

Ambient ionization mass spectrometry was developed as a sample preparation-free alternative to traditional MS-based workflows. Desorption electrospray ionization (DESI)-MS methods were demonstrated to allow the direct analysis of a broad range of samples including unaltered biological tissue specimens. In contrast to this advantageous feature, nowadays DESI-MS is almost exclusively used for sample preparation intensive mass spectrometric imaging (MSI) in the area of cancer research. As an alternative to MALDI, DESI-MSI offers matrix deposition-free experiment with improved signal in the lower (<500m/z) range. DESI-MSI enables the spatial mapping of tumor metabolism and has been broadly demonstrated to offer an alternative to frozen section histology for intraoperative tissue identification and surgical margin assessment. Rapid evaporative ionization mass spectrometry (REIMS) was developed exclusively for the latter purpose by the direct combination of electrosurgical devices and mass spectrometry. In case of the REIMS technology, aerosol particles produced by electrosurgical dissection are subjected to MS analysis, providing spectral information on the structural lipid composition of tissues. REIMS technology was demonstrated to give real-time information on the histological nature of tissues being dissected, deeming it an ideal tool for intraoperative tissue identification including surgical margin control. More recently, the method has also been used for the rapid lipidomic phenotyping of cancer cell lines as it was demonstrated in case of the NCI-60 cell line collection. © 2017 Elsevier Inc. All rights reserved.

Subchronic treatment with acai frozen pulp prevents the brain oxidative damage in rats with acute liver failure.

PubMed

de Souza Machado, Fernanda; Kuo, Jonnsin; Wohlenberg, Mariane Farias; da Rocha Frusciante, Marina; Freitas, Márcia; Oliveira, Alice S; Andrade, Rodrigo B; Wannmacher, Clovis M D; Dani, Caroline; Funchal, Claudia

2016-12-01

Acai has been used by the population due to its high nutritional value and its benefits to health, such as its antioxidant properties. The aim of this study was to evaluate the protective effect of acai frozen pulp on oxidative stress parameters in cerebral cortex, hippocampus and cerebellum of Wistar rats treated with carbon tetrachloride (CCl 4 ). Thirty male Wistar rats (90-day-old) were orally treated with water or acai frozen pulp for 14 days (7 μL/g). On the 15th day, half of the animals received treatment with mineral oil and the other half with CCl 4 (3.0 mL/kg). The cerebral cortex, hippocampus and cerebellum were dissected and used for analysis of creatine kinase activity (CK), thiobarbituric acid reactive substances (TBARS), carbonyl, sulfhydryl, and the activity of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). Statistical analysis was performed by ANOVA followed by Tukey's post-test. CCl 4 was able to inhibit CK activity in all tissues tested and to provoke lipid damage in cerebral cortex and cerebellum, and protein damage in the three tissues tested. CCl 4 enhanced CAT activity in the cerebral cortex, and inhibited CAT activity in the hippocampus and cerebellum and reduced SOD activity in all tissues studied. Acai frozen pulp prevented the inhibition of CK, TBARS, carbonyl and CAT activity in all brain structures and only in hippocampus for SOD activity. Therefore, acai frozen pulp has antioxidant properties and maybe could be useful in the treatment of some diseases that affect the central nervous system that are associated with oxidative damage.

Comparison of inferred fractions of n-3 and n-6 polyunsaturated fatty acids in feral domestic cat diets with those in commercial feline extruded diets.

PubMed

Backus, Robert C; Thomas, David G; Fritsche, Kevin L

2013-04-01

To compare presumed fatty acid content in natural diets of feral domestic cats (inferred from body fat polyunsatrated fatty acids content) with polyunsaturated fatty acid content of commercial feline extruded diets. Subcutaneous and intra-abdominal adipose tissue samples (approx 1 g) from previously frozen cadavers of 7 adult feral domestic cats trapped in habitats remote from human activity and triplicate samples (200 g each) of 7 commercial extruded diets representing 68% of market share obtained from retail stores. Lipid, triacylglycerol, and phospholipid fractions in adipose tissue samples and ether extracts of diet samples were determined by gas chromatography of methyl esters. Triacylglycerol and phospholipid fractions in the adipose tissue were isolated by thin-layer chromatography. Diet samples were also analyzed for proximate contents. For the adipose tissue samples, with few exceptions, fatty acids fractions varied only moderately with lipid fraction and site from which tissue samples were obtained. Linoleic, α-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acid fractions were 15.0% to 28.2%, 4.5% to 18.7%, 0.9% to 5.0%, < 0.1% to 0.2%, and 0.6% to 1.7%, respectively. As inferred from the adipose findings, dietary fractions of docosahexaenoic and α-linolenic acid were significantly greater than those in the commercial feline diets, but those for linoleic and eicosapentaenoic acids were not significantly different. The fatty acid content of commercial extruded feline diets differed from the inferred content of natural feral cat diets, in which dietary n-3 and possibly n-6 polyunsaturated fatty acids were more abundant. The impact of this difference on the health of pet cats is not known.

Sample storage-induced changes in the quantity and quality of soil labile organic carbon

PubMed Central

Sun, Shou-Qin; Cai, Hui-Ying; Chang, Scott X.; Bhatti, Jagtar S.

2015-01-01

Effects of sample storage methods on the quantity and quality of labile soil organic carbon are not fully understood even though their effects on basic soil properties have been extensively studied. We studied the effects of air-drying and frozen storage on cold and hot water soluble organic carbon (WSOC). Cold- and hot-WSOC in air-dried and frozen-stored soils were linearly correlated with those in fresh soils, indicating that storage proportionally altered the extractability of soil organic carbon. Air-drying but not frozen storage increased the concentrations of cold-WSOC and carbohydrate in cold-WSOC, while both increased polyphenol concentrations. In contrast, only polyphenol concentration in hot-WSOC was increased by air-drying and frozen storage, suggesting that hot-WSOC was less affected by sample storage. The biodegradability of cold- but not hot-WSOC was increased by air-drying, while both air-drying and frozen storage increased humification index and changed specific UV absorbance of both cold- and hot-WSOC, indicating shifts in the quality of soil WSOC. Our results suggest that storage methods affect the quantity and quality of WSOC but not comparisons between samples, frozen storage is better than air-drying if samples have to be stored, and storage should be avoided whenever possible when studying the quantity and quality of both cold- and hot-WSOC. PMID:26617054

Methods of human body odor sampling: the effect of freezing.

PubMed

Lenochova, Pavlina; Roberts, S Craig; Havlicek, Jan

2009-02-01

Body odor sampling is an essential tool in human chemical ecology research. However, methodologies of individual studies vary widely in terms of sampling material, length of sampling, and sample processing. Although these differences might have a critical impact on results obtained, almost no studies test validity of current methods. Here, we focused on the effect of freezing samples between collection and use in experiments involving body odor perception. In 2 experiments, we tested whether axillary odors were perceived differently by raters when presented fresh or having been frozen and whether several freeze-thaw cycles affected sample quality. In the first experiment, samples were frozen for 2 weeks, 1 month, or 4 months. We found no differences in ratings of pleasantness, attractiveness, or masculinity between fresh and frozen samples. Similarly, almost no differences between repeatedly thawed and fresh samples were found. We found some variations in intensity; however, this was unrelated to length of storage. The second experiment tested differences between fresh samples and those frozen for 6 months. Again no differences in subjective ratings were observed. These results suggest that freezing has no significant effect on perceived odor hedonicity and that samples can be reliably used after storage for relatively long periods.

A multicolor flow cytometric assay for measurement of platelet-derived microparticles.

PubMed

Mobarrez, Fariborz; Antovic, Jovan; Egberg, Nils; Hansson, Mona; Jörneskog, Gun; Hultenby, Kjell; Wallén, Håkan

2010-03-01

Flow cytometry (FCM) is the most commonly used method for detection of platelet-derived microparticles (PDMPs), but it is poorly standardized and mainly used for "bedside" analyses in fresh samples. If PDMPs could be analyzed in previously frozen samples it would increase the usefulness of the method. However, cell membrane fragments from contaminating cells created during freezing/thawing may cause artifacts and disturb measurements. PDMPs were labeled with monoclonal antibodies directed against CD42a and CD62P, or CD42a and CD142. The PDMP gate was determined using forward scatter (FSC) and CD42a expression. The mean fluorescence intensities (MFIs) of CD62P or CD142 positive particles were translated into MESF -values (Molecules of Equivalent Soluble Fluorochrome) using a standard curve. FITC-labeled phalloidin (which binds to intracellular actin) was used to detect destroyed cells/cell fragments. Phalloidin-positive particles were significantly more common in supernatants of frozen/thawed platelet rich and platelet poor plasma samples compared with supernatants of platelet free plasma. High-speed centrifugation was then used to obtain PDMP samples with low contamination of cell fragments. Electron microscopy showed that these samples contained numerous round stained particles with cellular membranes of a size of 100-700 nm. Reproducibility experiments using plasma samples from healthy individuals showed that the coefficients of variation (CVs) of MESF values of CD62P and CD142 (both intra- and interassay) were <10%, and the variation between two cytometers in two different laboratories was <5%. We also found that PDMP expression of CD142 (i.e. tissue factor [TF]) and CD62P (i.e P-selectin) was around two times higher in samples from type 1-diabetes patients compared with those from healthy controls (p<0.001). The use of MESF values to quantify PDMP expression of P-selectin and TF yields reproducible data and enables comparison of data between laboratories. If high-speed centrifugation is performed, contamination of cell fragments is low in frozen/thawed samples. (c) 2009 Elsevier Ltd. All rights reserved.

Humidity-controlled preparation of frozen-hydrated biological samples for cryogenic coherent x-ray diffraction microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takayama, Yuki; Nakasako, Masayoshi; RIKEN Harima Institute/SPring-8, 1-1-1 Kouto, Mikaduki, Sayo, Hyogo 679-5148

2012-05-15

Coherent x-ray diffraction microscopy (CXDM) has the potential to visualize the structures of micro- to sub-micrometer-sized biological particles, such as cells and organelles, at high resolution. Toward advancing structural studies on the functional states of such particles, here, we developed a system for the preparation of frozen-hydrated biological samples for cryogenic CXDM experiments. The system, which comprised a moist air generator, microscope, micro-injector mounted on a micromanipulator, custom-made sample preparation chamber, and flash-cooling device, allowed for the manipulation of sample particles in the relative humidity range of 20%-94%rh at 293 K to maintain their hydrated and functional states. Here, wemore » report the details of the system and the operation procedure, including its application to the preparation of a frozen-hydrated chloroplast sample. Sample quality was evaluated through a cryogenic CXDM experiment conducted at BL29XUL of SPring-8. Taking the performance of the system and the quality of the sample, the system was suitable to prepare frozen-hydrated biological samples for cryogenic CXDM experiments.« less

A simple cryogenic holder for tensile testing of soft biological tissues.

PubMed

Lepetit, J; Favier, R; Grajales, A; Skjervold, P O

2004-04-01

To overcome the difficulty of gripping soft biological materials for tensile test, a simple inexpensive cryogenic holder was developed which allows rapid (3 min) preparation of samples. It is made of 6 parts, built in a bakelite cloth, which is an excellent thermal isolant, and is used with rectangular (8x10(-2)x10(-2)x10(-2)m) samples. The holder with the sample inside is completely immersed in liquid nitrogen for 50 s. This duration allows the freezing of the sample ends on a 10(-2)m length and gives a very flat freezing surface throughout the sample cross section. The 6x10(-2)m central part of the sample remained at ambient temperature. Two parts of the holder help maintain the sample until its ends are vertically gripped in the tensile machine thus avoiding any sample deformation during this step. No pressure was applied on the frozen part of the sample by grips of the tensile machine and this avoids breaks in this region. The sample is fixed by adhesion forces (>1 kN) between its frozen parts and 2 pieces of the holder. The procedure has been successfully tested with bovine and salmon muscle samples and results show tensile breaks randomly distributed in the unfrozen region of the samples. Particular attention has been paid to obtain a very flat freezing surface so that the axial strain is equal throughout the sample and therefore any strain-related mechanical parameters can be accurately determined. The dimensions of the holder can be easily modified to fit other sample geometries and can be used with other biological materials.

Method for procuring specific populations of viable human prostate cells for research.

PubMed

Fischer, A H; Philips, A; Taysavang, P; McKenney, J K; Amin, M B

2001-04-01

A wider range of research can be conducted on viable tissue samples than on fixed or frozen samples. A major obstacle to studying viable prostate tissue samples is the inability to accurately identify cancer on direct examination of unembedded tissue. We used a dissecting microscope to identify cancer in unfixed prostate tissue samples stained on the cut surface with 0.5% aqueous toluidine blue. We measured the diagnostic accuracy of this technique in 25 consecutive prostatectomies, determined the viability of procured samples, and estimated the effect on final pathologic assessment. Both surfaces of a 3- to 5-mm thick cross-section taken midway between base and apex of the prostate were examined. A 4-mm punch biopsy was directed to one benign and one malignant area when clearly present. The dissecting microscope allowed clearcut recognition of carcinoma in 17 of the 25 cross-sections, and carcinoma was confirmed in all 17 (100%). In 8 of 25 cases, no procurement was attempted because no carcinoma was evident in the one cross-section studied. Twenty of 25 cross-sections were adequate for benign tissue procurement; five of the cross-sections were not suitable for procurement because of the presence of extensive carcinoma or atrophy. Seventeen of the 20 were accurately diagnosed as benign (85%); one showed pseudohyperplastic adenocarcinoma, one showed focal high-grade prostatic intraepithelial neoplasia, and one showed urothelial carcinoma in situ. Prostatic epithelium obtained with the technique remains viable and can be separated from stroma. The dissecting microscope technique appears to facilitate rather than interfere with accurate pathologic assessment: extraprostatic extension or positive margins were correctly identified during tissue procurement in three cases. The procedure takes only about 30 minutes.

Freezing and drying effects on potential plant contributions to phosphorus in runoff.

PubMed

Roberson, Tiffany; Bundy, Larry G; Andraski, Todd W

2007-01-01

Phosphorus (P) in runoff from landscapes can promote eutrophication of natural waters. Soluble P released from plant material can contribute significant amounts of P to runoff particularly after plant freezing or drying. This study was conducted to evaluate P losses from alfalfa or grass after freezing or drying as potential contributors to runoff P. Alfalfa (Medicago sativa L.) and grass (principally, Agropyron repens L.) plant samples were subjected to freezing and drying treatments to determine P release. Simulated rainfall runoff and natural runoff from established alfalfa fields and a grass waterway were collected to study P contributions from plant tissue to runoff. The effects of freezing and drying on P released from plant tissue were simulated by a herbicide treatment in selected experiments. Soluble reactive P (SP) extracted from alfalfa and grass samples was markedly increased by freezing or drying. In general, SP extracted from plant samples increased in the order fresh < frozen < frozen/thawed < dried, and averaged 1, 8, 14, and 26% of total P in alfalfa, respectively. Soluble reactive P extracted from alfalfa after freezing or drying increased with increasing soil test P (r(2) = 0.64 to 0.68), suggesting that excessive soil P levels increased the risk of plant P contributions to runoff losses. In simulated rainfall studies, paraquat (1,1'-dimethyl-4, 4''-bipyridinium ion) treatment of alfalfa increased P losses in runoff, and results suggested that this treatment simulated the effects of drying on plant P loss. In contrast to the simulated rainfall results, natural runoff studies over 2 yr did not show higher runoff P losses that could be attributed to P from alfalfa. Actual P losses likely depend on the timing and extent of plant freezing and drying and of precipitation events after freezing.

Post-sampling release of free fatty acids - effects of heat stabilization and methods of euthanasia.

PubMed

Jernerén, Fredrik; Söderquist, Marcus; Karlsson, Oskar

2015-01-01

The field of lipid research has made progress and it is now possible to study the lipidome of cells and organelles. A basic requirement of a successful lipid study is adequate pre-analytical sample handling, as some lipids can be unstable and postmortem changes can cause substantial accumulation of free fatty acids (FFAs). The aim of the present study was to investigate the effects of conductive heat stabilization and euthanasia methods on FFA levels in the rat brain and liver using liquid chromatography tandem mass spectrometry. The analysis of brain homogenates clearly demonstrated phospholipase activity and time-dependent post-sampling changes in the lipid pool of snap frozen non-stabilized tissue. There was a significant increase in FFAs already at 2min, which continued over time. Heat stabilization was shown to be an efficient method to reduce phospholipase activity and ex vivo lipolysis. Post-sampling effects due to tissue thawing and sample preparation induced a massive release of FFAs (up to 3700%) from non-stabilized liver and brain tissues compared to heat stabilized tissue. Furthermore, the choice of euthanasia method significantly influenced the levels of FFAs in the brain. The FFAs were decreased by 15-44% in the group of animals euthanized by pentobarbital injection compared with CO2 inhalation or decapitation. Our results highlight the importance of considering euthanasia methods and pre-analytical treatment in lipid analysis, factors which may otherwise interfere with the outcome of the experiments. Copyright © 2014 Elsevier Inc. All rights reserved.

Electron Tomography of Cryo-Immobilized Plant Tissue: A Novel Approach to Studying 3D Macromolecular Architecture of Mature Plant Cell Walls In Situ

PubMed Central

Sarkar, Purbasha; Bosneaga, Elena; Yap, Edgar G.; Das, Jyotirmoy; Tsai, Wen-Ting; Cabal, Angelo; Neuhaus, Erica; Maji, Dolonchampa; Kumar, Shailabh; Joo, Michael; Yakovlev, Sergey; Csencsits, Roseann; Yu, Zeyun; Bajaj, Chandrajit; Downing, Kenneth H.; Auer, Manfred

2014-01-01

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls. PMID:25207917

Characterization of human breast cancer by scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Chen, Di; Malyarenko, Eugene; Seviaryn, Fedar; Yuan, Ye; Sherman, Mark; Bandyopadhyay, Sudeshna; Gierach, Gretchen; Greenway, Christopher W.; Maeva, Elena; Strumban, Emil; Duric, Neb; Maev, Roman

2013-03-01

Objectives: The purpose of this study was to characterize human breast cancer tissues by the measurement of microacoustic properties. Methods: We investigated eight breast cancer patients using acoustic microscopy. For each patient, seven blocks of tumor tissue were collected from seven different positions around a tumor mass. Frozen sections (10 micrometer, μm) of human breast cancer tissues without staining and fixation were examined in a scanning acoustic microscope with focused transducers at 80 and 200 MHz. Hematoxylin and Eosin (H and E) stained sections from the same frozen breast cancer tissues were imaged by optical microscopy for comparison. Results: The results of acoustic imaging showed that acoustic attenuation and sound speed in cancer cell-rich tissue regions were significantly decreased compared with the surrounding tissue regions, where most components are normal cells/tissues, such as fibroblasts, connective tissue and lymphocytes. Our observation also showed that the ultrasonic properties were influenced by arrangements of cells and tissue patterns. Conclusions: Our data demonstrate that attenuation and sound speed imaging can provide biomechanical information of the tumor and normal tissues. The results also demonstrate the potential of acoustic microscopy as an auxiliary method for operative detection and localization of cancer affected regions.

Long-term frozen storage of urine samples: a trouble to get PCR results in Schistosoma spp. DNA detection?

PubMed

Fernández-Soto, Pedro; Velasco Tirado, Virginia; Carranza Rodríguez, Cristina; Pérez-Arellano, José Luis; Muro, Antonio

2013-01-01

Human schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples' storage or conditions for handling and DNA preservation and extraction methods. We evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patients urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template. Long-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA method of extraction used.

Optimization of a Widefield Structured Illumination Microscope for Non-Destructive Assessment and Quantification of Nuclear Features in Tumor Margins of a Primary Mouse Model of Sarcoma

PubMed Central

Fu, Henry L.; Mueller, Jenna L.; Javid, Melodi P.; Mito, Jeffrey K.; Kirsch, David G.; Ramanujam, Nimmi; Brown, J. Quincy

2013-01-01

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm−1 used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy. PMID:23894357

Primary cultures of astrocytes from fetal bovine brain.

PubMed

Ballarin, Cristina; Peruffo, Antonella

2012-01-01

We describe here a method to obtain primary cell cultures from the cerebral cortex and the hypothalamus of bovine fetuses. We report how tissue origin, developmental stages, and culture medium conditions influence cell differentiation and the prevalence of glial cells vs. neurons. We compare explants from early, middle, and late stages of development and two different fetal calf serum concentrations (1 and 10%) to identify the best conditions to obtain and grow viable astrocytes in culture. In addition, we describe how to cryopreserve and obtain viable cortical astrocytes from frozen fetal bovine brain samples.

Subepithelial corneal fibrosis partially due to epithelial-mesenchymal transition of ocular surface epithelium

PubMed Central

Kawashima, Motoko; Higa, Kazunari; Satake, Yoshiyuki; Omoto, Masahiro; Tsubota, Kazuo; Shimmura, Shigeto; Shimazaki, Jun

2010-01-01

Purpose To determine whether epithelial-mesenchymal transition is involved in the development of corneal subepithelial fibrosis (pannus). Methods Frozen samples of pannus tissue removed from human corneas with a diagnosis of total limbal stem cell deficiency were characterized by immunostaining for both epithelial and mesenchymal markers. We selected transformation-related protein 63 (p63) and pancytokeratin as epithelial markers and vimentin and α-smooth muscle actin (α-SMA) as mesenchymal markers. Immunostaining for β-catenin and E-cadherin was performed to determine wingless-Int (Wnt)-pathway activation. RT–PCR analysis was also performed on epithelial tissue obtained from pannus samples after dispase digestion. Results Immunohistochemistry revealed strong nuclear expression of p63 and weak intercellular expression of E-cadherin in epithelial basal cells of pannus tissue. Furthermore, translocation of β-catenin from intercellular junctions to the nucleus and cytoplasm was also observed. Double-positive cells for both p63 and α-SMA were observed in the subepithelial stroma of pannus tissue, which was supported by RT–PCR and cytospin analysis. Conclusions Epithelial-mesenchymal transition may be partially involved in the development of subepithelial corneal fibrosis due to total limbal stem cell deficiency. PMID:21179238

Curation of Frozen Samples

NASA Technical Reports Server (NTRS)

Fletcher, L. A.; Allen, C. C.; Bastien, R.

2008-01-01

NASA's Johnson Space Center (JSC) and the Astromaterials Curator are charged by NPD 7100.10D with the curation of all of NASA s extraterrestrial samples, including those from future missions. This responsibility includes the development of new sample handling and preparation techniques; therefore, the Astromaterials Curator must begin developing procedures to preserve, prepare and ship samples at sub-freezing temperatures in order to enable future sample return missions. Such missions might include the return of future frozen samples from permanently-shadowed lunar craters, the nuclei of comets, the surface of Mars, etc. We are demonstrating the ability to curate samples under cold conditions by designing, installing and testing a cold curation glovebox. This glovebox will allow us to store, document, manipulate and subdivide frozen samples while quantifying and minimizing contamination throughout the curation process.

Effect of Freezing Conditions on Distances and Their Distributions Derived from Double Electron Electron Resonance (DEER): A Study of Doubly-Spin-Labeled T4 Lysozyme

PubMed Central

Georgieva, Elka R.; Roy, Aritro S.; Grigoryants, Vladimir M.; Borbat, Petr P.; Earle, Keith A.; Scholes, Charles P.; Freed, Jack H.

2012-01-01

Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution. We compared the results from rapidly freeze-quenched (≤100 μs) samples to those from commonly shock-frozen (slow freeze, 1s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions. PMID:22341208

The Prostate Cancer Biorepository Network (PCBN)

DTIC Science & Technology

2016-10-01

site includes blood (serum, plasma, and buffy coat), prostatectomy tissues (frozen), biopsies and metastatic tissue from rapid autopsies (paraffin...embedded material and tissue microarrays (TMAs)), prostate cancer patient derived xenografts (PDX) and derived specimens (DNA and RNA) from prostate...Genitourinary Cancer Biorepository set up a rapid autopsy program to provide access to metastatic tissue and create patient derived xenograft (PDX

Resurrection of a bull by cloning from organs frozen without cryoprotectant in a -80 degrees c freezer for a decade.

PubMed

Hoshino, Yoichiro; Hayashi, Noboru; Taniguchi, Shunji; Kobayashi, Naohiko; Sakai, Kenji; Otani, Tsuyoshi; Iritani, Akira; Saeki, Kazuhiro

2009-01-01

Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a -80 degrees C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.

Resurrection of a Bull by Cloning from Organs Frozen without Cryoprotectant in a −80°C Freezer for a Decade

PubMed Central

Hoshino, Yoichiro; Hayashi, Noboru; Taniguchi, Shunji; Kobayashi, Naohiko; Sakai, Kenji; Otani, Tsuyoshi; Iritani, Akira; Saeki, Kazuhiro

2009-01-01

Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a −80°C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells. PMID:19129919

Long-Term Frozen Storage of Urine Samples: A Trouble to Get PCR Results in Schistosoma spp. DNA Detection?

PubMed Central

Fernández-Soto, Pedro; Velasco Tirado, Virginia; Carranza Rodríguez, Cristina; Pérez-Arellano, José Luis; Muro, Antonio

2013-01-01

Background Human schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples’ storage or conditions for handling and DNA preservation and extraction methods. Methodology/Principal Findings We evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patientś urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template. Conclusions/Significance Long-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA method of extraction used. PMID:23613907

Cryopreservation of ovarian tissue for fertility preservation in young female oncological patients.

PubMed

Andersen, Claus Yding; Kristensen, Stine Gry; Greve, Tine; Schmidt, Kirsten Tryde

2012-05-01

Girls and women suffering from a cancer that requires treatment with gonadotoxic drugs may experience cessation of reproductive function as a side effect due to obliteration of the ovarian pool of follicles. Techniques are now available for fertility preservation, such as cryopreservation of mature oocytes, embryos or ovarian cortical tissue. Whereas collection of mature oocytes and embryos requires at least a 2-week period, ovarian tissue may on short notice be frozen prior to treatment and can be transplanted back into women with ovarian failure. Transplanted frozen/thawed tissue supports survival and growth of follicles, giving rise to menstrual cycles and hormone production for several years. Worldwide, the procedure has resulted in the birth of 15 healthy children. Many cancer patients including girls and young women want fertility preservation, and the techniques are now being further developed and implemented in several centers.

Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla).

PubMed

O'Brien, J K; Stojanov, T; Crichton, E G; Evans, K M; Leigh, D; Maxwell, W M C; Evans, G; Loskutoff, N M

2005-08-01

We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa. Copyright 2005 Wiley-Liss, Inc.

Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction.

PubMed

Critser, J K; Huse-Benda, A R; Aaker, D V; Arneson, B W; Ball, G D

1987-04-01

Three experiments were conducted to evaluate effects of holding semen at +5.0 degrees C for 30 minutes or -5.0 degrees C for 10 minutes and ice crystal induction (seeding) on frozen-thawed human spermatozoa. In experiment 1, spermatozoa were frozen, and postthaw motility was evaluated immediately (0 hour) and 24 hours later. At both 0 and 24 hours, nonfrozen control samples had higher motility than all other treatment groups. At 0 hour postthaw, motility was higher in samples held at -5.0 degrees C for 10 minutes with no significant effect of seeding. At 24 hours, samples held at -5.0 degrees C for 10 minutes and seeded, but not samples held at -5.0 degrees C and not seeded, had higher motility than samples held at +5.0 degrees C. In experiment 2, semen samples were frozen, and fertilizability was evaluated in a zona-free hamster egg penetration assay. Seeded samples had a higher frequency of sperm penetration than either nonfrozen or nonseeded samples. In experiment 3, nonfrozen controls and frozen treatment groups were evaluated for the frequency of survival and acrosomal integrity. Seeded samples had higher frequencies of survival and loss of acrosomal integrity than nonseeded samples. All frozen-thawed samples had a lower frequency of survival and a higher frequency of loss of acrosomal integrity than nonfrozen controls. Although altered patterns of fertilizability and acrosomal integrity are induced, collectively these data suggest that incorporating a holding temperature of -5.0 degrees C for 10 minutes and seeding may result in a superior protocol for freezing human spermatozoa.

Application of non-lethal stable isotope analysis to assess feeding patterns of juvenile pallid sturgeon Scaphirhynchus albus: a comparison of tissue types and sample preservation methods

USGS Publications Warehouse

Andvik, R.T.; VanDeHey, J.A.; Fincel, M.J.; French, William E.; Bertrand, K.N.; Chipps, Steven R.; Klumb, Robert A.; Graeb, B.D.S.

2010-01-01

Traditional techniques for stable isotope analysis (SIA) generally require sacrificing animals to collect tissue samples; this can be problematic when studying diets of endangered species such as the pallid sturgeon Scaphirhynchus albus. Our objectives were to (i) determine if pectoral fin tissue (non-lethal) could be a substitute for muscle tissue (lethal) in SIA of juvenile pallid sturgeon, and (ii) evaluate the influence of preservation techniques on stable isotope values. In the laboratory, individual juvenile pallid sturgeon were held for up to 186 day and fed chironomids, fish, or a commercially available pellet diet. Significant, positive relationships (r² ≥ 0.8) were observed between fin and muscle tissues for both δ15N and δ13C; in all samples isotopes were enriched in fins compared to muscle tissue. Chironomid and fish based diets of juvenile pallid sturgeon were distinguishable for fast growing fish (0.3 mm day−1) using stable δ15N and δ13C isotopes. Frozen and preserved fin tissue δ15N isotopes were strongly related (r2 = 0.89) but δ13C isotopes were weakly related (r2 = 0.16). Therefore, freezing is recommended for preservation of fin clips to avoid the confounding effect of enrichment by ethanol. This study demonstrates the utility of a non-lethal technique to assess time integrated food habits of juvenile pallid sturgeon and should be applicable to other threatened or endangered species.

Image sampling in static telepathology for frozen section diagnosis.

PubMed

Della Mea, V; Cataldi, P; Boi, S; Finato, N; Dalla Palma, P; Beltrami, C A

1999-10-01

A frozen section diagnostic service is often not directly available in small rural or mountain hospitals. In these cases, it could be possible to provide frozen section diagnosis through telepathology systems. Telepathology is based on two main methods: static and dynamic. The former is less expensive, but involves the crucial problem of image sampling. To characterise the differences in image sampling for static telepathology when undertaken by pathologists with different experience. As a test field, a previously studied telepathology method based on multimedia email was adopted. Using this method, three pathologists with different levels of experience sampled images from 155 routine frozen sections and sent them to a distant pathology institute, where diagnoses were made on digital images. After the telepathology diagnoses, the glass slides of both the frozen sections and the definitive sections were sent to the remote pathologists for review. Four of 155 transmissions were considered inadequate by the remote pathologist. In the remaining 151 cases, the telepathology diagnosis agreed with the gold standard in 146 (96.7%). There was no significant divergence between the three pathologists in their sampling of the images. Each case comprised five images on average, acquired in four minutes. The overall time for transmission was about 19 minutes. The results suggest that in routine frozen section diagnosis an inexperienced pathologist can sample images sufficiently well to permit remote diagnosis. However, as expected, the internet is too unreliable for such a time dependent task. An improvement in the system would involve integrated real time features, so that there could be interaction between the two pathologists.

Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

PubMed

Preissl, Sebastian; Fang, Rongxin; Huang, Hui; Zhao, Yuan; Raviram, Ramya; Gorkin, David U; Zhang, Yanxiao; Sos, Brandon C; Afzal, Veena; Dickel, Diane E; Kuan, Samantha; Visel, Axel; Pennacchio, Len A; Zhang, Kun; Ren, Bing

2018-03-01

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.

Intracerebral Injections and Ultrastructural Analysis of High-Pressure Frozen Brain Tissue.

PubMed

Weil, Marie-Theres; Ruhwedel, Torben; Möbius, Wiebke; Simons, Mikael

2017-01-03

Intracerebral injections are an invasive method to bypass the blood brain barrier and are widely used to study molecular and cellular mechanisms of the central nervous system. The administered substances are injected directly at the site of interest, executing their effect locally. By combining injections in the rat brain with state-of-the-art electron microscopy, subtle changes in ultrastructure of the nervous tissue can be detected prior to overt damage or disease. The protocol presented here involves stereotactic injection into the corpus callosum of Lewis rats and the cryopreparation of freshly dissected tissue for electron microscopy. The localization of the injection site in tissue sections during the sample preparation for transmission electron microscopy is explained and possible artifacts of the method are indicated. With the help of this powerful combination of injections and electron microscopy, subtle effects of the applied substances on the biology of neural cells can be identified and monitored over time. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Artificial insemination of cranes with frozen semen

USGS Publications Warehouse

Gee, G.F.; Sexton, T.J.; Lewis, J.C.

1979-01-01

For the first time (1978) artificial insemination (AI) with frozen greater sandhill crane (Grus canadensis tabida) semen resulted in fertile eggs and chicks. During the 2 year (1977-78) study, 6 of 27 eggs produced were fertile. Three chicks hatched. Semen samples used for insemination were frozen and stored in liquid nitrogen for two months or less. Recent improvements in the laboratory indicated that a more effective sample can be prepared and greater fertility rates should be expected.

Gastric Cancer-Specific Protein Profile Identified Using Endoscopic Biopsy Samples via MALDI Mass Spectrometry

PubMed Central

Kim, Hark Kyun; Reyzer, Michelle L.; Choi, Il Ju; Kim, Chan Gyoo; Kim, Hee Sung; Oshima, Akira; Chertov, Oleg; Colantonio, Simona; Fisher, Robert J.; Allen, Jamie L.; Caprioli, Richard M.; Green, Jeffrey E.

2012-01-01

To date, proteomic analyses on gastrointestinal cancer tissue samples have been performed using surgical specimens only, which are obtained after a diagnosis is made. To determine if a proteomic signature obtained from endoscopic biopsy samples could be found to assist with diagnosis, frozen endoscopic biopsy samples collected from 63 gastric cancer patients and 43 healthy volunteers were analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. A statistical classification model was developed to distinguish tumor from normal tissues using half the samples and validated with the other half. A protein profile was discovered consisting of 73 signals that could classify 32 cancer and 22 normal samples in the validation set with high predictive values (positive and negative predictive values for cancer, 96.8% and 91.3%; sensitivity, 93.8%; specificity, 95.5%). Signals overexpressed in tumors were identified as α-defensin-1, α-defensin-2, calgranulin A, and calgranulin B. A protein profile was also found to distinguish pathologic stage Ia (pT1N0M0) samples (n = 10) from more advanced stage (Ib or higher) tumors (n = 48). Thus, protein profiles obtained from endoscopic biopsy samples may be useful in assisting with the diagnosis of gastric cancer and, possibly, in identifying early stage disease. PMID:20557134

Freezing of homogenized sputum samples for intermittent storage.

PubMed

Holz, O; Mücke, M; Zarza, P; Loppow, D; Jörres, R A; Magnussen, H

2001-08-01

Among the reasons that restrict the application of sputum induction in outpatient settings is the need for processing of samples within 2 h after induction. The aim of our study was to assess whether freezing is suitable for intermediate storage of sputum samples before processing. We compared differential cell counts between two sputum aliquots derived from the same sample. One aliquot was processed within 2 h after production and one, after it had been frozen under addition of dimethyl-sulfoxid (DMSO) and stored up to 10 days at -20 degrees C. Thirty-five samples were frozen immediately prior to preparation of cytospins, and 10 samples were frozen at an even earlier stage, directly after homogenization. In both sets of experiments we observed a significant relationship between frozen and native samples regarding macrophages, neutrophils and eosinophils, as indicated by respective intraclass correlation coefficients of 0.96, 0.96, and 0.93 in the first, and of 0.92, 0.96 and 0.77 in the second experiments. Our results indicate that the freezing of sputum samples at different stages of processing does not alter sputum morphology to an extent that affects the results of differential cell counts.

Sensory descriptive quantitative analysis of unpasteurized and pasteurized juçara pulp (Euterpe edulis) during long-term storage

PubMed Central

da Silva, Paula Porrelli Moreira; Casemiro, Renata Cristina; Zillo, Rafaela Rebessi; de Camargo, Adriano Costa; Prospero, Evanilda Teresinha Perissinotto; Spoto, Marta Helena Fillet

2014-01-01

This study evaluated the effect of pasteurization followed by storage under different conditions on the sensory attributes of frozen juçara pulp using quantitative descriptive analysis (QDA). Pasteurization of packed frozen pulp was performed by its immersion in stainless steel tank containing water (80°C) for 5 min, followed by storage under refrigerated and frozen conditions. A trained sensory panel evaluated the samples (6°C) on day 1, 15, 30, 45, 60, 75, and 90. Sensory attributes were separated as follows: appearance (foamy, heterogeneous, purple, brown, oily, and creamy), aroma (sweet and fermented), taste (astringent, bitter, and sweet), and texture (oily and consistent), and compared to a reference material. In general, unpasteurized frozen pulp showed the highest score for foamy appearance, and pasteurized samples showed highest scores to creamy appearance. Pasteurized samples remained stable regarding brown color development while unpasteurized counterparts presented increase. Color is an important attribute related to the product identity. All attributes related to taste and texture remained constant during storage for all samples. Pasteurization followed by storage under frozen conditions has shown to be the best conservation method as samples submitted to such process received the best sensory evaluation, described as foamy, slightly heterogeneous, slightly bitter, and slightly astringent. PMID:25473489

Sensory descriptive quantitative analysis of unpasteurized and pasteurized juçara pulp (Euterpe edulis) during long-term storage.

PubMed

da Silva, Paula Porrelli Moreira; Casemiro, Renata Cristina; Zillo, Rafaela Rebessi; de Camargo, Adriano Costa; Prospero, Evanilda Teresinha Perissinotto; Spoto, Marta Helena Fillet

2014-07-01

This study evaluated the effect of pasteurization followed by storage under different conditions on the sensory attributes of frozen juçara pulp using quantitative descriptive analysis (QDA). Pasteurization of packed frozen pulp was performed by its immersion in stainless steel tank containing water (80°C) for 5 min, followed by storage under refrigerated and frozen conditions. A trained sensory panel evaluated the samples (6°C) on day 1, 15, 30, 45, 60, 75, and 90. Sensory attributes were separated as follows: appearance (foamy, heterogeneous, purple, brown, oily, and creamy), aroma (sweet and fermented), taste (astringent, bitter, and sweet), and texture (oily and consistent), and compared to a reference material. In general, unpasteurized frozen pulp showed the highest score for foamy appearance, and pasteurized samples showed highest scores to creamy appearance. Pasteurized samples remained stable regarding brown color development while unpasteurized counterparts presented increase. Color is an important attribute related to the product identity. All attributes related to taste and texture remained constant during storage for all samples. Pasteurization followed by storage under frozen conditions has shown to be the best conservation method as samples submitted to such process received the best sensory evaluation, described as foamy, slightly heterogeneous, slightly bitter, and slightly astringent.

Real-Time Reverse-Transcription Quantitative Polymerase Chain Reaction Assay Is a Feasible Method for the Relative Quantification of Heregulin Expression in Non-Small Cell Lung Cancer Tissue.

PubMed

Kristof, Jessica; Sakrison, Kellen; Jin, Xiaoping; Nakamaru, Kenji; Schneider, Matthias; Beckman, Robert A; Freeman, Daniel; Spittle, Cindy; Feng, Wenqin

2017-01-01

In preclinical studies, heregulin ( HRG ) expression was shown to be the most relevant predictive biomarker for response to patritumab, a fully human anti-epidermal growth factor receptor 3 monoclonal antibody. In support of a phase 2 study of erlotinib ± patritumab in non-small cell lung cancer (NSCLC), a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay for relative quantification of HRG expression from formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples was developed and validated and described herein. Test specimens included matched FFPE normal lung and NSCLC and frozen NSCLC tissue, and HRG -positive and HRG -negative cell lines. Formalin-fixed paraffin-embedded tissue was examined for functional performance. Heregulin distribution was also analyzed across 200 NSCLC commercial samples. Applied Biosystems TaqMan Gene Expression Assays were run on the Bio-Rad CFX96 real-time PCR platform. Heregulin RT-qPCR assay specificity, PCR efficiency, PCR linearity, and reproducibility were demonstrated. The final assay parameters included the Qiagen FFPE RNA Extraction Kit for RNA extraction from FFPE NSCLC tissue, 50 ng of RNA input, and 3 reference (housekeeping) genes ( HMBS, IPO8 , and EIF2B1 ), which had expression levels similar to HRG expression levels and were stable among FFPE NSCLC samples. Using the validated assay, unimodal HRG distribution was confirmed across 185 evaluable FFPE NSCLC commercial samples. Feasibility of an RT-qPCR assay for the quantification of HRG expression in FFPE NSCLC specimens was demonstrated.

Effect of freezing on electrical properties and quality of thawed chicken breast meat

PubMed Central

Wei, Ran; Wang, Peng; Han, Minyi; Chen, Tianhao; Xu, Xinglian; Zhou, Guanghong

2017-01-01

Objective The objective of this research was to study the electrical properties and quality of frozen-thawed chicken breast meat and to investigate the relationship between these parameters at different times of frozen storage. Methods Thawed samples of chicken breast muscles were evaluated after being kept in frozen storage at −18°C for different periods of time (1, 2, 3, 4, 5, 6, 7, and 8 months). Results The results showed that water-holding capacity (WHC) and protein solubility decreased while thiobarbituric acid-reactive substances content increased with increasing storage time. The impedance module of samples decreased during 8-month frozen storage. Pearson correlation coefficients showed that the impedance change ratio (Q value) was significantly (p<0.05) related to pH, color, WHC, lipid oxidation and protein solubility, indicating a good relationship between the electrical properties and qualities of frozen-thawed chicken breast meat. Conclusion Impedance measurement has a potential to assess the quality of frozen chicken meat combining with quality indices. PMID:27554358

RCL2, a New Fixative, Preserves Morphology and Nucleic Acid Integrity in Paraffin-Embedded Breast Carcinoma and Microdissected Breast Tumor Cells

PubMed Central

Delfour, Christophe; Roger, Pascal; Bret, Caroline; Berthe, Marie-Laurence; Rochaix, Philippe; Kalfa, Nicolas; Raynaud, Pierre; Bibeau, Frédéric; Maudelonde, Thierry; Boulle, Nathalie

2006-01-01

Methacarn and RCL2, a new noncrosslinking fixative, were compared to formalin-fixed or frozen tissue samples of the same invasive breast carcinoma and were evaluated for their effects on tissue morphology and immunohistochemistry as well as DNA and RNA integrity. The histomorphology of methacarn- or RCL2-fixed paraffin-embedded tumors was similar to that observed with the matched formalin-fixed tissues. Immunohistochemistry using various antibodies showed comparable results with either fixative, leading to accurate breast tumor diagnosis and determination of estrogen and progesterone receptors, and HER2 status. Methacarn and RCL2 fixation preserved DNA integrity as demonstrated by successful amplification and sequencing of large DNA amplicons. Similarly, high-quality RNA could be extracted from methacarn- or RCL2-fixed paraffin-embedded MCF-7 cells, whole breast tumor tissues, or microdissected breast tumor cells, as assessed by electropherogram profiles and real-time reverse transcriptase-polymerase chain reaction quantification of various genes. Moreover, tissue morphology and RNA integrity were preserved after 8 months of storage. Altogether, these results indicate that methacarn, as previously shown, and RCL2, a promising new fixative, have great potential for performing both morphological and molecular analyses on the same fixed tissue sample, even after laser-capture microdissection, and can open new doors for investigating small target lesions such as premalignant breast lesions. PMID:16645201

Ex vivo validation of a stoichiometric dual energy CT proton stopping power ratio calibration

NASA Astrophysics Data System (ADS)

Xie, Yunhe; Ainsley, Christopher; Yin, Lingshu; Zou, Wei; McDonough, James; Solberg, Timothy D.; Lin, Alexander; Teo, Boon-Keng Kevin

2018-03-01

A major source of uncertainty in proton therapy is the conversion of Hounsfield unit (HU) to proton stopping power ratio relative to water (SPR). In this study, we measured and quantified the accuracy of a stoichiometric dual energy CT (DECT) SPR calibration. We applied a stoichiometric DECT calibration method to derive the SPR using CT images acquired sequentially at 80 kVp and 140 kVp . The dual energy index was derived based on the HUs of the paired spectral images and used to calculate the effective atomic number (Z eff), relative electron density ({{ρ }e} ), and SPRs of phantom and biological materials. Two methods were used to verify the derived SPRs. The first method measured the sample’s water equivalent thicknesses to deduce the SPRs using a multi-layer ion chamber (MLIC) device. The second method utilized Gafchromic EBT3 film to directly compare relative ranges between sample and water after proton pencil beam irradiation. Ex vivo validation was performed using five different types of frozen animal tissues with the MLIC and three types of fresh animal tissues using film. In addition, the residual ranges recorded on the film were used to compare with those from the treatment planning system using both DECT and SECT derived SPRs. Bland-Altman analysis indicates that the differences between DECT and SPR measurement of tissue surrogates, frozen and fresh animal tissues has a mean of 0.07% and standard deviation of 0.58% compared to 0.55% and 1.94% respectively for single energy CT (SECT) and SPR measurement. Our ex vivo study indicates that the stoichiometric DECT SPR calibration method has the potential to be more accurate than SECT calibration under ideal conditions although beam hardening effects and other image artifacts may increase this uncertainty.

The case study of drillbit and borehole frozen water of the subglacial Lake Vostok, East Antarctica for microbial content

NASA Astrophysics Data System (ADS)

Bulat, Sergey; Doronin, Maxim; Dominique, Marie; Lipenkov, Vladimir; Lukin, Valery; Karlov, Denis; Demchenko, Leonid; Khilchenko, Margarita

The objective was to estimate microbial content and diversity in the subglacial Lake Vostok (buried beneath 4-km thick East Antarctic ice sheet) by studying the uppermost water layer which entered the borehole upon lake entry (February 5, 2012) and then shortly frozen within. The samples of so-called drillbit water frozen on a drill bit upon lake enter (RAE57) along with re-drilled so-called borehole-frozen water (RAE58) were provided for the study with the ultimate goal to discover the life in this extreme icy environment. The comprehensive analyses (constrained by Ancient DNA research criteria) of the first lake water samples - drillbit- (one sample) and borehole-frozen (3 different depths 5G-2N-3425, 3429 et 3450m), are nearly got finished. If the drillbit water sample was heavily polluted with drill fluid (at ratio 1:1), re-drilled borehole-frozen samples were proved to be rather clean but still strongly smelling kerosene and containing numerous micro-droplets of drill fluid making the ice non-transparent. The cell concentrations measured by flow cytofluorometry showed 167 cells per ml in the drillbit water sample while in borehole-frozen samples ranged from 5.5 (full-cylinder 3429m deep frozen water ice core) to 38 cells per ml (freeze-centre of 3450m deep moon-shape ice core). DNA analyses came up with total 44 bacterial phylotypes discovered by sequencing of different regions (v3-v5, v4-v8, v4-v6 et full-gene) of 16S rRNA genes. Amongst them all but two were considered to be contaminants (were present in our contaminant library, including drill fluid findings). The 1st remaining phylotype successfully passing all contamination criteria proved to be hitherto-unknown type of bacterium (group of clones, 3 allelic variants) showing less than 86% similarity with known taxa. Its phylogenetic assignment to bacterial divisions or lineages was also unsuccessful despite of the RDP has classified it belonging to OD1 uncultured Candidate Division. The 2nd phylotype was less remarkable and still dubious in terms of contamination. It was presented by just one clone and showed 93% similarity with Janthinobacterium sp of Oxalobacteraceae (Beta-Proteobacteria) - well-known ‘water-loving’ bacteria. No archaea were detected in lake water frozen samples. Thus, the unidentified and unclassified bacterial w123-10 phylotype for the first time discovered in the uppermost water layer in subglacial Lake Vostok might represent ingenious cell populations in the lake, making the life in the lake less elusive. The proof may come (as well as novel phylotype discoveries) with farther analyses (e.g., sample screening with w123-10-specific primers, 16S rRNA v4 region amplicon sequencing) of existing and newly requested moon-shape samples of borehole-frozen water which are on a way to laboratories. We are deeply grateful to Jean Robert Petit and Jean Martins, UJF-CNRS, Grenoble (France) for assistance in conducting some analyses.

Structural locus of transmucosal albumin efflux in canine ileum. A fluorescent study.

PubMed

Granger, D N; Cook, B H; Taylor, A E

1976-12-01

This study demonstrates the effects of elevated intestinal venous pressure on the intestinal tissue spaces and the histological locus of the transmucosal albumin flux under such conditions. The authors were able to localize albumin in the tissues using an Evans blue-albumin fluorescence technique. This technique makes use of the fluorescence properties and albumin affinity of Evans blue dye (T-1824). Evans blue dye has a high affinity for albumin and emits a red-orange fluorescence at a wavelength of 720 nm. Evans blue was mixed with a solution of bovine serum albumin at concentrations that yield negligible amounts of free dye. Control ileal samples were obtained in order to visualize the natural tissue morphology and fluorescence. The Evans blue-albumin solution was injected and tissue samples were obtained 15 and 60 min postinjection, then venous outflow was occluded and after 15 and 60 min the tissues were sampled. Each sample was immediately frozen, freeze dried, embedded in paraffin, and 7-mu sections were made. The Evans blue-albumin was demonstrated histologically with a fluorescence microscope. No leakage sites were apparent at normal venous pressures. However, after elevation of venous pressure, Evans blue-albumin was observed in the interepithelial and/or intraepithelial spaces of villus tips, but no Evans blue-albumin was observed either between or within the epithelial cells of the crypts, or within the tubular crypt lumina. These results indicate that at elevated venous pressures, the transmucosal albumin flux occurs exclusively at the villus tip region, suggesting a great vulnerability of the cells found in this region to elevations in tissue pressure as compared to the crypt epithelial cells.

In vitro autoradiography of carcinoembryonic antigen in tissue from patients with colorectal cancer using multifunctional antibody TF2 and 67/68Ga-labeled haptens by pretargeting

PubMed Central

Hall, Håkan; Velikyan, Irina; Blom, Elisabeth; Ulin, Johan; Monazzam, Azita; Påhlman, Lars; Micke, Patrick; Wanders, Alkwin; McBride, William; Goldenberg, David M.; Långström, Bengt

2012-01-01

The carcinoembryonic antigen (CEA) was visualized in vitro in tissue from patients with colorectal cancer with trivalent bispecific antibody TF2 and two hapten molecules, [67/68Ga]Ga-IMP461 and [67/68Ga]Ga-IMP485 by means of pretargeting. Colorectal cancer tissue samples obtained from surgery at Uppsala University Hospital, were frozen fresh and cryosectioned. The two hapten molecules comprising 1,4,7-triazacyclononanetriacetic acid chelate moiety (NOTA) were labeled with 67Ga or 68Ga. The autoradiography was conducted by incubating the tissue samples with the bispecific antibody TF2, followed by washing and incubation with one of the radiolabeled hapten molecules. After washing, drying and exposure to phosphor imager plates, the autoradiograms were analyzed and compared to standard histochemistry (hematoxylin-eosin). Pronounced binding was found in the tissue from colorectal cancer using the bispecific antibody TF2 and either of the haptens [67/68Ga]Ga-IMP461 and [67/68Ga]Ga-IMP485. Distinct binding was also detected in the epithelium of most samples of neighboring tissue, taken at a minimum of 10 cm from the site of the tumor. It is concluded that pretargeting CEA with the bispecific antibody TF2 followed by the addition of 67/68Ga-labeled hapten is extremely sensitive for visualizing this marker for colorectal cancer. This methodology is therefore a very specific complement to other histochemical techniques in the diagnosis of biopsies or in samples taken from surgery. Use of the pretargeting technique in vivo may also be an advance in diagnosing patients with colorectal cancer, either using 67Ga and SPECT or 68Ga and PET. PMID:23133809

Extraction of high-quality DNA from ethanol-preserved tropical plant tissues.

PubMed

Bressan, Eduardo A; Rossi, Mônica L; Gerald, Lee T S; Figueira, Antonio

2014-04-24

Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species. Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA. Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue.

Extraction of high-quality DNA from ethanol-preserved tropical plant tissues

PubMed Central

2014-01-01

Background Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species. Results Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA. Conclusions Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue. PMID:24761774

A surrogate analyte-based liquid chromatography-tandem mass spectrometry method for the determination of endogenous cyclic nucleotides in rat brain.

PubMed

Chen, Jie; Tabatabaei, Ali; Zook, Doug; Wang, Yan; Danks, Anne; Stauber, Kathe

2017-11-30

A robust high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable isotopically labeled 3',5'-cyclic adenosine- 13 C 5 monophosphate ( 13 C 5 -cAMP) and 3',5'-cyclic guanosine- 13 C, 15 N 2 monophosphate ( 13 C 15 N 2 -cGMP) were used as surrogate analytes to measure endogenous 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Pre-weighed frozen rat brain samples were rapidly homogenized in 0.4M perchloric acid at a ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were analyzed using negative ion mode electrospray ionization LC-MS/MS. The calibration curves for both analytes ranged from 5 to 2000ng/g and showed excellent linearity (r 2 >0.996). Relative surrogate analyte-to-analyte LC-MS/MS responses were determined to correct concentrations derived from the surrogate curves. The intra-run precision (CV%) for 13 C 5 -cAMP and 13 C 15 N 2 -cGMP was below 6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively. The intra-run accuracy (Dev%) for 13 C 5 -cAMP and 13 C 15 N 2 -cGMP was <11.9% and 10.3%, respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover. Acceptable frozen storage, freeze/thaw, benchtop, processed sample and autosampler stability were shown in brain sample homogenates as well as post-processed samples. The method was found to be suitable for the analysis of rat brain tissue cAMP and cGMP levels in preclinical biomarker development studies. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Association between Propionibacterium acnes and frozen shoulder: a pilot study.

PubMed

Bunker, Tim D; Boyd, Matthew; Gallacher, Sian; Auckland, Cressida R; Kitson, Jeff; Smith, Chris D

2014-10-01

Frozen shoulder has not previously been shown to be associated with infection. The present study set out to confirm the null hypothesis that there is no relationship between infection and frozen shoulder using two modern scientific methods, extended culture and polymerase chain reaction (PCR) for bacterial nucleic acids. A prospective cohort of 10 patients undergoing arthroscopic release for stage II idiopathic frozen shoulder had two biopsies of tissue taken from the affected shoulder joint capsule at the time of surgery, along with control biopsies of subdermal fat. The biopsies and controls were examined with extended culture and PCR for microbial nucleic acid. Eight of the 10 patients had positive findings on extended culture in their shoulder capsule and, in six of these, Propionibacterium acnes was present. The findings mean that we must reject the null hypothesis that there is no relationship between infection and frozen shoulder. More studies are urgently needed to confirm or refute these findings. If they are confirmed, this could potentially lead to new and effective treatments for this common, painful and disabling condition. Could P. acnes be the Helicobacter of frozen shoulder?

Histological methods to determine blood flow distribution with fluorescent microspheres.

PubMed

Luchtel, D L; Boykin, J C; Bernard, S L; Glenny, R W

1998-11-01

We evaluated several histological methods and determined their advantages and disadvantages for histological studies of tissues and organs perfused with fluorescent microspheres. Microspheres retained their fluorescence in 7-10 microm serial sections with a change in the antimedium from toluene when samples were fixed in formalin and embedded in paraffin. Several antimedia allowed both wax infiltration of tissue and preservation of microsphere fluorescence. Histoclear II was the best substitute for toluene. When samples were fixed in formalin and embedded in glycol methacrylate, thinner (3-5 microm) sections provided greater histological detail but had fewer microspheres per section. Air dried lung tissue followed by Vibratome sectioning provided thick sections (100 microm) that facilitated rapid survey of large volumes of tissue for microspheres but limited histological detail, and the air drying procedure was restricted to lung tissue. Samples fixed in formalin followed by Vibratome sectioning of unembedded tissue provided better histological detail of lung tissue and was also useful for other organs. These sections were more difficult to handle and to mount on slides compared to air dried tissue, whereas fixed tissue embedded in gelatin provided better tissue support for Vibratome sectioning. Rapid freezing followed by cryo-microtome sectioning resulted in frozen sections that were relatively difficult to handle compared to embedded or unembedded tissue; they also deteriorated relatively rapidly with time. Paraffin sections were stained with hematoxylin and eosin or with aqueous methyl green, although tissue autofluorescence by itself was usually sufficient to identify histological features. Methacrylate sections quenched tissue autofluorescence, and Lee's stain or Richardson's stain were used for staining sections. Toluene based mountants such as Cytoseal quenched fluorescence, particularly the red fluorescent microspheres. Aqueous based mountants such as Aquamount, Crystal/Mount, Fluoromount-G were substituted, although such preparations were not as permanent as Cytoseal mounted coverglasses and tended to cause fading of stained sections.

Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)*

PubMed Central

Bader, Sabine; Zajac, Magdalena; Friess, Thomas; Ruge, Elisabeth; Rieder, Natascha; Gierke, Berthold; Heubach, Yvonne; Thomas, Marlene; Pawlak, Michael

2015-01-01

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues. PMID:26106084

Microbiological quality of five potato products obtained at retail markets.

PubMed Central

Duran, A P; Swartzentruber, A; Lanier, J M; Wentz, B A; Schwab, A H; Barnard, R J; Read, R B

1982-01-01

The microbiological quality of frozen hash brown potatoes, dried hash brown potatoes with onions, frozen french fried potatoes, dried instant mashed potatoes, and potato salad was determined by a national sampling at the retail level. A wide range of results was obtained, with most sampling units of each products having excellent microbiological quality. Geometric mean aerobic plate counts were as follows: dried hash brown potatoes, 270/g; frozen hash brown potatoes with onions, 580/g; frozen french fried potatoes 78/g; dried instant mashed potatoes, 1.1 x 10(3)/g; and potato salad, 3.6 x 10(3)/g. Mean values of coliforms, Escherichia coli, and Staphylococcus aureus were less than 10/g. PMID:6758695

An improved cryo-FIB method for fabrication of frozen hydrated lamella.

PubMed

Zhang, Jianguo; Ji, Gang; Huang, Xiaojun; Xu, Wei; Sun, Fei

2016-05-01

Cryo-electron tomography (cryo-ET) provides great insights into the ultrastructure of cells and tissues in their native state and provides a promising way to study the in situ 3D structures of macromolecular complexes. However, this technique has been limited on the very thin specimen, which is not applicable for most cells and tissues. Besides cryo-sectioning approach, cryo focused ion beam (cryo-FIB) appeared recently to achieve 'artifact-free' thin frozen hydrated lamella via fabrication. Considering that the current cryo-FIB methods need modified holders or cartridges, here, with a "D-shaped" molybdenum grid and a specific shutter system, we developed a simple cryo-FIB approach for thin frozen hydrated lamella fabrication, which fits both standard transmission cryo-electron microscopes with side-entry cryo-holders and state-of-the-art ones with AutoGrids. Our approach will expand the usage of cryo-FIB approach in many labs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A SIMPLE FREEZE-FRACTURE REPLICATION METHOD FOR ELECTRON MICROSCOPY

PubMed Central

Bullivant, Stanley; Ames, Adelbert

1966-01-01

A simple method to achieve results similar to the freeze-etching technique of Moor et al. (1961) is described. The frozen tissue is cut under liquid nitrogen with a razor blade outside the evaporator rather than inside with a cooled microtome. The conditions of the experiment do not favor sublimation, and it is proposed that the structure of the replica be explained by local faults in the cleavage plane which leaves structures, such as membranes, standing above the ice. Micrographs of replicas of glycerol-protected frozen small intestine of mouse prepared by the method are presented and the structural details they show are discussed. The problem of vapor-deposited contamination is discussed. It is concluded that this is a practical method for obtaining electron micrographs that are relatively free of artifact, and that further improvements may be expected from the use of rapidly frozen fresh tissue and a clean vacuum system, possibly of the ion-pumped type. PMID:5962938

Influence of the freezing method on the changes that occur in grape samples after frozen storage.

PubMed

Santesteban, Luis G; Miranda, Carlos; Royo, José B

2013-09-01

Sample freezing is frequently used in oenological laboratories as a compromise solution to increase the number of samples that can be analysed, despite the fact that some grape characteristics are known to change after frozen storage. However, freezing is usually performed using standard freezers, which provide a slow freezing. The aim of this work was to evaluate whether blast freezing would decrease the impact of standard freezing on grape composition. Grape quality parameters were assessed in fresh and in frozen stored samples that had been frozen using three different procedures: standard freezing and blast freezing using either a blast freezer or an ultra-freezer. The implications of frozen storage in grape samples reported in earlier research were observed for the three freezing methods evaluated. Although blast freezing improved repeatability for the most problematic parameters (tartaric acidity, TarA; total phenolics, TP), the improvement was not important from a practical point of view. However, TarA and TP were relatively repeatable among the three freezing procedures, which suggests that freezing had an effect on these parameters independently of the method used . According to our results, the salification potential of the must is probably implied in the changes observed for TarA, whereas for TP the precipitation of protoanthocyanins after association with cell wall material is hypothesized to cause the lack of repeatability between fresh and frozen grapes. Blast freezing would not imply a great improvement if implemented in oenological laboratories, at least for the parameters included in this study. © 2013 Society of Chemical Industry.

Whole slide imaging of unstained tissue using lensfree microscopy

NASA Astrophysics Data System (ADS)

Morel, Sophie Nhu An; Hervé, Lionel; Bordy, Thomas; Cioni, Olivier; Delon, Antoine; Fromentin, Catherine; Dinten, Jean-Marc; Allier, Cédric

2016-04-01

Pathologist examination of tissue slides provides insightful information about a patient's disease. Traditional analysis of tissue slides is performed under a binocular microscope, which requires staining of the sample and delays the examination. We present a simple cost-effective lensfree imaging method to record 2-4μm resolution wide-field (10 mm2 to 6 cm2) images of unstained tissue slides. The sample processing time is reduced as there is no need for staining. A wide field of view (10 mm2) lensfree hologram is recorded in a single shot and the image is reconstructed in 2s providing a very fast acquisition chain. The acquisition is multispectral, i.e. multiple holograms are recorded simultaneously at three different wavelengths, and a dedicated holographic reconstruction algorithm is used to retrieve both amplitude and phase. Whole tissue slides imaging is obtained by recording 130 holograms with X-Y translation stages and by computing the mosaic of a 25 x 25 mm2 reconstructed image. The reconstructed phase provides a phase-contrast-like image of the unstained specimen, revealing structures of healthy and diseased tissue. Slides from various organs can be reconstructed, e.g. lung, colon, ganglion, etc. To our knowledge, our method is the first technique that enables fast wide-field lensfree imaging of such unlabeled dense samples. This technique is much cheaper and compact than a conventional phase contrast microscope and could be made portable. In sum, we present a new methodology that could quickly provide useful information when a rapid diagnosis is needed, such as tumor margin identification on frozen section biopsies during surgery.

Aberrant Expression of PIWIL1 and PIWIL2 and Their Clinical Significance in Ductal Breast Carcinoma.

PubMed

Litwin, Monika; Szczepańska-Buda, Anna; Michałowska, Dagmara; Grzegrzółka, Jędrzej; Piotrowska, Aleksandra; Gomułkiewicz, Agnieszka; Wojnar, Andrzej; Dzięgiel, Piotr; Witkiewicz, Wojciech

2018-04-01

P-Element-induced wimpy testis (PIWI) proteins in complex with PIWI-interacting RNA (piRNA) are involved in epigenetic regulation of gene expression in germline cells. Aberrant expression of piRNA and PIWI proteins have been identified in various types of tumour cells. The aim of this study was to evaluate the expression profiles of PIWI-like protein-1, -2 (PIWIL1 and PIWIL2), their immunohistochemical (IHC) characteristics in ductal breast cancer, and determine their correlation with clinicopathological parameters of this type of cancer. Material for IHC studies comprised of 101 invasive ductal carcinoma (IDC) cases and 31 mastopathy tissues. Frozen fragments of paired tissue specimens (tumour and adjacent non-malignant breast tissue) taken from 55 patients with IDC and 18 samples of mastopathy were used for molecular studies using real-time polymerase chain reaction (RT-PCR). A statistically significantly higher level of PIWIL1 and PIWIL2 was found in IDC compared to mastopathy samples (p≤0.0001). Increased expression of PIWIL1 was correlated with increased PIWIL2 expression in breast cancer tissue. Surprisingly, PIWIL1 mRNA was detected only in cancer and mastopathy, but was not found in most normal breast tissues, although it is noteworthy that the PIWIL2 mRNA level was statistically significantly lower in mastopathy and IDC samples compared to normal breast tissues. Our results affirm the hypothesis that reactivation of PIWI expression in various caner types is crucial for cancer development. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Urine phenobarbital drug screening: potential use for compliance assessment in neonates.

PubMed

Guillet, Ronnie; Kwon, Jennifer M; Chen, Sixaio; McDermott, Michael P

2012-02-01

This study was done to determine if urine phenobarbital measurements provide a reliable indicator of presence of the drug in neonates. Urine was collected from neonates treated with phenobarbital for clinical indications within 4 to 6 hours of clinically indicated collection of serum phenobarbital levels. Urine samples were also collected from control neonates not treated with phenobarbital. One aliquot was assayed fresh, another frozen at -30°C and assayed 1 to 3 months later. Phenobarbital was assayed using the ONLINE TDM Roche/Hitachi automated clinical chemistry analyzer. Serum and urine concentrations were compared as were fresh and frozen urine measurements. Serum phenobarbital ranged from 5.6 to 52.7 μg/mL. Matched urine samples were 56.6 ± 12.5% of the serum level. Frozen samples were 98.3 ± 8.0% of the fresh samples. Urine phenobarbital concentrations, either fresh or frozen, can be used in neonates as a noninvasive estimate of drug levels.

Bacteria-killing ability of fresh blood plasma compared to frozen blood plasma

DOE PAGES

Jacobs, Anne C.; Fair, Jeanne Marie

2015-10-09

In recent years, the bacteria-killing assay (BKA) has become a popular technique among ecoimmunologists. New variations of that assay allow researchers to use smaller volumes of blood, an important consideration for those working on small-bodied animals. However, this version of the assay requires access to a lab with a nanodrop spectrophotometer, something that may not be available in the field. One possible solution is to freeze plasma for transport; however, this assumes that frozen plasma samples will give comparable results to fresh ones. Here, we tested this assumption using plasma samples from three species of birds: chickens (Gallus gallus), ash-throatedmore » flycatchers (Myiarchus cinerascens), and western bluebirds (Sialia mexicana). Chicken plasma samples lost most or all of their bacterial killing ability after freezing. This did not happen in flycatchers and bluebirds; however, frozen plasma did not produce results comparable to those obtained using fresh plasma. Finally, we caution researchers using the BKA to use fresh samples whenever possible, and to validate the use of frozen samples on a species-by-species basis.« less

Bacteria-killing ability of fresh blood plasma compared to frozen blood plasma.

PubMed

Jacobs, Anne C; Fair, Jeanne M

2016-01-01

In recent years, the bacteria-killing assay (BKA) has become a popular technique among ecoimmunologists. New variations of that assay allow researchers to use smaller volumes of blood, an important consideration for those working on small-bodied animals. However, this version of the assay requires access to a lab with a nanodrop spectrophotometer, something that may not be available in the field. One possible solution is to freeze plasma for transport; however, this assumes that frozen plasma samples will give comparable results to fresh ones. We tested this assumption using plasma samples from three species of birds: chickens (Gallus gallus), ash-throated flycatchers (Myiarchus cinerascens), and western bluebirds (Sialia mexicana). Chicken plasma samples lost most or all of their bacterial killing ability after freezing. This did not happen in flycatchers and bluebirds; however, frozen plasma did not produce results comparable to those obtained using fresh plasma. We caution researchers using the BKA to use fresh samples whenever possible, and to validate the use of frozen samples on a species-by-species basis. Copyright © 2015 Elsevier Inc. All rights reserved.

Bacteria-killing ability of fresh blood plasma compared to frozen blood plasma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacobs, Anne C.; Fair, Jeanne Marie

In recent years, the bacteria-killing assay (BKA) has become a popular technique among ecoimmunologists. New variations of that assay allow researchers to use smaller volumes of blood, an important consideration for those working on small-bodied animals. However, this version of the assay requires access to a lab with a nanodrop spectrophotometer, something that may not be available in the field. One possible solution is to freeze plasma for transport; however, this assumes that frozen plasma samples will give comparable results to fresh ones. Here, we tested this assumption using plasma samples from three species of birds: chickens (Gallus gallus), ash-throatedmore » flycatchers (Myiarchus cinerascens), and western bluebirds (Sialia mexicana). Chicken plasma samples lost most or all of their bacterial killing ability after freezing. This did not happen in flycatchers and bluebirds; however, frozen plasma did not produce results comparable to those obtained using fresh plasma. Finally, we caution researchers using the BKA to use fresh samples whenever possible, and to validate the use of frozen samples on a species-by-species basis.« less

MicroRNA Expression in Formalin-fixed Paraffin-embedded Cancer Tissue: Identifying Reference MicroRNAs and Variability.

PubMed

Boisen, Mogens Karsbøl; Dehlendorff, Christian; Linnemann, Dorte; Schultz, Nicolai Aagaard; Jensen, Benny Vittrup; Høgdall, Estrid Vilma Solyom; Johansen, Julia Sidenius

2015-12-29

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 of miRNA expression in FFPE tissue samples from patients with colorectal (CRC) and pancreatic (PC) cancer and to quantify the variability associated with sample age and fixation. High-throughput miRNA profiling results from 203 CRC and 256 PC FFPE samples as well as from 37 paired frozen/FFPE samples from nine other CRC tumors (methodological samples) were used. Candidate reference miRNAs were identified by their correlation with global mean expression. The stability of reference genes was analyzed according to published methods. The association between sample age and global mean miRNA expression was tested using linear regression. Variability was described using correlation coefficients and linear mixed effects models. Normalization effects were determined by changes in standard deviation and by hierarchical clustering. We created lists of 20 miRNAs with the best correlation to global mean expression in each cancer type. Nine of these miRNAs were present in both lists, and miR-103a-3p was the most stable reference miRNA for both CRC and PC FFPE tissue. The optimal number of reference miRNAs was 4 in CRC and 10 in PC. Sample age had a significant effect on global miRNA expression in PC (50% reduction over 20 years) but not in CRC. Formalin fixation for 2-6 days decreased miRNA expression 30-65%. Normalization using global mean expression reduced variability for technical and biological replicates while normalization using the expression of the identified reference miRNAs reduced variability only for biological replicates. Normalization only had a minor impact on clustering results. We identified suitable reference miRNAs for future miRNA expression experiments using CRC- and PC FFPE tissue samples. Formalin fixation decreased miRNA expression considerably, while the effect of increasing sample age was estimated to be negligible in a clinical setting.

Relative IGF-1 and IGF-2 gene expression in maternal and fetal tissues from diabetic swine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolverton, C.K.; Leaman, D.W.; White, M.E.

1990-02-26

Fourteen pregnant, crossbred gilts were utilized in this study. Seven gilts were injected with alloxan (50 mg/kg) at day 75 of gestation to induce diabetes. Gilts underwent caesarean section on day 105 of gestation. Samples were collected from maternal skeletal muscle, adipose tissue, uterus and endometrium; and from fetal skeletal muscle, adipose tissue, placenta, liver, lung, kidney, heart, brain and spleen. Tissues were frozen in liquid nitrogen for later analysis of IGF-1 and IGF-2 gene expression. Samples were pooled and total RNA was isolated using the guanidine isothiocynate method. Total mRNA was analyzed by dot blot hybridization. Blots were probedmore » with {sup 32}P-cDNA for porcine IGF-1 and rat IGF-2. IGF-1 gene expression in maternal tissues was unaffected by diabetes. Maternal diabetes increased IGF-2 mRNA in maternal adipose tissue but exhibited no effect in muscle or uterus. Expression of IGF-2 by maternal endometrium was decreased by diabetes. Maternal diabetes induced an increase in IGF-1 gene expression in muscle and placenta while causing an increase in IGF-2 expression in fetal liver and placenta. IGF-2 mRNA was lower in lung from fetuses of diabetic mothers than in controls. These results suggest that maternal diabetes alters IGF-1 and IGF-2 gene expression in specific tissues and differential regulation of these genes appears to exist in the mother and developing fetus.« less

Stimulation Induced Changes in Frog Neuromuscular Junctions: A Quantitative Ultrastructural Comparison of Rapid-Frozen and Chemically Fixed Nerve Terminals

DTIC Science & Technology

1984-03-06

study was conducted to determine the presynaptic morphological changes due to neural activity in rapidly stimulated neuromuscular junctions...Control preparations were unstimulated and preserved either by chemical fixation or rapid-freezing. This study provides evidence that most of the...tissue. The rapid-frozen preparations in the present study showed, in addition, that rapid stimulation produces an increase in synaptic vesicle

Freezing-thawing and sub-sampling influence the marination performance of chicken breast meat.

PubMed

Bowker, B; Zhuang, H

2017-09-01

Vacuum-tumbling marination is often used to improve the yield and quality of whole or portioned broiler breast fillets. The relationship between the marination performance of whole Pectoralis major muscles and breast fillet sub-samples is not well understood. The objective was to determine the effects of sub-sampling and freezing-thawing on the marination performance and cook loss of broiler breast meat. Paired right and left breast fillets were marinated as whole fillets or sub-samples (cranial and mid-caudal portions). Samples were marinated at 48 h postmortem (fresh) or stored at -20°C and then thawed prior to marination (frozen-thawed). Samples were vacuum-tumbled in 20% wt/wt brine (5% NaCl, 3% STP) and weighed pre-marination, during marination (15, 30, and 45 min), and 24 h post-marination. Samples were then cooked to 75°C for determination of cook loss. Marinade uptake was greater in caudal sub-samples than intact fillets and cranial sub-samples after 15 min of marination (P < 0.0001). After 30 min, marinade uptake was greater in caudal sub-samples and intact fillets than cranial sub-samples (P < 0.05). After 45 min, marinade uptake for fresh samples was greatest in intact fillets and lowest in cranial sub-samples. For frozen-thawed samples, marinade uptake at 45 min was greater in caudal sub-samples and intact fillets than cranial sub-samples (P < 0.0001). Marinade uptake in sub-samples at 30 min was greater in frozen-thawed versus fresh fillets (P < 0.05). Differences in marinade retention were not observed. Cook loss was similar between fresh and frozen-thawed samples but was greater in sub-samples compared to intact fillets (P < 0.0001). Correlations between marinade uptake in intact fillets and cranial sub-samples were greater in fresh (r = 0.64 to 0.78) than frozen-thawed samples (r = 0.39 to 0.59). Correlations between marinade uptake in intact fillets and caudal sub-samples were greater in frozen-thawed (r = 0.79 to 0.82) than fresh samples (r = 0.46 to 0.63). Data suggest that the relationships between marination performance of whole breast fillets and fillet sub-samples are dependent upon prior sample handling and intra-fillet sampling location. Published by Oxford University Press on behalf of Poultry Science Association 2017.

Cryosurgery with pulsed electric fields.

PubMed

Daniels, Charlotte S; Rubinsky, Boris

2011-01-01

This study explores the hypothesis that combining the minimally invasive surgical techniques of cryosurgery and pulsed electric fields will eliminate some of the major disadvantages of these techniques while retaining their advantages. Cryosurgery, tissue ablation by freezing, is a well-established minimally invasive surgical technique. One disadvantage of cryosurgery concerns the mechanism of cell death; cells at high subzero temperature on the outer rim of the frozen lesion can survive. Pulsed electric fields (PEF) are another minimally invasive surgical technique in which high strength and very rapid electric pulses are delivered across cells to permeabilize the cell membrane for applications such as gene delivery, electrochemotherapy and irreversible electroporation. The very short time scale of the electric pulses is disadvantageous because it does not facilitate real time control over the procedure. We hypothesize that applying the electric pulses during the cryosurgical procedure in such a way that the electric field vector is parallel to the heat flux vector will have the effect of confining the electric fields to the frozen/cold region of tissue, thereby ablating the cells that survive freezing while facilitating controlled use of the PEF in the cold confined region. A finite element analysis of the electric field and heat conduction equations during simultaneous tissue treatment with cryosurgery and PEF (cryosurgery/PEF) was used to study the effect of tissue freezing on electric fields. The study yielded motivating results. Because of decreased electrical conductivity in the frozen/cooled tissue, it experienced temperature induced magnified electric fields in comparison to PEF delivered to the unfrozen tissue control. This suggests that freezing/cooling confines and magnifies the electric fields to those regions; a targeting capability unattainable in traditional PEF. This analysis shows how temperature induced magnified and focused PEFs could be used to ablate cells in the high subzero freezing region of a cryosurgical lesion.

Iodine-131 dose-dependent gene expression: alterations in both normal and tumour thyroid tissues of post-Chernobyl thyroid cancers.

PubMed

Abend, M; Pfeiffer, R M; Ruf, C; Hatch, M; Bogdanova, T I; Tronko, M D; Hartmann, J; Meineke, V; Mabuchi, K; Brenner, A V

2013-10-15

A strong, consistent association between childhood irradiation and subsequent thyroid cancer provides an excellent model for studying radiation carcinogenesis. We evaluated gene expression in 63 paired RNA specimens from frozen normal and tumour thyroid tissues with individual iodine-131 (I-131) doses (0.008-8.6 Gy, no unirradiated controls) received from Chernobyl fallout during childhood (Ukrainian-American cohort). Approximately half of these randomly selected samples (32 tumour/normal tissue RNA specimens) were hybridised on 64 whole-genome microarrays (Agilent, 4 × 44 K). Associations between I-131 dose and gene expression were assessed separately in normal and tumour tissues using Kruskal-Wallis and linear trend tests. Of 155 genes significantly associated with I-131 after Bonferroni correction and with ≥2-fold increase per dose category, we selected 95 genes. On the remaining 31 RNA samples these genes were used for validation purposes using qRT-PCR. Expression of eight genes (ABCC3, C1orf9, C6orf62, FGFR1OP2, HEY2, NDOR1, STAT3, and UCP3) in normal tissue and six genes (ANKRD46, CD47, HNRNPH1, NDOR1, SCEL, and SERPINA1) in tumour tissue was significantly associated with I-131. PANTHER/DAVID pathway analyses demonstrated significant over-representation of genes coding for nucleic acid binding in normal and tumour tissues, and for p53, EGF, and FGF signalling pathways in tumour tissue. The multistep process of radiation carcinogenesis begins in histologically normal thyroid tissue and may involve dose-dependent gene expression changes.

Frozen yogurt with added inulin and isomalt.

PubMed

Isik, U; Boyacioglu, D; Capanoglu, E; Erdil, D Nilufer

2011-04-01

The objective of this study was to produce a frozen yogurt containing low fat and no added sugar. Samples containing 5% polydextrose, 0.065% aspartame and acesulfame-K mixture, and different levels of inulin and isomalt (5.0, 6.5, and 8.0%) were produced at pilot scale and analyzed for their physical and chemical properties including proximate composition, viscosity, acidity, overrun, melting rate, heat shock stability, as well as sensory characteristics, and viability of lactic acid bacteria. With the addition of inulin and isomalt, viscosity increased by 19 to 52% compared with that of sample B (reduced-fat control). The average calorie values of samples substituted with sweeteners were about 43% lower than that of original sample. Low-calorie frozen yogurt samples melted about 33 to 48% slower than the reduced-fat control sample at 45 min. Based on quantitative descriptive profile test results, statistically significant differences among products were observed for hardness, iciness, foamy melting, whey separation, and sweetness characteristics. The results of principal component analysis showed that the sensory properties of the sample containing 6.5% inulin and 6.5% isomalt were similar to those of control. Lactic acid bacteria counts of frozen yogurt were found to be between 8.12 and 8.49 log values, 3 mo after the production. The overall results showed that it is possible to produce an attractive frozen yogurt product with the incorporation of inulin and isomalt with no added sugar and reduced fat. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Laser Capture and Single Cell Genotyping from Frozen Tissue Sections.

PubMed

Kroneis, Thomas; Ye, Jody; Gillespie, Kathleen

2016-01-01

There is an increasing requirement for genetic analysis of individual cells from tissue sections. This is particularly the case for analysis of tumor cells but is also a requirement for analysis of cells in pancreas from individuals with type 1 diabetes where there is evidence of viral infection or in the analysis of chimerism in pancreas; either post-transplant or as a result of feto-maternal cell transfer.This protocol describes a strategy to isolate cells using laser microdissection and to run a 17plex PCR to discriminate between cells of haplo-identical origin (i.e., fetal and maternal cells) in pancreas tissue but other robust DNA tests could be used. In short, snap-frozen tissues are cryo-sectioned and mounted onto membrane-coated slides. Target cells are harvested from the tissue sections by laser microdissection and pressure catapulting (LMPC) prior to DNA profiling. This is based on amplification of highly repetitive yet stably inherited loci (short tandem repeats, STR) as well as the amelogenin locus for sex determination and separation of PCR products by capillary electrophoresis.

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

PubMed Central

Bobik, Krzysztof; Dunlap, John R.; Burch-Smith, Tessa M.

2014-01-01

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality. PMID:25350384

Use of virtual slide system for quick frozen intra-operative telepathology diagnosis in Kyoto, Japan.

PubMed

Tsuchihashi, Yasunari; Takamatsu, Terumasa; Hashimoto, Yukimasa; Takashima, Tooru; Nakano, Kooji; Fujita, Setsuya

2008-07-15

We started to use virtual slide (VS) and virtual microscopy (VM) systems for quick frozen intra-operative telepathology diagnosis in Kyoto, Japan. In the system we used a digital slide scanner, VASSALO by CLARO Inc., and a broadband optic fibre provided by NTT West Japan Inc. with the best effort capacity of 100 Mbps. The client is the pathology laboratory of Yamashiro Public Hospital, one of the local centre hospitals located in the south of Kyoto Prefecture, where a full-time pathologist is not present. The client is connected by VPN to the telepathology centre of our institute located in central Kyoto. As a result of the recent 15 test cases of VS telepathology diagnosis, including cases judging negative or positive surgical margins, we could estimate the usefulness of VS in intra-operative remote diagnosis. The time required for the frozen section VS file making was found to be around 10 min when we use x10 objective and if the maximal dimension of the frozen sample is less than 20 mm. Good correct focus of VS images was attained in all cases and all the fields of each tissue specimen. Up to now the capacity of best effort B-band appears to be sufficient to attain diagnosis on time in intra-operation. Telepathology diagnosis was achieved within 5 minutes in most cases using VS viewer provided by CLARO Inc. The VS telepathology system was found to be superior to the conventional still image telepathology system using a robotic microscope since in the former we can observe much greater image information than in the latter in a certain limited time of intra-operation and in the much more efficient ways. In the near future VS telepathology will replace conventional still image telepathology with a robotic microscope even in quick frozen intra-operative diagnosis.

Use of virtual slide system for quick frozen intra-operative telepathology diagnosis in Kyoto, Japan

PubMed Central

Tsuchihashi, Yasunari; Takamatsu, Terumasa; Hashimoto, Yukimasa; Takashima, Tooru; Nakano, Kooji; Fujita, Setsuya

2008-01-01

We started to use virtual slide (VS) and virtual microscopy (VM) systems for quick frozen intra-operative telepathology diagnosis in Kyoto, Japan. In the system we used a digital slide scanner, VASSALO by CLARO Inc., and a broadband optic fibre provided by NTT West Japan Inc. with the best effort capacity of 100 Mbps. The client is the pathology laboratory of Yamashiro Public hospital, one of the local centre hospitals located in the south of Kyoto Prefecture, where a fulltime pathologist is not present. The client is connected by VPN to the telepathology centre of our institute located in central Kyoto. As a result of the recent 15 test cases of VS telepathology diagnosis, including cases judging negative or positive surgical margins, we could estimate the usefulness of VS in intra-operative remote diagnosis. The time required for the frozen section VS file making was found to be around 10 min when we use ×10 objective and if the maximal dimension of the frozen sample is less than 20 mm. Good correct focus of VS images was attained in all cases and all the fields of each tissue specimen. Up to now the capacity of best effort B-band appears to be sufficient to attain diagnosis on time in intra-operation. Telepathology diagnosis was achieved within 5 minutes in most cases using VS viewer provided by CLARO Inc. The VS telepathology system was found to be superior to the conventional still image telepathology system using a robotic microscope since in the former we can observe much greater image information than in the latter in a certain limited time of intra-operation and in the much more efficient ways. In the near future VS telepathology will replace conventional still image telepathology with a robotic microscope even in quick frozen intra-operative diagnosis. PMID:18673520

OMNY—A tOMography Nano crYo stage

NASA Astrophysics Data System (ADS)

Holler, M.; Raabe, J.; Diaz, A.; Guizar-Sicairos, M.; Wepf, R.; Odstrcil, M.; Shaik, F. R.; Panneels, V.; Menzel, A.; Sarafimov, B.; Maag, S.; Wang, X.; Thominet, V.; Walther, H.; Lachat, T.; Vitins, M.; Bunk, O.

2018-04-01

For many scientific questions gaining three-dimensional insight into a specimen can provide valuable information. We here present an instrument called "tOMography Nano crYo (OMNY)," dedicated to high resolution 3D scanning x-ray microscopy at cryogenic conditions via hard X-ray ptychography. Ptychography is a lens-less imaging method requiring accurate sample positioning. In OMNY, this in achieved via dedicated laser interferometry and closed-loop position control reaching sub-10 nm positioning accuracy. Cryogenic sample conditions are maintained via conductive cooling. 90 K can be reached when using liquid nitrogen as coolant, and 10 K is possible with liquid helium. A cryogenic sample-change mechanism permits measurements of cryogenically fixed specimens. We compare images obtained with OMNY with older measurements performed using a nitrogen gas cryo-jet of stained, epoxy-embedded retina tissue and of frozen-hydrated Chlamydomonas cells.

Volatile generation in bell peppers during frozen storage and thawing using selected ion flow tube mass spectrometry (SIFT-MS).

PubMed

Wampler, Brendan; Barringer, Sheryl A

2012-06-01

To determine volatile formation during storage and thawing, whole, pureed, blanched, and raw green and red bell peppers (Capsicum annuum) were frozen quickly or slowly then stored at -18 °C for up to 7 mo, with and without SnCl(2) addition during thawing. Headspace analysis was performed by a Selected Ion Flow Tube Mass Spectrometer (SIFT-MS). After blanching, (Z)-3-hexenal had a large significant decrease in concentration since it is a heat labile compound while most other volatiles did not change in concentration. The freezing process increased volatile levels in the puree only. Slow freeze peppers had higher levels of some LOX generated volatiles during storage than quick freeze. During frozen storage of blanched samples (E)-2-hexenal, (Z and E)-hexen-1-ol, and (E)-2-pentenal increased likely because of nonenzymatic autoxidation of fatty acids while other volatiles remained constant. In Raw Whole peppers, (Z)-3-hexenal, hexanal, and 2-pentylfuran were generated during storage likely because the LOX enzyme is still active during frozen storage. However, blanched samples had higher concentrations of (E)-2-hexenal, (Z and E)-hexen-1-ol, 1-penten-3-one, and (E)-2-heptenal because of enzymatic destruction of these volatiles in the raw samples. The levels of many of the volatiles in the raw samples, including (Z)-3-hexenal, (E)-2-hexenal, (Z and E)-hexen-1-ol, hexanal, (E)-2-pentenal, and 2-pentylfuran, appeared to peak around 34 d after freezing. Pureed samples had significantly higher levels of volatiles than the whole samples, and volatiles peaked earlier. Green bell pepper volatile levels were always higher than red bell pepper. Significantly higher volatile formation occurred during thawing than it did during frozen storage. Studying and monitoring the headspace volatiles with a SIFT-MS can give information that will help manufacturers better understand how the volatiles in bell peppers change during frozen storage. This will give valuable information to processors on how to minimize volatile changes during storage of frozen peppers. © 2012 Institute of Food Technologists®

Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins

PubMed Central

Chen, Yi; Fisher, Kate J.; Lloyd, Mark; Wood, Elizabeth R.; Coppola, Domenico; Siegel, Erin; Shibata, David; Chen, Yian A.; Koomen, John M.

2017-01-01

Quantitative evaluation of protein expression across multiple cancer-related signaling pathways (e.g. Wnt/β-catenin, TGF-β, receptor tyrosine kinases (RTK), MAP kinases, NF-κB, and apoptosis) in tumor tissues may enable the development of a molecular profile for each individual tumor that can aid in the selection of appropriate targeted cancer therapies. Here, we describe the development of a broadly applicable protocol to develop and implement quantitative mass spectrometry assays using cell line models and frozen tissue specimens from colon cancer patients. Cell lines are used to develop peptide-based assays for protein quantification, which are incorporated into a method based on SDS-PAGE protein fractionation, in-gel digestion, and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). This analytical platform is then applied to frozen tumor tissues. This protocol can be broadly applied to the study of human disease using multiplexed LC-MRM assays. PMID:28808993

Assessment of frozen storage duration effect on quality characteristics of various horse muscles.

PubMed

Seong, Pil Nam; Seo, Hyun Woo; Kim, Jin-Hyoung; Kang, Geun Ho; Cho, Soo-Hyun; Chae, Hyun Seok; Park, Beom Young; Van Ba, Hoa

2017-12-01

The study aimed at assessing the effects of frozen storage duration on quality characteristics, lipid oxidation and sensory quality of various horse muscles. Five representative muscles: longissimus dorsi (LD), gluteus medius (GM), semimembranosus (SM), biceps femoris (BF), and triceps brachii (TB) at 24 h post-mortem obtained from 28-mo-old Jeju female breed horses (n = 8) were used in the present investigation. The muscles were vacuum-packaged and frozen at -20°C for 120, 240, and 360 days. All the samples were analyzed for thawing and cooking losses, pH, Warner-Bratzler shear forces (WBSF), color traits, total volatile basic nitrogen (TVBN), thiobarbituric acid reactive substances (TBARS) and sensory traits. The muscle samples analyzed on day 0 of frozen storage (fresh, non-frozen) were used for comparison. Results revealed that thawing and cooking losses significantly (p<0.05) increased in all the muscles after 120 days and then remained unchanged up to 360 days of frozen storage. The TBARS and TVBN contents significantly increased as increasing frozen storage time up to 360 days (p<0.05). While, significant decreases in WBSF values were observed for all the muscles with increased frozen storage time (p<0.05). Frozen storage variously affected the color traits of the muscles for instance; the redness of LD, GM, and BF muscles showed a decreasing tendency during frozen storage while it was not changed in TB and SM muscles. Furthermore, the frozen storage did not produce detrimental effects on sensory quality as it did not cause flavor and juiciness defects whereas it partially improved the tenderness of all the muscles studied. Based on the results obtained from our work, it is concluded that frozen storage could be applied to increase the long-term shelf life of horsemeat while still retaining its sensory quality.

Effect of Irradiation on Tissue Penetration Depth of Doxorubicin after Pressurized Intra-Peritoneal Aerosol Chemotherapy (PIPAC) in a Novel Ex-Vivo Model.

PubMed

Khosrawipour, Veria; Giger-Pabst, Urs; Khosrawipour, Tanja; Pour, Yousef Hedayat; Diaz-Carballo, David; Förster, Eckart; Böse-Ribeiro, Hugo; Adamietz, Irenäus Anton; Zieren, Jürgen; Fakhrian, Khashayar

2016-01-01

This study was performed to assess the impact of irradiation on the tissue penetration depth of doxorubicin delivered during Pressurized Intra-Peritoneal Aerosol Chemotherapy (PIPAC). Fresh post mortem swine peritoneum was cut into 10 proportional sections. Except for 2 control samples, all received irradiation with 1, 2, 7 and 14 Gy, respectively. Four samples received PIPAC 15 minutes after irradiation and 4 other after 24 hours. Doxorubicin was aerosolized in an ex-vivo PIPAC model at 12 mmHg/36°C. In-tissue doxorubicin penetration was measured using fluorescence microscopy on frozen thin sections. Doxorubicin penetration after PIPAC (15 minutes after irradiation) was 476 ± 74 µm for the control sample, 450 ± 45µm after 1 Gy (p > 0.05), 438 ± 29 µm after 2 Gy (p > 0.05), 396 ± 32 µm after 7 Gy (p = 0.005) and 284 ± 57 after 14 Gy irradiation (p < 0.001). The doxorubicin penetration after PIPAC (24 hours after irradiation) was 428 ± 77 µm for the control sample, 393 ± 41 µm after 1 Gy (p > 0.05), 379 ± 56 µm after 2 Gy (p > 0.05), 352 ± 53 µm after 7 Gy (p = 0.008) and 345 ± 53 after 14 Gy irradiation (p = 0.001). Higher (fractional) radiation dose might reduce the tissue penetration depth of doxorubicin in our ex-vivo model. However, irradiation with lower (fractional) radiation dose does not affect the tissue penetration negatively. Further studies are warranted to investigate if irradiation can be used safely as chemopotenting agent for patients with peritoneal metastases treated with PIPAC.

Association between Propionibacterium acnes and frozen shoulder: a pilot study

PubMed Central

Bunker, Tim D; Gallacher, Sian; Auckland, Cressida R; Kitson, Jeff; Smith, Chris D

2014-01-01

Background Frozen shoulder has not previously been shown to be associated with infection. The present study set out to confirm the null hypothesis that there is no relationship between infection and frozen shoulder using two modern scientific methods, extended culture and polymerase chain reaction (PCR) for bacterial nucleic acids. Methods A prospective cohort of 10 patients undergoing arthroscopic release for stage II idiopathic frozen shoulder had two biopsies of tissue taken from the affected shoulder joint capsule at the time of surgery, along with control biopsies of subdermal fat. The biopsies and controls were examined with extended culture and PCR for microbial nucleic acid. Results Eight of the 10 patients had positive findings on extended culture in their shoulder capsule and, in six of these, Propionibacterium acnes was present. Conclusions The findings mean that we must reject the null hypothesis that there is no relationship between infection and frozen shoulder. More studies are urgently needed to confirm or refute these findings. If they are confirmed, this could potentially lead to new and effective treatments for this common, painful and disabling condition. Could P. acnes be the Helicobacter of frozen shoulder? PMID:27582943

Quantitative Method for Analysis of Hydrocodone, Hydromorphone and Norhydrocodone in Human Plasma by Liquid Chromatography-tandem Mass Spectrometry

DTIC Science & Technology

2013-03-01

ratio ranges obtained for the six standards. Twelve samples were analyzed to demonstrate the efficiency of the extraction procedure. Drug and internal...frozen (−70 ◦C). Refrigerated samples were tested after 2 months of storage ; frozen samples were tested for up to 1 year from stor- age date. The...freeze–thaw stability was evaluated by analyzing three subject samples with known drug concentrations and two quality control samples at concentrations

Inner ear tissue preservation by rapid freezing: Improving fixation by high-pressure freezing and hybrid methods

PubMed Central

Bullen, A.; Taylor, R.R.; Kachar, B.; Moores, C.; Fleck, R.A.; Forge, A.

2014-01-01

In the preservation of tissues in as ‘close to life’ state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40 μm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues. PMID:25016142

Testing an aflatoxin B1 gene signature in rat archival tissues.

PubMed

Merrick, B Alex; Auerbach, Scott S; Stockton, Patricia S; Foley, Julie F; Malarkey, David E; Sills, Robert C; Irwin, Richard D; Tice, Raymond R

2012-05-21

Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures.

Merkel cell carcinoma: histopathologic and prognostic features according to the immunohistochemical expression of Merkel cell polyomavirus large T antigen correlated with viral load.

PubMed

Leroux-Kozal, Valérie; Lévêque, Nicolas; Brodard, Véronique; Lesage, Candice; Dudez, Oriane; Makeieff, Marc; Kanagaratnam, Lukshe; Diebold, Marie-Danièle

2015-03-01

Merkel cell carcinoma (MCC) is a neuroendocrine skin malignancy frequently associated with Merkel cell polyomavirus (MCPyV), which is suspected to be oncogenic. In a series of MCC patients, we compared clinical, histopathologic, and prognostic features according to the expression of viral large T antigen (LTA) correlated with viral load. We evaluated the LTA expression by immunohistochemistry using CM2B4 antibody and quantified viral load by real-time polymerase chain reaction. We analyzed formalin-fixed, paraffin-embedded (FFPE) tissue samples (n = 36) and corresponding fresh-frozen biopsies when available (n = 12), of the primary tumor and/or metastasis from 24 patients. MCPyV was detected in 88% and 58% of MCC patients by real-time polymerase chain reaction and immunohistochemistry, respectively. The relevance of viral load measurements was demonstrated by the strong consistency of viral load level between FFPE and corresponding frozen tissues as well as between primary tumor and metastases. From FFPE samples, 2 MCC subgroups were distinguished based on a viral load threshold defined by the positivity of CM2B4 immunostaining. In the LTA-negative subgroup with no or low viral load (nonsignificant), tumor cells showed more anisokaryosis (P = .01), and a solar elastosis around the tumor was more frequently observed (P = .03). LTA-positive MCCs with significant viral load had a lower proliferation index (P = .03) and a longer survival of corresponding patients (P = .008). Depending on MCPyV involvement, 2 MCC subgroups can be distinguished on histopathologic criteria, and the CM2B4 antibody is able to differentiate them reliably. Furthermore, the presence of a significant viral load in tumors is predictive of better prognosis. Copyright © 2015 Elsevier Inc. All rights reserved.

The role of topically applied L-ascorbic acid in ex-vivo examination of burn-injured human skin

NASA Astrophysics Data System (ADS)

Pielesz, Anna; Biniaś, Dorota; Bobiński, Rafał; Sarna, Ewa; Paluch, Jadwiga; Waksmańska, Wioletta

2017-10-01

Wound treatment and healing is complex and is comprised of an elaborate set of processes including cellular, spectroscopic and biochemical ones as well as the ;reaction; of local tissue to thermal injury. Vitamin C as L-ascorbic acid (LA) prevents injurious effects of oxidants because it reduces reactive oxygen species to stable molecules, it becomes oxidized to the short-lived ascorbyl radical. As a result, antioxidant treatment may contribute to minimizing injury in burn patients. The aim of this study is to assess changes in molecular structure of collagen extracted from human epidermis burn wound scab during incubation of the epidermis in L-ascorbic acid solution. The study will be performed using FTIR and FT Raman spectroscopies. During this research it was observed that the intensity of Raman peaks increased where healing was being modified by LA. The intensity of the amide III band at 1247 cm- 1 relative to the intensity at 1326 cm- 1 was used to test tissue repair degree at the incision site. FTIR spectra were recorded from frozen specimens of serum modified by LA; an analysis of shifts in the amide I band position was conducted. The appearance of a new band for frozen samples modified by LA was observed around 1149-1220 cm- 1. The above conclusions confirmed the creation of hydrogen bonds between Nsbnd H stretch and Cdbnd O. Samples being incubated in solutions of L-ascorbic acid demonstrated the absence of electrophoretic bands of albumin. Alterations in the surface of the skin incubated in L-ascorbic acid were investigated with the use of Scanning Electron Microscopy (SEM). A decrease in external symptoms of burn injury was noted in the damaged epidermis incubated in L-ascorbic acid.

Differentiating cancerous from normal breast tissue by redox imaging

NASA Astrophysics Data System (ADS)

Xu, He N.; Tchou, Julia; Feng, Min; Zhao, Huaqing; Li, Lin Z.

2015-02-01

Abnormal metabolism can be a hallmark of cancer occurring early before detectable histological changes and may serve as an early detection biomarker. The current gold standard to establish breast cancer (BC) diagnosis is histological examination of biopsy. Previously we have found that pre-cancer and cancer tissues in animal models displayed abnormal mitochondrial redox state. Our technique of quantitatively measuring the mitochondrial redox state has the potential to be implemented as an early detection tool for cancer and may provide prognostic value. We therefore in this present study, investigated the feasibility of quantifying the redox state of tumor samples from 16 BC patients. Tumor tissue aliquots were collected from both normal and cancerous tissue from the affected cancer-bearing breasts of 16 female patients (5 TNBC, 9 ER+, 2 ER+/Her2+) shortly after surgical resection. All specimens were snap-frozen with liquid nitrogen on site and scanned later with the Chance redox scanner, i.e., the 3D cryogenic NADH/oxidized flavoprotein (Fp) fluorescence imager. Our preliminary results showed that both NADH and Fp (including FAD, i.e., flavin adenine dinucleotide) signals in the cancerous tissues roughly tripled to quadrupled those in the normal tissues (p<0.05) and the redox ratio Fp/(NADH+Fp) was about 27% higher in the cancerous tissues than in the normal ones (p<0.05). Our findings suggest that the redox state could differentiate between cancer and non-cancer breast tissues in human patients and this novel redox scanning procedure may assist in tissue diagnosis in freshly procured biopsy samples prior to tissue fixation. We are in the process of evaluating the prognostic value of the redox imaging indices for BC.

Oxidative status of human milk and its variations during cold storage.

PubMed

Miranda, María; Muriach, María; Almansa, Inmaculada; Jareño, Enrique; Bosch-Morell, Francisco; Romero, Francisco J; Silvestre, Dolores

2004-01-01

Breastfeeding and human milk are widely accepted as optimal for human infants' nutrition. Nowadays lifestyle often makes it difficult to maintain or even initiate human lactation. This situation is mostly related to the workload of women away from home. New approaches are needed to enable maternal lactation under these circumstances. Human breastmilk storage for differed use is one possibility. The aim of this study was to assess changes in glutathione peroxidase (GPx) activity and in the concentration of the lipid peroxidation marker, malondialdehyde (MDA), when human milk was kept refrigerated or frozen. Thirty-two human milk samples were assayed for GPx activity and MDA concentration. Samples were divided in three aliquot portions, the first to be immediately analysed, the second to be refrigerated at 4 degrees C and analysed 24 h thereafter, and the third to be frozen at -20 degrees C and assayed after 10 days. GPx activity was significantly decreased in refrigerated and in frozen milk, when compared to their control samples. MDA was increased only in refrigerated milk but not in frozen samples. Thus, freezing seems better than refrigeration in order to prevent lipid peroxidation in stored human milk samples.

Oxidative stability of red palm oils blended chicken nuggets during frozen storage

NASA Astrophysics Data System (ADS)

Kamaruzaman, Nurkhuzaiah; Babji, Abdul Salam

2014-09-01

The effects of red palm oils known as Naturally Vitamin Rich Oil (NVRO) on the lipid stability of chicken nuggets were determined. Lipid oxidation was analyzed during frozen storage (-18 °C) for up to 4 months. Thiobarbituric acid (TBA) values and peroxide value (PV) for all samples chicken nuggets increased throughout 3 months of frozen storage and then start to decrease thereafter. Chicken nuggets formulated with NVRO, NVRO-100 and NVRO-50 showed lower TBA values and PV compared to the samples prepared with chicken fat. However, among NVRO, there were not significantly different for most of the months. It was concluded that the utilization of red palm oils in chicken nuggets significantly reduced the oxidation of lipid, which was indicated by the PV throughout 4 months of frozen storage.

System for maintaining materials at freezer temperatures for shipping

DOEpatents

Schabron, John F [Laramie, WY; Sorini-Wong, Susan S [Laramie, WY

2007-08-28

At least one embodiment of the inventive technology relates to a frozen environmental sample temperature control system that comprises a frozen formulation having water in an amount from substantially 87% to 78% by weight of the formulation, and salt in an amount from substantially 13% to 22% by weight of the formulation, the system further including at least one container containing the frozen formulation; and a cooler having insulating material disposed between an outer wall and an inner surface that defines an inner chamber into which the at least one container and the at least one frozen environmental sample may be placed for storage and/or transport. Various embodiments may incorporate specific types of insulating material and/or adaptations to an inner surface of the cooler to enhance the insulation effected thereby.

Survival and energetic costs of repeated cold exposure in the Antarctic midge, Belgica antarctica: a comparison between frozen and supercooled larvae.

PubMed

Teets, Nicholas M; Kawarasaki, Yuta; Lee, Richard E; Denlinger, David L

2011-03-01

In this study, we examined the effects of repeated cold exposure (RCE) on the survival, energy content and stress protein expression of larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae). Additionally, we compared results between larvae that were frozen at -5°C in the presence of water during RCE and those that were supercooled at -5°C in a dry environment. Although >95% of larvae survived a single 12 h bout of freezing at -5°C, after five cycles of RCE survival of frozen larvae dropped below 70%. Meanwhile, the survival of control and supercooled larvae was unchanged, remaining around 90% for the duration of the study. At the tissue level, frozen larvae had higher rates of cell mortality in the midgut than control and supercooled larvae. Furthermore, larvae that were frozen during RCE experienced a dramatic reduction in energy reserves; after five cycles, frozen larvae had 25% less lipid, 30% less glycogen and nearly 40% less trehalose than supercooled larvae. Finally, larvae that were frozen during RCE had higher expression of hsp70 than those that were supercooled, indicating a higher degree of protein damage in the frozen group. Results were similar between larvae that had accumulated 60 h of freezing at -5°C over five cycles of RCE and those that were frozen continuously for 60 h, suggesting that the total time spent frozen determines the physiological response. Our results suggest that it is preferable, both from a survival and energetic standpoint, for larvae to seek dry microhabitats where they can avoid inoculative freezing and remain unfrozen during RCE.

Indication and method of frozen section in vaginal radical trachelectomy.

PubMed

Chênevert, Jacinthe; Têtu, Bernard; Plante, Marie; Roy, Michel; Renaud, Marie-Claude; Grégoire, Jean; Grondin, Katherine; Dubé, Valérie

2009-09-01

Vaginal radical trachelectomy (VRT) is a fertility-sparing surgical technique used as an alternative to radical hysterectomy in early stage cervical carcinoma. With the advent of VRT, preoperative evaluation of the surgical margin has become imperative, because if the tumor is found within 5 mm of the endocervical margin, additional surgical resection is required. In a study published earlier from our center, we came to the conclusion that a frozen section should be conducted only when a cancerous lesion is grossly visible, and that it could be omitted in normal-looking specimens or VRT with nonspecific lesions. Since then, 53 VRT have been performed in our center, and frozen sections were conducted according to these recommendations. Fifteen VRT were grossly normal, 24 had a nonspecific lesion and 14 showed a grossly visible lesion. Final margins were satisfactory on all 15 grossly normal specimens. Of the 24 VRT with nonspecific lesions, 2 cases for which no frozen section was performed had unsatisfactory final margins (<5 mm). Of the 14 VRT with grossly visible lesions, 3 cases were inadequately evaluated by frozen section due to sampling errors, which led to unsatisfactory final margin assessment. These results confirm that a frozen section can be omitted on normal looking VRT specimens, but contrary to results published earlier, we recommend that a frozen section be performed on all VRT with nonspecific lesions. As for VRT with a grossly visible lesion, frozen section evaluation is still warranted, and we recommend increasing the sampling to improve the adequacy of frozen sections.

Comparative performance of isolation methods using Preston broth, Bolton broth and their modifications for the detection of Campylobacter spp. from naturally contaminated fresh and frozen raw poultry meat.

PubMed

Seliwiorstow, T; De Zutter, L; Houf, K; Botteldoorn, N; Baré, J; Van Damme, I

2016-10-03

The performance of different isolation methods was evaluated for the detection of Campylobacter from naturally contaminated raw poultry meat. Therefore, fresh and frozen poultry meat samples were analysed using the standard procedure (ISO 10272-1:2006), enrichment in Preston broth, and enrichment in modified Bolton broth (supplemented with (i) potassium clavulanate (C-BB), (ii) triclosan (T-BB), (iii) polymyxin B (P-BB)). The enrichment cultures were streaked onto both modified charcoal cefoperazone deoxycholate agar (mCCDA) and RAPID'Campylobacter agar (RCA). Moreover, direct plating on mCCDA and RCA was performed to quantify Campylobacter. In total, 33 out of 59 fresh retail meat samples (55.9%) were Campylobacter positive. For both fresh and frozen poultry meat samples, enrichment in Bolton broth (ISO 10272-1:2006) resulted in a higher number of positive samples than enrichment in Preston broth. Supplementation of Bolton broth with potassium clavulanate (C-BB) and triclosan (T-BB) enhanced the Campylobacter recovery from fresh poultry meat compared to non-supplemented Bolton broth, although the use of C-BB was less applicable than T-BB for Campylobacter recovery from frozen samples. Additionally, the use of RCA resulted in a higher isolation rate compared to mCCDA. The present study demonstrates the impact of culture medium on the recovery of Campylobacter from fresh and frozen naturally contaminated poultry meat samples and can support laboratories in choosing the most appropriate culturing method to detect Campylobacter. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimization of immunohistochemical and fluorescent antibody techniques for localization of foot-and-mouth disease virus in animal tissues

USDA-ARS?s Scientific Manuscript database

Immunohistochemical (IHC) and immunofluorescent (IF) techniques were optimized for the detection of foot-and-mouth disease virus (FMDV) structural and non-structural proteins in frozen and paraformaldehyde-fixed paraffin embedded (PFPE) tissues of bovine and porcine origin. Immunohistochemical local...

Colloid centrifugation of fresh stallion semen before cryopreservation decreased microorganism load of frozen-thawed semen without affecting seminal kinetics.

PubMed

Guimarães, T; Lopes, G; Pinto, M; Silva, E; Miranda, C; Correia, M J; Damásio, L; Thompson, G; Rocha, A

2015-01-15

Freezability of equine semen may be influenced by microorganism population of semen. The objective of this study was to verify the effect of single-layer density gradient centrifugation (SLC) of fresh semen before cryopreservation on semen's microbial load (ML) and sperm cells kinetics after freezing-thawing. For that, one ejaculate was collected from 20 healthy stallions and split into control (C) samples (cryopreserved without previous SLC) and SLC samples (subjected to SLC). Semen cryopreservation was performed according to the same protocol in both groups. Microbial load of each microorganism species and total microbial load (TML) expressed in colony-forming units (CFU/mL) as well as frozen-thawed sperm kinetics were assessed in both groups. Additional analysis of the TML was performed, subdividing the frozen-thawed samples in "suitable" (total motility ≥ 30%) and "unsuitable" (total motility < 30%) semen for freezing programs, and comparing the C and SLC groups within these subpopulations. After thawing, SLC samples had less (P < 0.05) TML (88.65 × 10(2) ± 83.8 × 10(2) CFU/mL) than C samples (155.69 × 10(2) ± 48.85 × 10(2) CFU/mL), mainly due to a reduction of Enterococcus spp. and Bacillus spp. A relationship between post-thaw motility and SLC effect on ML was noted, as only in samples with more than 30% total motility was ML reduced (P < 0.05) by SLC (from 51.33 × 10(2) ± 33.26 × 10(2) CFU/mL to 26.68 × 10(2) ± 12.39 × 10(2) CFU/mL in "suitable" frozen-thawed semen vs. 240.90 × 10(2) ± 498.20 × 10(2) to 139.30 × 10(2) ± 290.30 × 10(2) CFU/mL in "unsuitable" frozen-thawed semen). The effect of SLC on kinetics of frozen-thawed sperm cells was negligible. Copyright © 2015 Elsevier Inc. All rights reserved.

Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies

PubMed Central

Saylor, Karen L.; Anver, Miriam R.; Salomon, David S.; Golubeva, Yelena G.

2016-01-01

Laser capture microdissection (LCM) of tissue is an established tool in medical research for collection of distinguished cell populations under direct microscopic visualization for molecular analysis. LCM samples have been successfully analyzed in a number of genomic and proteomic downstream molecular applications. However, LCM sample collection and preparation procedure has to be adapted to each downstream analysis platform. In this present manuscript we describe in detail the adaptation of LCM methodology for the collection and preparation of fresh frozen samples for NanoString analysis based on a study of a model of mouse mammary gland carcinoma and its lung metastasis. Our adaptation of LCM sample preparation and workflow to the requirements of the NanoString platform allowed acquiring samples with high RNA quality. The NanoString analysis of such samples provided sensitive detection of genes of interest and their associated molecular pathways. NanoString is a reliable gene expression analysis platform that can be effectively coupled with LCM. PMID:27077656

Diagnosing breast cancer using Raman spectroscopy: prospective analysis

NASA Astrophysics Data System (ADS)

Haka, Abigail S.; Volynskaya, Zoya; Gardecki, Joseph A.; Nazemi, Jon; Shenk, Robert; Wang, Nancy; Dasari, Ramachandra R.; Fitzmaurice, Maryann; Feld, Michael S.

2009-09-01

We present the first prospective test of Raman spectroscopy in diagnosing normal, benign, and malignant human breast tissues. Prospective testing of spectral diagnostic algorithms allows clinicians to accurately assess the diagnostic information contained in, and any bias of, the spectroscopic measurement. In previous work, we developed an accurate, internally validated algorithm for breast cancer diagnosis based on analysis of Raman spectra acquired from fresh-frozen in vitro tissue samples. We currently evaluate the performance of this algorithm prospectively on a large ex vivo clinical data set that closely mimics the in vivo environment. Spectroscopic data were collected from freshly excised surgical specimens, and 129 tissue sites from 21 patients were examined. Prospective application of the algorithm to the clinical data set resulted in a sensitivity of 83%, a specificity of 93%, a positive predictive value of 36%, and a negative predictive value of 99% for distinguishing cancerous from normal and benign tissues. The performance of the algorithm in different patient populations is discussed. Sources of bias in the in vitro calibration and ex vivo prospective data sets, including disease prevalence and disease spectrum, are examined and analytical methods for comparison provided.

Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

NASA Astrophysics Data System (ADS)

Chandra, Subhash

2008-12-01

Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2+) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells provided a rule-of-thumb criterion for the validation of sample preparation. The fractured freeze-dried cells allowed 3-D SIMS imaging and localization of 13C 15N labeled molecules and therapeutic drugs containing an elemental tag. Examples are shown to demonstrate that both diffusible elements and molecules are prone to artifact-induced relocation at subcellular scale if the sample preparation is compromised. The sample preparation is problem dependent and may vary widely between the diverse sample types of biological systems and the type of instrument used for SIMS analysis. The sample preparation, however, must be validated so that SIMS can be applied with confidence in biology and medicine.

TL and ESR based identification of gamma-irradiated frozen fish using different hydrolysis techniques

NASA Astrophysics Data System (ADS)

Ahn, Jae-Jun; Akram, Kashif; Shahbaz, Hafiz Muhammad; Kwon, Joong-Ho

2014-12-01

Frozen fish fillets (walleye Pollack and Japanese Spanish mackerel) were selected as samples for irradiation (0-10 kGy) detection trials using different hydrolysis methods. Photostimulated luminescence (PSL)-based screening analysis for gamma-irradiated frozen fillets showed low sensitivity due to limited silicate mineral contents on the samples. Same limitations were found in the thermoluminescence (TL) analysis on mineral samples isolated by density separation method. However, acid (HCl) and alkali (KOH) hydrolysis methods were effective in getting enough minerals to carry out TL analysis, which was reconfirmed through the normalization step by calculating the TL ratios (TL1/TL2). For improved electron spin resonance (ESR) analysis, alkali and enzyme (alcalase) hydrolysis methods were compared in separating minute-bone fractions. The enzymatic method provided more clear radiation-specific hydroxyapatite radicals than that of the alkaline method. Different hydrolysis methods could extend the application of TL and ESR techniques in identifying the irradiation history of frozen fish fillets.

Low prevalence of human papillomavirus in head and neck squamous cell carcinoma in the northwest region of the Philippines.

PubMed

Albano, Pia Marie; Holzinger, Dana; Salvador, Christianne; Orosa, Jose; Racelis, Sheryl; Leaño, Modesty; Sanchez, Danilo; Angeles, Lara Mae; Halec, Gordana; Schmitt, Markus; Ramos, John Donnie; Pawlita, Michael

2017-01-01

Geographic heterogeneity of human papillomavirus (HPV) involvement in head and neck squamous cell carcinoma (HNSCC) has been observed over the last few years. This trend has not been evaluated in the Philippines. Hence, this study aims to provide for the first time a data on the prevalence of HPV in HNSCC in the northwestern region of the Philippines. Two hundred one (201) biopsy samples (179 formalin fixed paraffin embedded and 22 fresh frozen) from 163 Filipino HNSCC cases (oral cavity = 88; larynx = 60; oropharynx = 15) diagnosed between 2003 to 2013 were initially included in this study. HPV DNA was detected by two methods: (1) BSGP5+/6+-PCR/ multiplex human papillomavirus genotyping and (2) TaqMan probes-based real-time qPCR. Presence of HPV type-specific transcripts were also analyzed by reverse transcription-PCR with subsequent hybridization to oligonucleotide probes coupled to Luminex beads. Co-amplification of the β-globin and ubiquitin C genes served as internal positive controls for DNA and RNA analyses, respectively. Of the 163, 82 (50.3%) cases had at least one tissue sample that was valid for molecular analysis. Only two of the DNA valid cases (2.4%) were HPV DNA-positive (HPV11 and HPV33). All HPV mRNA assays rendered negative results except for HPV11 transcripts. Results of this study may indicate that there is probably very low prevalence of HPV-associated HNSCC among Filipino adults living in a rural region of the Philippines. This study could serve as a benchmark for designing follow-up studies that would assess possible changes in trends of HNSCC among Filipinos in different ethnic regions of the country, especially urban areas in which the population is expected to adapt Western style sexual behavior. A prospective sampling of fresh frozen tissue is also highly recommended to ensure better molecular analyses.

Expression of programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase (IDO) in the tumor microenvironment and in tumor-draining lymph nodes of breast cancer.

PubMed

Ye, Qian; Wang, Chenglong; Xian, Jie; Zhang, Ming; Cao, Yijia; Cao, Youde

2018-05-01

Programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase (IDO) are both immunosuppressive proteins. Here, we investigated the relationship between PD-1 and IDO in the tumor microenvironment (TME) and in tumor-draining lymph nodes (TDLNs) in breast cancer patients. First, the protein and mRNA expression levels of PD-1 and IDO in 20 frozen tissues were examined using Western blotting and real-time polymerase chain reaction. Second, 151 paraffin-embedded breast samples and 52 lymph node samples were analyzed by immunohistochemistry. Third, correlation and survival data for PD-1 and IDO in 963 breast tumor patients were mined using the cBio Cancer Genomics Portal. We found that the protein expression level of IDO was significantly increased in frozen tumor tissues (P = .005). From paraffin-embedded samples in the TME, PD-1 + cells were only located in the stroma, while IDO was expressed in myoepithelial, stromal, and tumor cells. PD-1 and stromal IDO in the TME showed increased expression in tumors (P< .001 and P < .001, respectively). In TDLNs, PD-1 + cells were primarily located in the germinal centers (GCs), and IDO + cells were primarily located in the paracortex. Normal lymph nodes expressed PD-1 and IDO at the same level as non-metastatic and metastatic lymph nodes (P = .151 and P = .812, respectively). According to cBioPortal, the correlation analysis showed that IDO and PD-1 had high correlation coefficients (r = 0.83). These findings suggest that there is a positive correlation between the expression of PD-1 and IDO and that blocking both PD-1 and IDO pathways may represent an attractive therapeutic strategy in breast cancer treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

Frozen cultured sheets of epidermal keratinocytes in reepithelialization and repair of the cornea after photorefractive keratectomy.

PubMed

Castro-Muñozledo, Federico; Ozorno-Zarate, Jorge; Naranjo-Tackman, Ramon; Kuri-Harcuch, Walid

2002-09-01

To determine whether frozen cultured sheets of human allogeneic epidermal keratinocytes (CEAK) improved wound repair after experimental corneal ablation by photorefractive keratectomy (PRK). Hospital "Luis Sanchez Bulnes" de la Asociación para Evitar la Ceguera en Mexico, I.A.P, and Department of Cell Biology, CINVESTAV-IPN, Mexico City, Mexico. Transepithelial PRK was performed in the right eye of male albino rabbits to obtain a 112 microm deep and 6.0 mm diameter ablation zone. In 17 eyes, the ablations were covered with frozen CEAK; in 11 eyes, the ablations were covered with a disposable contact lens without the cultured sheets; and in the control group (13 eyes), the ablations were not covered. Subepithelial fibrosis and reepithelialization of the ablated zone were evaluated in serial paraffin-embedded tissue sections from all wounds. Treatment with CEAK reduced fibroblast proliferation and the inflammatory response beneath the ablated zone and produced better organization of the newly formed epithelium by eliminating significant hyperplasia or discontinuities in the periodic acid Shiff-stained basement membrane. It also led to accelerated reepithelialization. The use of frozen CEAK as a biologically active wound dressing improved tissue repair at 1 month in corneas ablated by transepithelial PRK in the male albino rabbit model. Treatment with CEAK could improve the outcome of PRK in humans.

Comparison of techniques for stabilizing hemoglobins of rainbow trout (Salmo gairdneri) during frozen storage

USGS Publications Warehouse

Reinitz, G.L.

1976-01-01

1. The stability of hemoglobin of rainbow trout under frozen conditions in oxyform, carboxyform, and cyanometform was examined.2. Carboxyhemoglobin retained its original electrophoretic banding pattern after 14 days of frozen storage, whereas oxyform and cyanometform hemoglobins did not.3. Banding patterns changed in some samples in all treatment groups after 21 days of storage.

Fresh frozen cadaver workshops for advanced vascular surgical training.

PubMed

Jansen, Shirley; Cowie, Margaret; Linehan, John; Hamdorf, Jeffery M

2014-11-01

Reduction in working hours, streamlined training schemes and increasing use of endovascular techniques has meant a reduction in operative experience for newer vascular surgical trainees, especially those exposures which are not routinely performed such as thoracoabdominal, thoracotomy and retroperitoneal aortic, for example. This paper describes an Advanced Anatomy of Exposure course which was designed and convened at the Clinical Training & Evaluation Centre in Western Australia and uses fresh frozen cadavers. Feedback was obtained from the participants who attended over three courses by questionnaire. Feedback was strongly positive for the course meeting both its learning outcomes and personal learning objectives, and in addition, making a significant contribution to specialty skills. Most participants thought the fresh frozen cadaveric model significantly improved the learning objectives for training. The fresh frozen cadaver is an excellent teaching model highly representative of the living open surgical scenario where advanced trainees and newly qualified consultants can improve their operative confidence and consequently patient safety in vascular surgery. An efficient fresh frozen cadaver teaching programme can benefit many health professionals simultaneously maximizing the use of donated human tissue. © 2013 Royal Australasian College of Surgeons.

Prevalence of Listeria spp and Listeria monocytogenes in meat and fermented fish in Malaysia.

PubMed

Hassan, Z; Purwati, E; Radu, S; Rahim, R A; Rusul, G

2001-06-01

Fermented fish and meat samples were purchased from supermarket and wet market for microbiological analysis of Listeria species and Listeria monocytogenes contamination. Listeria species were isolated from 17 (73.9%) of 23 samples of imported frozen beef, 10 (43.5%) of the 23 samples of local beef and 14 (56%) of the 25 samples of fermented fish from wet market. Listeria monocytogenes occurred in 15 (75%) of the frozen beef samples, 6 (30.4%) of the 23 samples of local meat and 3 (12%) of the 25 samples from fermented fish. Listeria species was not isolated from any of the 23 samples of imported frozen beef from supermarket and from the 5 samples of buffalo meat examined. This highlights the possibility of Listeria spp or L. monocytogenes to persist in meat and fermented fish in wet market and raises the problem of illness due to the handling and consumption of Listeria-contaminated meat or fermented fish are likely as evidence by the high contamination rates of samples sold at the wet market.

Effect of ELISA kit manufacturing process and incubation time on progesterone concentration measured in dog serum for ovulation diagnosis - Short communication.

PubMed

Thuróczy, Julianna; Reiczigel, Jenő; Balogh, Lajos

2016-09-01

Twenty-two serum samples of healthy bitches were tested with the frozen and lyophilised version of the same ELISA kit (Quanticheck, Faculty of Veterinary Science, Budapest, Hungary). Samples were chosen on the basis of their progesterone (P4) concentrations, which were between 1.00 and 20.00 ng/mL. As it is well known, this range has the highest clinical relevance in ovulation diagnosis. Both types of microplates were read at 15-min intervals from the 15th until the 90th minute (min) of incubation, and the results were compared with those of frozen plates at 60 min of incubation as 100 percent. Lyophilised microplates gave on average 18 percent higher results than the frozen version at equal incubation times. The highest difference between lyophilised and frozen samples was observed at 45 and 60 min of incubation. Ninety-four percent of the reaction in the frozen microplate occurred in the first 15 min, and during the subsequent 30 min the reaction seemingly stopped. After the 45th min of incubation, this 94 percent increased to 108 percent in the subsequent 30 min, which remained the final approximate result at the end of the 90 min of incubation. In contrast to the frozen microplate, the measured concentration increased continuously in the lyophilised version and reached the highest level at the 60th min. The results of the lyophilised microplate reached the same level at 30 min of incubation as those of the frozen version at 60 min. In conclusion, a mechanical increase or decrease of the incubation time does not generate a linear change in the test results. This study demonstrated that the results of a series of samples collected from the same bitch cannot be compared if they are measured with different laboratory methods or different ELISA kits.

Freeze chromatography method and apparatus

DOEpatents

Scott, C.D.

1987-04-16

A freeze chromatography method and apparatus are provided which enable separation of the solutes contained in a sample. The apparatus includes an annular column construction comprising cylindrical inner and outer surfaces defining an annular passage therebetween. One of the surfaces is heated and the other cooled while passing an eluent through the annular passageway so that the eluent in contact with the cooled surface freezes and forms a frozen eluent layer thereon. A mixture of solutes dissolved in eluent is passed through the annular passageway in contact with the frozen layer so that the sample solutes in the mixture will tend to migrate either toward or away the frozen layer. The rate at which the mixture flows through the annular passageway is controlled so that the distribution of the sample solutes approaches that at equilibrium and thus a separation between the sample solutes occurs. 3 figs.

[Adaptability of sweet corn ears to a frozen process].

PubMed

Ramírez Matheus, Alejandra O; Martínez, Norelkys Maribel; de Bertorelli, Ligia O; De Venanzi, Frank

2004-12-01

The effects of frozen condition on the quality of three sweet corn ears (2038, 2010, 2004) and the pattern (Bonanza), were evaluated. Biometrics characteristics like ear size, ear diameter, row and kernel deep were measured as well as chemical and physical measurement in fresh and frozen states. The corn ears were frozen at -95 degrees C by 7 minutes. The yield and stability of the frozen ears were evaluated at 45 and 90 days of frozen storage (-18 degrees C). The average commercial yield as frozen corn ear for all the hybrids was 54.2%. The industry has a similar value range of 48% to 54%. The ear size average was 21.57 cm, row number was 15, ear diameter 45.54 mm and the kernel corn deep was 8.57 mm. All these measurements were found not different from commercial values found for the industry. All corn samples evaluated showed good stability despites the frozen processing and storage. Hybrid 2038 ranked higher in quality.

Anatomical Distribution of Lipids in Human Brain Cortex by Imaging Mass Spectrometry

NASA Astrophysics Data System (ADS)

Veloso, Antonio; Astigarraga, Egoitz; Barreda-Gómez, Gabriel; Manuel, Iván; Ferrer, Isidro; Teresa Giralt, María; Ochoa, Begoña; Fresnedo, Olatz; Rodríguez-Puertas, Rafael; Fernández, José A.

2011-02-01

Molecular mass images of tissues will be biased if differences in the physicochemical properties of the microenvironment affect the intensity of the spectra. To address this issue, we have performed—by means of MALDI-TOF mass spectrometry—imaging on slices and lipidomic analysis in extracts of frontal cortex, both from the same postmortem tissue samples of human brain. An external calibration was used to achieve a mass accuracy of 10 ppm (1 σ) in the spectra of the extracts, although the final assignment was based on a comparison with previously reported species. The spectra recorded directly from tissue slices (imaging) show excellent s/n ratios, almost comparable to those obtained from the extracts. In addition, they retain the information about the anatomical distribution of the molecular species present in autopsied frozen tissue. Further comparison between the spectra from lipid extracts devoid of proteins and those recorded directly from the tissue unambiguously show that the differences in lipid composition between gray and white matter observed in the mass images are not an artifact due to microenvironmental influences of each anatomical area on the signal intensity, but real variations in the lipid composition.

A multiple-well method for immunohistochemical testing of many reagents on a single microscopic slide.

PubMed

McKeever, P E; Letica, L H; Shakui, P; Averill, D R

1988-09-01

Multiple wells (M-wells) have been made over tissue sections on single microscopic slides to simultaneously localize binding specificity of many antibodies. More than 20 individual 4-microliter wells over tissue have been applied/slide, representing more than a 5-fold improvement in wells/slide and a 25-fold reduction in reagent volume over previous methods. More than 30 wells/slide have been applied over cellular monolayers. To produce the improvement, previous strategies of placing specimens into wells were changed to instead create wells over the specimen. We took advantage of the hydrophobic properties of paint to surround the wells and to segregate the various different primary antibodies. Segregation was complete on wells alternating with and without primary monoclonal antibody. The procedure accommodates both frozen and paraffin sections, yielding slides which last more than a year. After monoclonal antibody detection, standard histologic stains can be applied as counterstains. M-wells are suitable for localizing binding of multiple reagents or sample unknowns (polyclonal or monoclonal antibodies, hybridoma supernatants, body fluids, lectins) to either tissues or cells. Their small sample volume and large number of sample wells/slide could be particularly useful for early screening of hybridoma supernatants and for titration curves in immunohistochemistry (McKeever PE, Shakui P, Letica LH, Averill DR: J Histochem Cytochem 36:931, 1988).

3D Reconstruction of Frozen Plant Tissue: a unique histological analysis to image post-freeze responses

USDA-ARS?s Scientific Manuscript database

Winter hardiness in plants is the result of a complex interaction between genes, the tissue where those genes are expressed and the environment. The light microscope is a valuable tool to understand this complexity which will ultimately help researchers improve the tolerance of plants to freezing st...

A simple method for collecting eggs of taeniid cestodes from fresh, frozen or ethanol-fixed segments.

PubMed

Takemoto, Y; Negita, T; Ohnishi, K; Suzuki, M; Ito, A

1995-04-01

A simple method was devised for collecting eggs of Taenia taeniaeformis and T. saginata. All gravid segments, either fresh or frozen or 70% ethanol-fixed, were gently scraped using a pestle on a 150 mesh stainless steel sieve. Eggs and tissue debris were washed out all together with mouse tonicity phosphate buffered saline (MTPBS) through the 150 mesh sieve into a glass beaker. Egg suspension with a huge amount of tissue debris in MTPBS was centrifuged 5 min at 3000 r.p.m. (x 1600 g) and the pellet of eggs and tissue debris was resuspended with 1 vol. of MTPBS and 2 vol. of Percoll (Pharmacia) and centrifuged 60 min at 3000 r.p.m. More than 90% of eggs sedimented in the pellet. The supernatant covered with tissue debris was decanted, and the egg pellet was resuspended and centrifuged several times with MTPBS to remove Percoll. It is suggested that this simple method may prove useful for preparation of eggs of biohazardous taeniid cestodes, such as Taenia solium and Echinococcus spp.

Effect of freezing, irradiation, and frozen storage on survival of Salmonella in concentrated orange juice.

PubMed

Niemira, Brendan A; Sommers, Christopher H; Boyd, Glenn

2003-10-01

Six strains of Salmonella (Anatum F4317, Dublin 15480, Enteritidis 13076, Enteritidis WY15159, Stanley H0588, and Typhimurium 14028) were individually inoculated into orange juice concentrate (OJC) and frozen to -20 degrees C. The frozen samples were treated with 0 (nonirradiated), 0.5, 1.0, or 2.0 kGy of gamma radiation and held frozen for 1 h, and the surviving bacterial population was assessed. The strains showed significant variability in their response to freezing and to freezing in combination with irradiation. The response was dose dependent. Relative to the nonfrozen, nonirradiated control, the reduction following the highest dose (2.0 kGy) ranged from 1.29 log CFU/ml (Salmonella Typhimurium) to 2.17 log CFU/ml (Salmonella Stanley). Samples of OJC inoculated with Salmonella Enteritidis WY15159 and irradiated were stored at -20 degrees C for 1, 2, 7, or 14 days, and the surviving population was determined. Relative to the nonfrozen, nonirradiated control, after 14 days, the population was reduced by 1.2 log CFU/ml in the nonirradiated samples and by 3.3 log CFU/ml following treatment with 2.0 kGy. The combination of frozen storage plus irradiation resulted in greater overall reductions than either process alone.

Photochemical Production of Singlet Oxygen from Dissolved Organic Matter in Ice.

PubMed

Fede, Alexis; Grannas, Amanda M

2015-11-03

Dissolved natural organic matter (DOM) is a ubiquitous component of natural waters and an important photosensitizer. A variety of reactive oxygen species (ROS) are known to be produced from DOM photochemistry, including singlet oxygen, 1O2. Recently, it has been determined that humic-like substances and unknown organic chromophores are significant contributors to sunlight absorption in snowpack; however, DOM photochemistry in snow/ice has received little attention in the literature. We recently showed that DOM plays an important role in indirect photolysis processes in ice, producing ROS and leading to the efficient photodegradation of a probe hydrophobic organic pollutant, aldrin.1 ROS scavenger experiments indicated that 1O2 played a significant role in the indirect photodegradation of aldrin. Here we quantitatively examine 1O2 photochemically produced from DOM in frozen and liquid aqueous solutions. Steady-state 1O2 production is enhanced up to nearly 1000 times in frozen DOM samples compared to liquid samples. 1O2 production is dependent on the concentration of DOM, but the nature of the DOM source (terrestrial vs microbial) does not have a significant effect on 1O2 production in liquid or frozen samples, with different source types producing similar steady-state concentrations of 1O2. The temperature of frozen samples also has a significant effect on steady-state 1O2 production in the range of 228-262 K, with colder samples producing more steady-state 1O2. The large enhancement in 1O2 in frozen samples suggests that it may play a significant role in the photochemical processes that occur in snow and ice, and DOM could be a significant, but to date poorly understood, oxidant source in snow and ice.

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

PubMed Central

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. PMID:28498833

Modulation of Stem Cell Differentiation and Myostatin as an Approach to Counteract Fibrosis in Muscle Dystrophy and Regeneration After Injury. Addendum

DTIC Science & Technology

2012-03-01

considerable increase in central nuclei in the regenerating myofibers, and molsidomine supplementation appears to have upregulated the overall stem cell...the increase of central nuclei as indicator of muscle repair observed in the SC group in comparison to the UT group, in frozen tissue sections...treatments on myofiber repair will be better defined by the counting of central nuclei on hematoxylin/eosin stained frozen sections as in Fig 3, and the

Sensitivity of frozen section histology for identifying Propionibacterium acnes infections in revision shoulder arthroplasty.

PubMed

Grosso, Matthew J; Frangiamore, Salvatore J; Ricchetti, Eric T; Bauer, Thomas W; Iannotti, Joseph P

2014-03-19

Propionibacterium acnes is a clinically relevant pathogen with total shoulder arthroplasty. The purpose of this study was to determine the sensitivity of frozen section histology in identifying patients with Propionibacterium acnes infection during revision total shoulder arthroplasty and investigate various diagnostic thresholds of acute inflammation that may improve frozen section performance. We reviewed the results of forty-five patients who underwent revision total shoulder arthroplasty. Patients were divided into the non-infection group (n = 15), the Propionibacterium acnes infection group (n = 18), and the other infection group (n = 12). Routine preoperative testing was performed and intraoperative tissue culture and frozen section histology were collected for each patient. The histologic diagnosis was determined by one pathologist for each of the four different thresholds. The absolute maximum polymorphonuclear leukocyte concentration was used to construct a receiver operating characteristics curve to determine a new potential optimal threshold. Using the current thresholds for grading frozen section histology, the sensitivity was lower for the Propionibacterium acnes infection group (50%) compared with the other infection group (67%). The specificity of frozen section was 100%. Using a receiver operating characteristics curve, an optimized threshold was found at a total of ten polymorphonuclear leukocytes in five high-power fields (400×). Using this threshold, the sensitivity of frozen section for Propionibacterium acnes was increased to 72%, and the specificity remained at 100%. Using current histopathology grading systems, frozen sections were specific but showed low sensitivity with respect to the Propionibacterium acnes infection. A new threshold value of a total of ten or more polymorphonuclear leukocytes in five high-power fields may increase the sensitivity of frozen section, with minimal impact on specificity.

A prospective, multicentre study of moxifloxacin concentrations in the sinus mucosa tissue of patients undergoing elective surgery of the sinus.

PubMed

Gehanno, P; Darantière, S; Dubreuil, C; Chobaut, J C; Bobin, S; Pages, J C; Renou, G; Bobin, F; Arvis, P; Stass, H

2002-05-01

A pharmacokinetic study was carried out to determine moxifloxacin concentrations in sinus tissue, after oral moxifloxacin 400 mg once daily for 5 days to patients with chronic sinusitis, undergoing elective sinus surgery. Patients were randomly allocated to one of seven treatment groups, in which tissues were sampled 2, 3, 4, 6, 12, 24 or 36 h post-dose. A control group with non-infected nasal polyps was also included. Forty-eight patients (13 female, 35 male, mean age 47.1 years) were allocated to one of each active treatment group (n = 42) or to the control group (n = 6). Tissue and plasma samples were taken simultaneously and stored frozen until assayed by HPLC. Thirty-nine patients were fully valid for pharmacokinetic analysis. The geometric mean moxifloxacin plasma concentration increased from 2.32 mg/L at 2 h to a maximum of 3.37 mg/L at 4 h post-dose, decreasing to 0.37 mg/L at 36 h post-dose. The moxifloxacin concentration in sinus mucosa was consistently greater than that in plasma being 4.56-5.73 mg/kg from 2 to 6 h and 2.81-1.25 mg/kg from 12 to 36 h post-dose. The elimination rates in plasma and sinus tissues were similar. The tissue/plasma ratio was c. 200% between 2 and 6 h, and up to 328.9% at 36 h. Results were similar whatever the site of tissue sampling (maxillary sinus, anterior ethmoid sinus or nasal polyps). Tissue levels exceeded the MIC(90) of all pathogens commonly causing acute sinusitis (e.g. 5-30 x MIC for Streptococcus pneumoniae: 0.25 mg/L). These results sup-port the use of moxifloxacin 400 mg once daily as a regimen for the treatment of sinus infections.

Structural requirements of research tissue banks derived from standardized project surveillance.

PubMed

Herpel, E; Koleganova, N; Schreiber, B; Walter, B; Kalle, C V; Schirmacher, P

2012-07-01

Tissue banks constitute decisive and rate-limiting resource and technology platforms for basic and translational biomedical research, notably in the area of cancer. Thus, it is essential to plan and structure tissue banking and allocate resources according to research needs, but essential requirements are still incompletely defined. The tissue bank of the National Center of Tumor Diseases Heidelberg (NCT) was founded with the intention to provide tissues of optimal quality and to prioritize the realization of research projects. We analysed its structure and prospective project management registration as well as tracking records for all projects of the NCT tissue bank as of its start in 2005 in order to obtain information that may be relevant for tissue bank planning. All project proposals submitted to the NCT tissue bank (n = 681) were included in the study. For a detailed evaluation of provided services, only projects that were completed until July 2011 (n = 605) were analysed. For these 605 projects, NCT tissue bank provided 769 specific services. In all projects/services, we recorded project leader, type and amount of material provided, type of research (basic/translational), work load of project and project completion. Furthermore, all completed projects were tracked after 90 days according to a standard protocol to determine principal investigators' (PI) satisfaction and quality of the provided material. Until July 2011, 605 projects had been successfully completed as documented by material transfer agreement. Of the projects, 72.7 % addressed basic research, 22.3 % were translational research projects and 3 % concerned epidemiological research; 91 % (n = 546) concerned a single PI and the NTC tissue bank. For these projects, 769 specific services were provided. Of these services, 288 concerned providing formalin-fixed and paraffin-embedded (FFPE) tissue (extracts, full size sections), 126 providing fresh frozen materials (including fresh frozen sections), 137 providing tissue micro-array (TMA)-based sections and 199 providing immunohistochemical services. Project tracking demonstrated that all projects had started within 90 days after reception of the material by the PIs, and PI satisfaction with provided material exceeded 97 %. Standardized registration and tracking provides valuable structural information for planning and financing of tissue banks and allocation of resources. The high number of completed projects as well as high user satisfaction demonstrates that structuring of tissue banks should be preferably research-oriented and highly efficient. The comparable number of requests for FFPE and fresh frozen tissue as well as TMA-based services underpins the need for a broad approach in terms of methods and material types in order to fulfil research needs.

Combined quantitative and qualitative two-channel optical biopsy technique for discrimination of tumor borders

NASA Astrophysics Data System (ADS)

Bocher, Thomas; Beuthan, Juergen; Scheller, M.; Hopf, Juergen U. G.; Linnarz, Marietta; Naber, Rolf-Dieter; Minet, Olaf; Becker, Wolfgang; Mueller, Gerhard J.

1995-12-01

Conventional laser-induced fluorescence spectroscopy (LIFS) of endogenous chromophores like NADH (Nicotineamide Adenine Dinucleotide, reduced form) and PP IX (Protoporphyrin IX) provides information about the relative amounts of these metabolites in the observed cells. But for diagnostic applications the concentrations of these chromophores have to be determined quantitatively to establish tissue-independent differentiation criterions. It is well- known that the individually and locally varying optical tissue parameters are major obstacles for the determination of the true chromophore concentrations by simple fluorescence spectroscopy. To overcome these problems a fiber-based, 2-channel technique including a rescaled NADH-channel (delivering quantitative values) and a relative PP IX-channel was developed. Using the accumulated information of both channels can provide good tissue state separation. Ex-vivo studies with resected and frozen samples (with LN2) of squamous cells in the histologically confirmed states: normal, tumor border, inflammation and hyperplasia were performed. Each state was represented in this series with at least 7 samples. At the identical tissue spot both, the rescaled NADH-fluorescence and the relative PP IX- fluorescence, were determined. In the first case a nitrogen laser (337 nm, 500 ps, 200 microjoule, 10 Hz) in the latter case a diode laser (633 nm, 15 mW, cw) were used as excitation sources. In this ex-vivo study a good separation between the different tissue states was achieved. With a device constructed for clinical usage one quantitative, in-vivo NADH- measurement was done recently showing similar separation capabilities.

Cryosurgery with Pulsed Electric Fields

PubMed Central

Daniels, Charlotte S.; Rubinsky, Boris

2011-01-01

This study explores the hypothesis that combining the minimally invasive surgical techniques of cryosurgery and pulsed electric fields will eliminate some of the major disadvantages of these techniques while retaining their advantages. Cryosurgery, tissue ablation by freezing, is a well-established minimally invasive surgical technique. One disadvantage of cryosurgery concerns the mechanism of cell death; cells at high subzero temperature on the outer rim of the frozen lesion can survive. Pulsed electric fields (PEF) are another minimally invasive surgical technique in which high strength and very rapid electric pulses are delivered across cells to permeabilize the cell membrane for applications such as gene delivery, electrochemotherapy and irreversible electroporation. The very short time scale of the electric pulses is disadvantageous because it does not facilitate real time control over the procedure. We hypothesize that applying the electric pulses during the cryosurgical procedure in such a way that the electric field vector is parallel to the heat flux vector will have the effect of confining the electric fields to the frozen/cold region of tissue, thereby ablating the cells that survive freezing while facilitating controlled use of the PEF in the cold confined region. A finite element analysis of the electric field and heat conduction equations during simultaneous tissue treatment with cryosurgery and PEF (cryosurgery/PEF) was used to study the effect of tissue freezing on electric fields. The study yielded motivating results. Because of decreased electrical conductivity in the frozen/cooled tissue, it experienced temperature induced magnified electric fields in comparison to PEF delivered to the unfrozen tissue control. This suggests that freezing/cooling confines and magnifies the electric fields to those regions; a targeting capability unattainable in traditional PEF. This analysis shows how temperature induced magnified and focused PEFs could be used to ablate cells in the high subzero freezing region of a cryosurgical lesion. PMID:22087224

Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping

PubMed Central

Wulfkuhle, Julia D.; Berg, Daniela; Wolff, Claudia; Langer, Rupert; Tran, Kai; Illi, Julie; Espina, Virginia; Pierobon, Mariaelena; Deng, Jianghong; DeMichele, Angela; Walch, Axel; Bronger, Holger; Becker, Ingrid; Waldhör, Christine; Höfler, Heinz; Esserman, Laura; Liotta, Lance A.; Becker, Karl-Friedrich; Petricoin, Emanuel F.

2017-01-01

Purpose Targeting of the HER2 protein in human breast cancer represents a major advance in oncology, but relies on measurements of total HER2 protein and not HER2 signaling network activation. We utilized reverse phase protein microarrays (RPMAs) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity. Experimental Design Three independent study sets, comprising a total of 415 individual patient samples from flash frozen core biopsy samples and FFPE surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pre-treatment core biopsy samples and whole tissue lysates from 288 FFPE samples and these results were compared to FISH and IHC. Results RPMA measurements of total HER2 were highly concordant (> 90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC+ population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC- tumors and, identical to that seen with FISH/IHC+ tumors, the HER2 activation was concordant with EGFR and HER3 phosphorylation and downstream signaling endpoint activation. Conclusions Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC-/FISH-/pHER2+ tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR. PMID:23045247

Analytical evaluation of the automated galectin-3 assay on the Abbott ARCHITECT immunoassay instruments.

PubMed

Gaze, David C; Prante, Christian; Dreier, Jens; Knabbe, Cornelius; Collet, Corinne; Launay, Jean-Marie; Franekova, Janka; Jabor, Antonin; Lennartz, Lieselotte; Shih, Jessie; del Rey, Jose Manuel; Zaninotto, Martina; Plebani, Mario; Collinson, Paul O

2014-06-01

Galectin-3 is secreted from macrophages and binds and activates fibroblasts forming collagen. Tissue fibrosis is central to the progression of chronic heart failure (CHF). We performed a European multicentered evaluation of the analytical performance of the two-step routine and Short Turn-Around-Time (STAT) galectin-3 immunoassay on the ARCHITECT i1000SR, i2000SR, and i4000SR (Abbott Laboratories). We evaluated the assay precision and dilution linearity for both routine and STAT assays and compared serum and plasma, and fresh vs. frozen samples. The reference interval and biological variability were also assessed. Measurable samples were compared between ARCHITECT instruments and between the routine and STAT assays and also to a galectin-3 ELISA (BG Medicine). The total assay coefficient of variation (CV%) was 2.3%-6.2% and 1.7%-7.4% for the routine and STAT assays, respectively. Both assays demonstrated linearity up to 120 ng/mL. Galectin-3 concentrations were higher in plasma samples than in serum samples and correlated well between fresh and frozen samples (R=0.997), between the routine and STAT assays, between the ARCHITECT i1000 and i2000 instruments and with the galectin-3 ELISA. The reference interval on 627 apparently healthy individuals (53% male) yielded upper 95th and 97.5th percentiles of 25.2 and 28.4 ng/mL, respectively. Values were significantly lower in subjects younger than 50 years. The galectin-3 routine and STAT assays on the Abbott ARCHITECT instruments demonstrated good analytical performance. Further clinical studies are required to demonstrate the diagnostic and prognostic potential of this novel marker in patients with CHF.

First pregnancies, live birth, and in vitro fertilization outcomes after transplantation of frozen-banked ovarian tissue with a human extracellular matrix scaffold using robot-assisted minimally invasive surgery.

PubMed

Oktay, Kutluk; Bedoschi, Giuliano; Pacheco, Fernanda; Turan, Volkan; Emirdar, Volkan

2016-01-01

Ovarian tissue cryopreservation is an experimental fertility preservation method and the transplantation techniques are still evolving. We attempted to improve the technique with the utility of a human decellularized extracellular tissue matrix (ECTM) scaffold, robot-assisted minimally invasive surgery, and perioperative pharmacological support. We prospectively studied 2 subjects with hemophagocytic lymphohistiocytosis (patient A) and non-Hodgkin lymphoma (patient B) who underwent ovarian tissue cryopreservation at the age of 23 years, before receiving preconditioning chemotherapy for hematopoietic stem cell transplantation. Both experienced ovarian failure postchemotherapy and we transplanted ovarian cortical tissues to the contralateral menopausal ovary 7 and 12 years later, using a human ECTM scaffold and robotic assistance. The ECTM scaffold tissue compatibility was shown in preclinical studies. Patients also received estrogen supplementation and baby aspirin preoperatively to aid in the revascularization process. Ovarian follicle development was observed approximately 10 (patient A) and 8 (patient B) weeks after ovarian tissue transplantation. Following 8 and 7 cycles of in vitro fertilization, 9 and 10 day-3 embryos were cryopreserved (patients A and B, respectively). While the baseline follicle-stimulating hormone (range 3.6-15.4 mIU/mL) levels near normalized by 7 months and remained steady postovarian transplantation in patient A, patient B showed improved but elevated follicle-stimulating hormone levels throughout (range 21-31 mIU/mL). Highest follicle yield was achieved 14 (8 follicles; patient A) and 11 (6 follicles; patient B) months postintervention. Patient A experienced a chemical pregnancy after the third frozen embryo transfer attempt. She then conceived following her first fresh in vitro fertilization embryo transfer and the pregnancy is currently ongoing. Patient B conceived after the first frozen embryo transfer attempt and delivered a healthy girl at term. We report the first pregnancies after the minimally invasive transplantation of previously cryopreserved ovarian tissue with an ECTM scaffold. This approach seems to be associated with steady ovarian function after a follow-up of up to 2 years. Copyright © 2016 Elsevier Inc. All rights reserved.

Psychiatric Brain Banking: Three Perspectives on Current Trends and Future Directions

PubMed Central

Deep-Soboslay, Amy; Benes, Francine M.; Haroutunian, Vahram; Ellis, Justin K.; Kleinman, Joel E.; Hyde, Thomas M.

2011-01-01

Introduction The study of postmortem human brain tissue is central to the advancement of the neurobiological studies of psychiatric illness, particularly for the study of brain-specific isoforms and molecules. Methods The state-of-the-art methods and recommendations for maintaining a successful brain bank for psychiatric disorders are discussed, using the convergence of viewpoints from three brain collections, the National Institute of Mental Health Brain Collection (NIMH), the Harvard Brain Tissue Resource Center (HBTRC), and the Mt. Sinai School of Medicine Brain Bank (MSSM-BB), with diverse research interests and divergent approaches to tissue acquisition. Results While the NIMH obtains donations from medical examiners for its collection, and places particular emphasis on clinical diagnosis, toxicology, and building lifespan control cohorts, the HBTRC is uniquely designed as a repository whose sole purpose is to collect large-volume, high quality brain tissue from community-based donors based on relationships across an expansive nationwide network, and places emphasis on the accessibility of its bank in disseminating tissue and related data to research groups worldwide. The MSSM-BB collection has shown that, with dedication, prospective recruitment is a successful approach to tissue donation, and places particular emphasis on rigorous clinical diagnosis through antemortem contact with donors. The MSSM-BB places great importance on stereological tissue sampling methods for neuroanatomical studies, and frozen tissue sampling approaches that enable multiple assessments (RNA, DNA, protein, enzyme activity, binding, etc.) of the same tissue block. Promising scientific approaches for elucidating the molecular and cellular pathways in brain that may contribute to schizophrenia and/or bipolar disorder, such as cell culture techniques and microarray-based gene expression and genotyping studies are briefly discussed. Conclusions Despite unique perspectives from three established brain collections, there is a consensus that (1) diverse strategies for tissue acquisition, (2) rigor in tissue and diagnostic characterization, (3) the importance of sample accessibility, and (4) continual application of innovative scientific approaches to the study of brain tissue are all integral to the success and future of psychiatric brain banking. The future of neuropsychiatric research depends upon in the availability of high quality brain specimens from large numbers of subjects, including non-psychiatric controls. PMID:20673875

Effect of inulin on the physicochemical properties, flow behavior and probiotic survival of frozen yogurt.

PubMed

Rezaei, Rahil; Khomeiri, Morteza; Aalami, Mehran; Kashaninejad, Mahdi

2014-10-01

This study investigated the effect of inulin (0, 1 and 2 %), on some physicochemical properties of frozen yogurt, as well as its effect on flow behavior and probiotic survival. The results showed that the addition of inulin improved overrun, viscosity and melting properties significantly (p < 0.05); when added at 2 % level, it also had significant effect on pH. Total acceptability of samples revealed that frozen yogurt with 2 % inulin had the most appealing sensory characteristics. The flow behavior of all samples showed their pseudoplastic nature; power law was the best model to predict their flow behavior. In terms of probiotic survival, the sample with 2 % inulin significantly improved the viability of Lactobacillus acidophilus and Bifidobacterium lactis.

Logistics and quality control for DNA sampling in large multicenter studies.

PubMed

Nederhand, R J; Droog, S; Kluft, C; Simoons, M L; de Maat, M P M

2003-05-01

To study associations between genetic variation and disease, large bio-banks need to be created in multicenter studies. Therefore, we studied the effects of storage time and temperature on DNA quality and quantity in a simulation experiment with storage up to 28 days frozen, at 4 degrees C and at room temperature. In the simulation experiment, the conditions did not influence the amount or quality of DNA to an unsatisfactory level. However, the amount of extracted DNA was decreased in frozen samples and in samples that were stored for > 7 days at room temperature. In a sample of patients from 24 countries of the EUROPA trial obtained by mail with transport times up to 1 month DNA yield and quality were adequate. From these results we conclude that transport of non-frozen blood by ordinary mail is usable and practical for DNA isolation for polymerase chain reaction in clinical and epidemiological studies.

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study

PubMed Central

Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I.; Dwyer, Karen M.; Saffery, Richard

2018-01-01

Aim To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. Background DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. Methods QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Results Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0–0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0–9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0–17.7μg/mL and 0–1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. Conclusion High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease. PMID:29462136

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study.

PubMed

Lecamwasam, Ashani; Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I; Dwyer, Karen M; Saffery, Richard

2018-01-01

To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0-0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0-9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0-17.7μg/mL and 0-1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease.

Cryo-isolation: a novel method for enzyme-free isolation of pancreatic islets involving in situ cryopreservation of islets and selective destruction of acinar tissue.

PubMed

Taylor, M J; Baicu, S

2011-11-01

A critical component of treating type I diabetes by transplantation is the availability of sufficient high-quality islets. Currently, islets can be obtained only by reliance on an expensive, inconsistent, and toxic enzyme digestion process. As an alternative, we hypothesize that cryobiologic techniques can be used for differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ. Pancreases were procured from juvenile pigs with the use of approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water (or saline solution) to fully distend the gland; 3) freezing the entire pancreas to -160°C, and stored in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen pancreas into small fragments; and 5) thawing, filtering and washing the frozen fragments with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and samples were taken for static glucose-stimulated insullin release assessment. As predicted the cryo-isolated contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets. The degree of cleavage of the cryoprotected islets from the freeze-destroyed exocrine cells, was variable. Islets were typically larger than their counterparts isolated from juvenile pigs with conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7 (n = 3). An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a novel method that avoids the problems associated with conventional collagenase digestion methods. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of Frozen Human Epidermis Storage Duration and Cryoprotectant on Barrier Function using Two Model Compounds

PubMed Central

Barbero, Ana M.; Frasch, H. Frederick

2015-01-01

Skin is commonly stored frozen and then thawed prior to use for in-vitro permeation experiments. Does frozen storage of skin alter its barrier property? Numerous studies have found contradictory answers to this question. In this study, the steady state flux and lag time of diethyl phthalate (DEP) were measured for fresh human skin and skin frozen at −85 °C for 1, 2, 3, 6, 9, 12, and 18 months, with 10% glycerol as cryoprotective agent. No significant differences in steady state flux were found between fresh and previously frozen samples (P = 0.6). For lag time, a significant (P = 0.002) difference was found among all groups but comparisons with fresh skin were not significant. Does glycerol have a cryoprotective effect? The steady state flux and lag time of DEP and caffeine were measured through human skin stored at −85 °C for up to 12 months with and without 10 % glycerol. No significant differences in steady state flux or lag time were found between samples stored with or without glycerol for either DEP or caffeine (P ≥ 0.17). These findings support the use of frozen skin to measure the passive permeation of chemicals in studies unconcerned with viability and metabolism. PMID:26606593

Aberrant expression of stress-induced phosphoprotein 1 in colorectal cancer and its clinicopathologic significance.

PubMed

Zhang, Zhimei; Ren, Hui; Yang, Liang; Zhang, Xinhua; Liang, Wei; Wu, Hui; Huang, Leilei; Kang, Jihui; Xu, Jianbo; Zhai, Ertao; Cai, Shirong; He, Yulong

2018-06-04

Stress-Inducible Phosphoprotein1 (STIP1) is an adaptor protein that bridges HSP70 and HSP90 folding and a secretory protein that regulates malignant tumor progression. The aim of the present study was to demonstrate the clinicopathological significance and prognostic role of STIP1 in colorectal cancer (CRC). We used data from The Cancer Genome Atlas (TCGA) to analyze STIP1 expression in CRC and utilized 8 pairs of fresh-frozen tissue samples to investigate STIP1 expression in CRC tissues and adjacent normal tissues using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays. We also used immunohistochemical staining to detect STIP1 expression in 144 formalin-fixed, paraffin-embedded (FFPE) CRC tissue samples and determine the clinical significance of STIP1 expression in CRC. The results of bioinformatics analysis, qRT-PCR, and western blot showed that STIP1 expression was higher in CRC tissues than in adjacent normal tissues. High STIP1 expression was significantly correlated with advanced T stage (P=.01), N stage (P=.001), M stage (P﹤0.001), and TNM stage (P﹤0.001). Moreover, Kaplan-Meier analyses indicated that higher STIP1 expression predicted a worse prognosis in patients with CRC, and Cox regression analysis revealed that STIP1 was an independent prognostic factor for overall survival and disease-free survival in patients with CRC. In conclusion, our results suggest that STIP1 acts as an oncogene in CRC and can therefore serve as a biomarker for the prognosis of patients with CRC. Copyright © 2018. Published by Elsevier Inc.

Evaluation of different pulverisation methods for RNA extraction in squash fruit: lyophilisation, cryogenic mill and mortar grinding.

PubMed

Román, Belén; González-Verdejo, Clara I; Peña, Francisco; Nadal, Salvador; Gómez, Pedro

2012-01-01

Quality and integrity of RNA are critical for transcription studies in plant molecular biology. In squash fruit and other high water content crops, the grinding of tissue with mortar and pestle in liquid nitrogen fails to produce a homogeneous and fine powered sample desirable to ensure a good penetration of the extraction reagent. To develop an improved pulverisation method to facilitate the homogenisation process of squash fruit tissue prior to RNA extraction without reducing quality and yield of the extracted RNA. Three methods of pulverisation, each followed by the same extraction protocol, were compared. The first approach consisted of the lyophilisation of the sample in order to remove the excess of water before grinding, the second one used a cryogenic mill and the control one a mortar grinding of frozen tissue. The quality of the isolated RNA was tested by carrying out a quantitative real time downstream amplification. In the three situations considered, mean values for A(260) /A(280) indicated minimal interference by proteins and RNA quality indicator (RQI) values were considered appropriate for quantitative real-time polymerase chain reaction (qRT-PCR) amplification. Successful qRT-PCR amplifications were obtained with cDNA isolated with the three protocols. Both apparatus can improve and facilitate the grinding step in the RNA extraction process in zucchini, resulting in isolated RNA of high quality and integrity as revealed by qRT-PCR downstream application. This is apparently the first time that a cryogenic mill has been used to prepare fruit samples for RNA extraction, thereby improving the sampling strategy because the fine powder obtained represents a homogeneous mix of the organ tissue. Copyright © 2012 John Wiley & Sons, Ltd.

Inner ear tissue preservation by rapid freezing: improving fixation by high-pressure freezing and hybrid methods.

PubMed

Bullen, A; Taylor, R R; Kachar, B; Moores, C; Fleck, R A; Forge, A

2014-09-01

In the preservation of tissues in as 'close to life' state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40 μm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Epidemiologic study of soft tissue rheumatism in Shantou and Taiyuan, China.

PubMed

Zeng, Qing-yu; Zang, Chang-hai; Lin, Ling; Chen, Su-biao; Li, Xiao-feng; Xiao, Zheng-yu; Dong, Hai-yuan; Zhang, Ai-lian; Chen, Ren

2010-08-05

Soft tissue rheumatism is a group of common rheumatic disorders reported in many countries. For investigating the prevalence rate of soft tissue rheumatism in different population in China, we carried out a population study in Shantou rural and Taiyuan urban area. Samples of 3915 adults in an urban area of Taiyuan, Shanxi Province, and 2350 in a rural area of Shantou, Guangdong Province were surveyed. Modified International League of Association for Rheumatology (ILAR)-Asia Pacific League of Association for Rheumatology (APLAR) Community Oriented Program for Control of Rheumatic Diseases (COPCORD) core questionnaire was implemented as screening tool. The positive responders were then all examined by rheumatologists. Prevalence rate of soft tissue rheumatism was 2.0% in Taiyuan, and 5.3% in Shantou. Rotator cuff (shoulder) tendinitis, adhesive capsulitis (frozen shoulder), lateral epicondylitis (tennis elbow), and digital flexor tenosynovitis (trigger finger) were the commonly seen soft tissue rheumatism in both areas. Tatarsalgia, plantar fasciitis, and De Quervain's tenosynovitis were more commonly seen in Shantou than that in Taiyuan. Only 1 case of fibromyalgia was found in Taiyuan and 2 cases in Shantou. The prevalence of soft tissue rheumatism varied with age, sex and occupation. Soft tissue rheumatism is common in Taiyuan and Shantou, China. The prevalence of soft tissue rheumatism was quite different with different geographic, environmental, and socioeconomic conditions; and varying with age, sex, and occupation. The prevalence of fibromyalgia is low in the present survey.

Distinction of brain tissue, low grade and high grade glioma with time-resolved fluorescence spectroscopy

PubMed Central

Yong, William H.; Butte, Pramod V.; Pikul, Brian K.; Jo, Javier A.; Fang, Qiyin; Papaioannou, Thanassis; Black, Keith L.; Marcu, Laura

2010-01-01

Neuropathology frozen section diagnoses are difficult in part because of the small tissue samples and the paucity of adjunctive rapid intraoperative stains. This study aims to explore the use of time-resolved laser-induced fluorescence spectroscopy as a rapid adjunctive tool for the diagnosis of glioma specimens and for distinction of glioma from normal tissues intraoperatively. Ten low grade gliomas, 15 high grade gliomas without necrosis, 6 high grade gliomas with necrosis and/or radiation effect, and 14 histologically uninvolved “normal” brain specimens are spectroscopicaly analyzed and contrasted. Tissue autofluorescence was induced with a pulsed Nitrogen laser (337 nm, 1.2 ns) and the transient intensity decay profiles were recorded in the 370-500 nm spectral range with a fast digitized (0.2 ns time resolution). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site were used for tissue characterization. A linear discriminant analysis diagnostic algorithm was used for tissue classification. Both low and high grade gliomas can be distinguished from histologically uninvolved cerebral cortex and white matter with high accuracy (above 90%). In addition, the presence or absence of treatment effect and/or necrosis can be identified in high grade gliomas. Taking advantage of tissue autofluorescence, this technique facilitates a direct and rapid investigation of surgically obtained tissue. PMID:16368511

Optimized protocol for quantitative multiple reaction monitoring-based proteomic analysis of formalin-fixed, paraffin embedded tissues

PubMed Central

Kennedy, Jacob J.; Whiteaker, Jeffrey R.; Schoenherr, Regine M.; Yan, Ping; Allison, Kimberly; Shipley, Melissa; Lerch, Melissa; Hoofnagle, Andrew N.; Baird, Geoffrey Stuart; Paulovich, Amanda G.

2016-01-01

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin embedded (FFPE) tissues. While the feasibility of using targeted, multiple reaction monitoring-mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e. 9 processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R2 = 0.94) and immuno-MRM (R2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

Raman molecular imaging of brain frozen tissue sections.

PubMed

Kast, Rachel E; Auner, Gregory W; Rosenblum, Mark L; Mikkelsen, Tom; Yurgelevic, Sally M; Raghunathan, Aditya; Poisson, Laila M; Kalkanis, Steven N

2014-10-01

Raman spectroscopy provides a molecular signature of the region being studied. It is ideal for neurosurgical applications because it is non-destructive, label-free, not impacted by water concentration, and can map an entire region of tissue. The objective of this paper is to demonstrate the meaningful spatial molecular information provided by Raman spectroscopy for identification of regions of normal brain, necrosis, diffusely infiltrating glioma and solid glioblastoma (GBM). Five frozen section tissues (1 normal, 1 necrotic, 1 GBM, and 2 infiltrating glioma) were mapped in their entirety using a 300-µm-square step size. Smaller regions of interest were also mapped using a 25-µm step size. The relative concentrations of relevant biomolecules were mapped across all tissues and compared with adjacent hematoxylin and eosin-stained sections, allowing identification of normal, GBM, and necrotic regions. Raman peaks and peak ratios mapped included 1003, 1313, 1431, 1585, and 1659 cm(-1). Tissue maps identified boundaries of grey and white matter, necrosis, GBM, and infiltrating tumor. Complementary information, including relative concentration of lipids, protein, nucleic acid, and hemoglobin, was presented in a manner which can be easily adapted for in vivo tissue mapping. Raman spectroscopy can successfully provide label-free imaging of tissue characteristics with high accuracy. It can be translated to a surgical or laboratory tool for rapid, non-destructive imaging of tumor margins.

Description of the EuroTARGET cohort: A European collaborative project on TArgeted therapy in renal cell cancer-GEnetic- and tumor-related biomarkers for response and toxicity.

PubMed

van der Zanden, Loes F M; Vermeulen, Sita H; Oskarsdottir, Arna; Maurits, Jake S F; Diekstra, Meta H M; Ambert, Valentin; Cambon-Thomsen, Anne; Castellano, Daniel; Fritsch, Achim; Garcia Donas, Jesus; Guarch Troyas, Rosa; Guchelaar, Henk-Jan; Hartmann, Arndt; Hulsbergen-van de Kaa, Christina; Jaehde, Ulrich; Junker, Kerstin; Martinez-Cardus, Anna; Masson, Gisli; Oosterwijk-Wakka, Jeannette; Radu, Marius T; Rafnar, Thorunn; Rodriguez-Antona, Cristina; Roessler, Max; Ruijtenbeek, Rob; Stefansson, Kari; Warren, Anne; Wessels, Lodewyk; Eisen, Tim; Kiemeney, Lambertus A L M; Oosterwijk, Egbert

2017-08-01

For patients with metastatic renal cell cancer (mRCC), treatment choice is mainly based on clinical parameters. With many treatments available and the limited response to treatment and associated toxicities, there is much interest in identifying better biomarkers for personalized treatment. EuroTARGET aims to identify and characterize host- and tumor-related biomarkers for prediction of response to tyrosine kinase inhibitor therapy in mRCC. Here, we describe the EuroTARGET mRCC patient cohort. EuroTARGET is a European collaborative project designed as an observational study for which patients with mRCC were recruited prospectively in 62 centers. In addition, 462 patients with mRCC from previous studies were included. Detailed clinical information (baseline and follow-up) from all patients was entered in web-based case record forms. Blood was collected for germline DNA and pharmacokinetic/pharmacodynamic analyses and, where available, fresh-frozen tumor material was collected to perform tumor DNA, RNA, kinome, and methylome analyses. In total, 1,210 patients with mRCC were included. Of these, 920 received a tyrosine kinase inhibitor as first-line targeted treatment (sunitinib [N = 713, 78%], sorafenib [N = 41, 4%], or pazopanib [N = 166, 18%]) and had at least 6 months of outcome assessment (median follow-up 15.3 months [interquartile range: 8.5-30.2 months]). Germline DNA samples were available from 824 of these patients, fresh-frozen tumor material from 142 patients, fresh-frozen normal kidney tissue from 95 patients, and tissue microarrays created from formalin-fixed paraffin-embedded tumor material from 247 patients. Of the 920 patients, germline DNA variant chip data were successfully generated for 811 patients (Illumina HumanOmniExpress BeadChip). For 80 patients, next-generation exome sequencing of germline and tumor DNA was performed, tumor RNA sequencing was performed for 124 patients, kinome activity measured and processed for 121 patients (PamChip), and methylome data (Illumina Infinium HumanMethylation450 BeadChip) were created for 116 RCC tissues (and 23 normal kidney tissues). For 73 out of the 920 patients, all platform data types were generated. In addition, 40 patients were included in a pharmacokinetic/pharmacodynamic phase IV substudy. Analysis of EuroTARGET cohort data will contribute to personalization of therapy for patients with mRCC. The extensive clinical data and multiplatform EuroTARGET data will be freely available. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Differentiation of fresh and frozen-thawed fish samples using Raman spectroscopy coupled with chemometric analysis.

PubMed

Velioğlu, Hasan Murat; Temiz, Havva Tümay; Boyaci, Ismail Hakki

2015-04-01

The potential of Raman spectroscopy was investigated in terms of its capability to discriminate the species of the fish samples and determine their freshness according to the number of freezing/thawing cycles they exposed. Species discrimination analysis was carried out on sixty-four fish samples from six different species, namely horse mackerel (Trachurus trachurus), European anchovy (Engraulis encrasicolus), red mullet (Mullus surmuletus), Bluefish (Pomatamus saltatrix), Atlantic salmon (Salmo salar) and flying gurnard (Trigla lucerna). Afterwards, fish samples were exposed to different numbers of freezing/thawing cycles and separated into three batches, namely (i) fresh, (ii) once frozen-thawed (OF) and (iii) twice frozen-thawed (TF) samples, in order to perform the freshness analysis. Raman data collected were used as inputs for chemometric analysis, which enabled us to develop two main PCA models to successfully terminate the studies for both species discrimination and freshness determination analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Production of healthy cloned mice from bodies frozen at -20 degrees C for 16 years.

PubMed

Wakayama, Sayaka; Ohta, Hiroshi; Hikichi, Takafusa; Mizutani, Eiji; Iwaki, Takamasa; Kanagawa, Osami; Wakayama, Teruhiko

2008-11-11

Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the "resurrection" of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at -20 degrees C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to "resurrect" animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation.

Production of healthy cloned mice from bodies frozen at −20°C for 16 years

PubMed Central

Wakayama, Sayaka; Ohta, Hiroshi; Hikichi, Takafusa; Mizutani, Eiji; Iwaki, Takamasa; Kanagawa, Osami; Wakayama, Teruhiko

2008-01-01

Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the “resurrection” of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at −20 °C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to “resurrect” animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation. PMID:18981419

Coherent Raman Scattering Microscopy for Evaluation of Head and Neck Carcinoma.

PubMed

Hoesli, Rebecca C; Orringer, Daniel A; McHugh, Jonathan B; Spector, Matthew E

2017-09-01

Objective We aim to describe a novel, label-free, real-time imaging technique, coherent Raman scattering (CRS) microscopy, for histopathological evaluation of head and neck cancer. We evaluated the ability of CRS microscopy to delineate between tumor and nonneoplastic tissue in tissue samples from patients with head and neck cancer. Study Design Prospective case series. Setting Tertiary care medical center. Subjects and Methods Patients eligible were surgical candidates with biopsy-proven, previously untreated head and neck carcinoma and were consented preoperatively for participation in this study. Tissue was collected from 50 patients, and after confirmation of tumor and normal specimens by hematoxylin and eosin (H&E), there were 42 tumor samples and 42 normal adjacent controls. Results There were 42 confirmed carcinoma specimens on H&E, and CRS microscopy identified 37 as carcinoma. Of the 42 normal specimens, CRS microscopy identified 40 as normal. This resulted in a sensitivity of 88.1% and specificity of 95.2% in distinguishing between neoplastic and nonneoplastic images. Conclusion CRS microscopy is a unique label-free imaging technique that can provide rapid, high-resolution images and can accurately determine the presence of head and neck carcinoma. This holds potential for implementation into standard practice, allowing frozen margin evaluation even at institutions without a histopathology laboratory.

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

NASA Technical Reports Server (NTRS)

Choi, S. Y.; Lai, S.; Klotz, R.; Popova, Y.; Chakravarty, K.; Beegle, J. E.; Wigley, C. L.; Globus, R. K.

2014-01-01

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development. Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science return. Livers and spleens from mice were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection procedures including storage at minus 80 degrees Centigrade for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RNA Integrity Number (RIN) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNA, later) and liver (fast-freezing) at various time points post-euthanasia (from 5 minutes up to 105 minutes). The RNA recovered was of high quality (spleen, RIN greater than 8; liver, RIN greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at 105 minutes. Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) High-quality RNA samples from many different tissues can be recovered by dissection following prolonged storage of the tissue in situ at minus 80 degrees Centigrade. These findings have relevance both to high-value, ground-based experiments when sample collection capability is severely constrained, and to future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

Effect of prostatic fluid on the quality of fresh and frozen-thawed canine epididymal spermatozoa.

PubMed

Korochkina, E; Johannisson, A; Goodla, Lavanya; Morrell, J M; Axner, E

2014-12-01

Canine epididymal spermatozoa have a low freeze-tolerance ability compared with ejaculated spermatozoa, which could arise from the absence of prostatic fluid (PF). Therefore, the purpose of this work was to elucidate the influence of PF on the quality of canine epididymal sperm before and after freezing. Caudae epididymides were retrieved from eight dogs after routine castration. Spermatozoa were released by slicing the tissue and were extended in either Tris solution or PF before freezing. Frozen sperm samples were thawed at 70 °C for 8 seconds in a waterbath. Sperm concentration, motility using computer-assisted sperm analysis, morphology, plasma membrane, acrosome and chromatin integrity were assessed in the fresh sperm samples (after 20 minutes incubation) and at 0 and 4 hours after thawing. Progressive motility, distance straight line, distance average path, average path velocity, curvilinear velocity, straight line velocity, straightness, linearity, wobble, and beat cross frequency were significantly increased after extraction into PF. There was a higher proportion of spermatozoa with DNA damage in the PF treatment group at 4 hours after thawing than in the Tris treatment group (15.8% vs. 6.7%, P < 0.05). These results suggest that the addition of PF to canine spermatozoa activates sperm motility in fresh spermatozoa but has a negative effect on chromatin integrity after freezing-thawing. Copyright © 2014 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Skyhar, M.J.; Danzig, L.A.; Hargens, A.R.

Twelve freshly killed mature male rabbits were used to study the effects of continuous passive motion (CPM) on regional and overall nonvascular nutritional pathways of the anterior cruciate ligament (ACL). One hundred fifty microcuries of /sup 35/S-sulfate was injected intraarticularly into each knee joint. The right knee underwent CPM for 1 hour, while the left knee remained immobilized. Both knee joints were then isolated and immediately frozen. The ACLs were removed while still mostly frozen, and sectioned into anterior, middle, and posterior thirds for the six rabbits in Group 1, and proximal, middle, and distal thirds for the six rabbitsmore » in Group 2. In addition, quadriceps tendon samples were harvested from each limb of three rabbits. After appropriate processing, all samples were counted in a scintillation counter, and counts per minute per milligram of tissue were calculated. There was significantly higher uptake in rest extremity ACLs compared to CPM extremity ACLs (P = 0.0001). No significant difference was demonstrated in regional uptake comparing respective thirds of the ACL in either Group 1 or Group 2. Quadriceps tendon uptake trended higher in the limbs exposed to CPM compared to those maintained at rest (P = 0.14). The ACL uses diffusion as a primary nutrient pathway. CPM does not increase nutrient uptake by the ACL in this avascular model, but CPM may facilitate transport of metabolites out of the joint. No regional differences in uptake within the ACL occurred in either group.« less

Histology as a Valid Tool To Differentiate Fresh from Frozen-Thawed Marinated Fish.

PubMed

Meistro, Serena; Pezzolato, Marzia; Muscolino, Daniele; Giarratana, Filippo; Baioni, Elisa; Panebianco, Antonio; Bozzetta, Elena

2016-08-01

European Commission Regulation (EU) 1276/2011 requires that fishery products intended for raw consumption be frozen at -20°C for not less than 24 h or at -35°C for at least 15 h in order to kill viable parasites other than trematodes. But because marinating processes are not always effective in destroying nematode larvae, raw marinated fish preparations should be frozen before consumption. This study evaluated the performance of a standardized histological method to distinguish between fresh and frozen-thawed raw marinated fish. Sixty anchovy (Engraulis encrasicolus) fillets were sampled: 30 were marinated at +4°C for 24 h, and 30 were frozen at -20°C for 24 h before being marinated for 24 h. All 60 samples were fixed in formalin, processed for paraffin embedding, cut, and stained with hematoxylin and eosin. The slide preparations were examined microscopically by three independent histopathologists and classified as frozen-thawed or negative according to standard operating procedure criteria in use at our laboratory. Performance evaluation of the method showed 100% sensitivity (95% confidence interval [CI], 88.4 to 100%) and 100% specificity (95% CI, 88.4 to 100%), and the interrater agreement (Cohen's kappa) was 1 (95% CI, 0.85 to 1). Histology proved a valid and reliable tool to distinguish fresh from frozen-thawed marinated fish. It can be applied to deliver safe raw fishery products to consumers in order to minimize the risk of anisakidosis.

Colorectal cancer detection by hyperspectral imaging using fluorescence excitation scanning

NASA Astrophysics Data System (ADS)

Leavesley, Silas J.; Deal, Joshua; Hill, Shante; Martin, Will A.; Lall, Malvika; Lopez, Carmen; Rider, Paul F.; Rich, Thomas C.; Boudreaux, Carole W.

2018-02-01

Hyperspectral imaging technologies have shown great promise for biomedical applications. These techniques have been especially useful for detection of molecular events and characterization of cell, tissue, and biomaterial composition. Unfortunately, hyperspectral imaging technologies have been slow to translate to clinical devices - likely due to increased cost and complexity of the technology as well as long acquisition times often required to sample a spectral image. We have demonstrated that hyperspectral imaging approaches which scan the fluorescence excitation spectrum can provide increased signal strength and faster imaging, compared to traditional emission-scanning approaches. We have also demonstrated that excitation-scanning approaches may be able to detect spectral differences between colonic adenomas and adenocarcinomas and normal mucosa in flash-frozen tissues. Here, we report feasibility results from using excitation-scanning hyperspectral imaging to screen pairs of fresh tumoral and nontumoral colorectal tissues. Tissues were imaged using a novel hyperspectral imaging fluorescence excitation scanning microscope, sampling a wavelength range of 360-550 nm, at 5 nm increments. Image data were corrected to achieve a NIST-traceable flat spectral response. Image data were then analyzed using a range of supervised and unsupervised classification approaches within ENVI software (Harris Geospatial Solutions). Supervised classification resulted in >99% accuracy for single-patient image data, but only 64% accuracy for multi-patient classification (n=9 to date), with the drop in accuracy due to increased false-positive detection rates. Hence, initial data indicate that this approach may be a viable detection approach, but that larger patient sample sizes need to be evaluated and the effects of inter-patient variability studied.

Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons

NASA Technical Reports Server (NTRS)

Chalmers, G. R.; Edgerton, V. R.

1989-01-01

The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.

A new rapid immunohistochemical staining technique using the EnVision antibody complex.

PubMed

Kämmerer, U; Kapp, M; Gassel, A M; Richter, T; Tank, C; Dietl, J; Ruck, P

2001-05-01

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3'diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623-630, 2001)

Mining the archives: a cross-platform analysis of gene ...

EPA Pesticide Factsheets

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 of nucleic acids. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues using two DNA microarray protocols and two whole transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other three methods by having the highest correlations of differentially expressed genes (DEGs) and best overlap of pathways between FRO and FFPE groups. We next tested the effect of sample time in formalin (18 hours or 3 weeks) on gene expression profiles. Hierarchical clustering of the datasets indicated that test article treatment, and not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18-hour and 3-week FFPE samples compared to FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of age in FFPE block on genomic profiles. RNA-seq analysis of 8-, 19-, and 26-year-old control blocks using the ribo-depletion protocol resulted in comparable quality metrics, inc

Irradiation Does Not Increase the Penetration Depth of Doxorubicin in Normal Tissue After Pressurized Intra-peritoneal Aerosol Chemotherapy (PIPAC) in an Ex Vivo Model.

PubMed

Khosrawipour, Veria; Bellendorf, Alexander; Khosrawipour, Carolina; Hedayat-Pour, Yousef; Diaz-Carballo, David; Förster, Eckart; Mücke, Ralph; Kabakci, Burak; Adamietz, Irenäus Anton; Fakhrian, Khashayar

To compare the impact of single fractional with bi-fractional irradiation on the depth of doxorubicin penetration into the normal tissue after pressurized intra-peritoneal aerosol chemotherapy (PIPAC) in our ex vivo model. Fresh post mortem swine peritoneum was cut into 12 proportional sections. Two control samples were treated with PIPAC only (no irradiation), one sample on day 1, the other on day 2. Five samples were irradiated with 1, 2, 4, 7 or 14 Gy followed by PIPAC. Four samples were treated on day one with 0.5, 1, 2, 3.5 or 7 Gy and with the same radiation dose 24 h later followed by PIPAC. Doxorubicin was aerosolized in an ex vivo PIPAC model at 12 mmHg/36°C. In-tissue doxorubicin penetration was measured using fluorescence microscopy on frozen thin sections. Doxorubicin penetration (DP) after PIPAC for the control samples was 407 μm and 373 μm, respectively. DP for samples with single fraction irradiation was 396 μm after 1 Gy, 384 μm after 2 Gy, 327 μm after 4 Gy, 280 μm after 7 Gy and 243 μm after 14 Gy. DP for samples with 2 fractions of irradiation was 376 μm after 0.5+0.5 Gy, 363 μm after 1+1 Gy, 372 μm after 2+2 Gy, 341 μm after 3.5+3.5 and 301 μm after 7+7 Gy irradiation. Fractionating of the irradiation did not significantly change DP into normal tissue. Irradiation does not increase the penetration depth of doxorubicin into the normal tissue but might have a limiting impact on penetration and distribution of doxorubicin. Further studies are warranted to investigate the impact of addition of irradiation to PIPAC of tumor cells and to find out if irradiation can be used safely as chemopotenting agent for patients with peritoneal metastases treated with PIPAC. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Cryopreserved and frozen hyaline cartilage imaged by environmental scanning electron microscope. An experimental and prospective study.

PubMed

Sastre, Sergi; Suso, Santiago; Segur, Josep-Maria; Bori, Guillem; Carbonell, José-Antonio; Agustí, Elba; Nuñez, Montse

2008-08-01

To obtain images of the articular surface of osteochondral grafts (fresh, frozen, and cryopreserved in RPMI) using an environmental scanning electron microscope (ESEM). To evaluate and compare the main morphological aspects of the chondral surface of the fresh, frozen, and cryopreserved grafts as visualized via ESEM. The study was based on osteochondral fragments from the internal condyle of the knee joint of New Zealand rabbits, corresponding to the chondral surface from fresh, frozen, and cryopreserved samples. One hundred ESEM images were obtained from each group and then classified according to a validated system. The kappa index and the corresponding concordance index were calculated, and the groups were compared by Pearson's chi-squared test (p < 0.05). The articular surface of cryopreserved osteochondral grafts had fewer even surfaces and filled lacunae and a higher number of empty lacunae as compared to fresh samples; these differences correspond to images of cell membrane lesions that lead to destruction of the chondrocyte. Frozen grafts showed more hillocky and knobby surfaces than did fresh grafts; they also had a greater number of empty chondrocyte lacunae. ESEM is useful for obtaining images of the surface of osteochondral grafts. When compared to fresh samples, cryopreservation in RPMI medium produces changes in the surface of hyaline cartilage, but to a lesser extent than those produced by freezing.

Improved selection of cortical ovarian strips for autotransplantation of ovarian tissue using full-field optical coherence tomography (FFOCT)

NASA Astrophysics Data System (ADS)

Stegehuis, Paulien L.; Peters, Inge T. A.; Eggermont, Jeroen; Kuppen, Peter J. K.; Trimbos, J. Baptist; Lelieveldt, Boudewijn P. F.; van de Velde, Cornelis J. H.; Bosse, Tjalling; Dijkstra, Jouke; Vahrmeijer, Alexander L.

2016-02-01

Premature ovarian failure is a major concern in women of reproductive age who undergo gonadotoxic cancer treatment. Autotransplantation of frozen-thawed cortical ovarian tissue allows the immediate start of cancer treatment, but risks reintroduction of cancer. Current tumor detection methods compromise the ovarian tissue's viability and can therefore only be used to exclude the presence of metastases in the cortical ovarian strips that are not transplanted. A non-invasive method is needed that can be used to exclude metastases in the actual ovarian autografts without affecting the tissue's viability. In this study we applied FFOCT - a non-fixative technique that uses white light interferometry to make highresolution images (1μm isotropic) of fresh tissue - to study healthy and malignant ovarian tissue. We created an image atlas of healthy ovarian tissues from premenopausal patients and ovarian tissues with breast cancer metastases. To get the best possible match between hematoxylin-and-eosin stained slides and FFOCT images formalinfixed paraffin-embedded tissue samples were deparaffinized and FFOCT images were acquired within a few minutes. FFOCT images were compared with histology images. All normal structures such as follicles in all phases, inclusion cysts, blood vessels, corpora lutea, and corpora albicantia were clearly recognizable. Ovarian metastases could be well distinguished from normal ovarian tissue. FFOCT is a promising technique in the field of fertility preservation: metastases can be detected and additionally cortical ovarian strips can be selected on the basis of high follicle density.

United States Transuranium and Uranium Registries. Annual Report, October 1, 1993--September 30, 1994

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kathren, R.L.; Harwick, L.A.

1995-08-01

This report summarizes the salient activities and progress of the United States Transuranium. and Uranium Registries for the period October 1, 1993 through September 30, 1994, along with details of specific programs areas including the National Human Radiobiology Tissue Repository (NHRTR) and tissue radiochemistry analysis project. Responsibility for tissue radioanalysis was transferred from Los Alamos National Laboratory to Washington State University in February 1994. The University of Washington was selected as the Quality Assurance/Quality Control laboratory and a three way intercomparison with them and LANL has been initiated. The results of the initial alpha spectrometry intercomparison showed excellent agreement amongmore » the laboratories and are documented in full in the Appendices to the report. The NHRTR serves as the initial point of receipt for samples received from participants in the USTUR program. Samples are weighed, divided, and reweighed, and a portion retained by the NHRTR as backup or for use in other studies. Tissue specimens retained in the NHRTR are maintained frozen at -70 C and include not only those from USTUR registrants but also those from the radium dial painter and thorium worker studies formerly conducted by Argonne National Laboratory. In addition, there are fixed tissues and a large collection of histopathology slides from all the studies, plus about 20,000 individual solutions derived from donated tissues. These tissues and tissue related materials are made available to other investigators for legitimate research purposes. Ratios of the concentration of actinides in various tissues have been used to evaluate the biokinetics, and retention half times of plutonium and americium. Retention half times for plutonium in various soft tissues range from 10-20 y except for the testes for which a retention half time of 58 y was observed. For americium, the retention half time in various soft tissues studied was 2.2-3.5 y.« less

Fluorescence confocal mosaicing microscopy of basal cell carcinomas ex vivo: demonstration of rapid surgical pathology with high sensitivity and specificity

NASA Astrophysics Data System (ADS)

Gareau, Daniel S.; Karen, Julie K.; Dusza, Stephen W.; Tudisco, Marie; Nehal, Kishwer S.; Rajadhyaksha, Milind

2009-02-01

Mohs surgery, for the precise removal of basal cell carcinomas (BCCs), consists of a series of excisions guided by the surgeon's examination of the frozen histology of the previous excision. The histology reveals atypical nuclear morphology, identifying cancer. The preparation of frozen histology is accurate but labor-intensive and slow. Nuclear pathology can be achieved by staining with acridine orange (1 mM, 20 s) BCCs in Mohs surgical skin excisions within 5-9 minutes, compared to 20-45 for frozen histology. For clinical utility, images must have high contrast and high resolution. We report tumor contrast of 10-100 fold over the background dermis and submicron (diffraction limited) resolution over a cm field of view. BCCs were detected with an overall sensitivity of 96.6%, specificity of 89.2%, positive predictive value of 93.0% and negative predictive value of 94.7%. The technique was therefore accurate for normal tissue as well as tumor. We conclude that fluorescence confocal mosaicing serves as a sensitive and rapid pathological tool. Beyond Mohs surgery, this technology may be extended to suit other pathological needs with the development of new contrast agents. The technique reported here accurately detects all subtypes of BCC in skin excisions, including the large nodular, small micronodular, and tiny sclerodermaform tumors. However, this technique may be applicable to imaging tissue that is larger, more irregular and of various mechanical compliances with further engineering of the tissue mounting and staging mechanisms.

[Establishment of the experimental animal model of Babesia microti].

PubMed

Lu, Yan; Cai, Yu-Chun; Chen, Shao-Hong; Chen, Jia-Xu; Guo, Jian; Chen, Mu-Xin; Ai, Lin; Chu, Yan-Hong; Chen, Zhuo; Zhou, Xiao-Nong

2012-12-30

To establish the experimental animal model for the study of Babesia microti. BALB/c mice, immunosuppressive BALB/c mice, SCID mice and NOD-SCID mice were inoculated with B. microti-infected red blood cells (RBC) by intraperitoneal injection respectively. After inoculation, thin blood smears were prepared every day, stained with Giemsa staining and examined for the presence of parasitemia. Three mice were dissected to examine the infectivity in bone marrow, brain, spleen, heart, lung, kidney and liver tissues. The infection rate of erythrocytes in different tissues was recorded, and the relationship between the infectivity of tissues and infection rate in peripheral blood was analyzed. Blood samples infected with B. microti were preserved in liquid nitrogen with dimethyl sulfoxide (DMSO) for 2 months. The thawed parasitized blood was injected into the BALB/c mice by same route and the parasitemia was monitored. The four kinds of mice were all infected by B. microti with parasitemia. The percentage of parasitized red blood cells from peripheral blood were 82.4% (BALB/c mice, d7), 73.2% (immunosuppressive BALB/c mice, d5), 86.4% (SCID mice, d8) and 72.5% (NOD-SCID mice, d8) at the maximum, respectively. Parasitemia decreased rapidly in BALB/c mice, whereas decreased slowly in immunosuppressive BALB/c mice. Only the parasitemia in SCID mice and NOD-SCID mice decreased significantly and tended to picking up again. The parasites were observed in RBCs from bone marrow, brain, spleen, heart, lung, kidney and liver tissues. The infection rate of erythrocytes in tissues increased with an increase of infection in peripheral blood. After cryopreservation, the parasites proliferated in BALB/c mice. Parasitemia appeared after inoculation with frozen infected blood two days later than that of fresh infected blood. The infection rate reached its peak after inoculation with frozen infected blood one day later than that of fresh infected blood. The experimental animal model of B. microti has been established. The infection rate of erythrocytes is related to the immune status of the host mice.

Disparities in Intratumoral Steroidogenesis

DTIC Science & Technology

2013-07-01

Tuesday shipment only) by overnight express for next day delivery on dry ice. Frozen specimens will be shipped on dry ice to the following address...Samples will be labeled with the study subject number and date of surgery. Frozen samples will be batch shipped (Monday and Tuesday shipment only) by... Morris MJ, de Bono JS, Ryan CJ, Denmeade SR, Smith MR, et al. Phase II multicenter study of abiraterone acetate plus prednisone therapy in patients

A rapid and efficient DNA extraction protocol from fresh and frozen human blood samples.

PubMed

Guha, Pokhraj; Das, Avishek; Dutta, Somit; Chaudhuri, Tapas Kumar

2018-01-01

Different methods available for extraction of human genomic DNA suffer from one or more drawbacks including low yield, compromised quality, cost, time consumption, use of toxic organic solvents, and many more. Herein, we aimed to develop a method to extract DNA from 500 μL of fresh or frozen human blood. Five hundred microliters of fresh and frozen human blood samples were used for standardization of the extraction procedure. Absorbance at 260 and 280 nm, respectively, (A 260 /A 280 ) were estimated to check the quality and quantity of the extracted DNA sample. Qualitative assessment of the extracted DNA was checked by Polymerase Chain reaction and double digestion of the DNA sample. Our protocol resulted in average yield of 22±2.97 μg and 20.5±3.97 μg from 500 μL of fresh and frozen blood, respectively, which were comparable to many reference protocols and kits. Besides yielding bulk amount of DNA, our protocol is rapid, economical, and avoids toxic organic solvents such as Phenol. Due to unaffected quality, the DNA is suitable for downstream applications. The protocol may also be useful for pursuing basic molecular researches in laboratories having limited funds. © 2017 Wiley Periodicals, Inc.

The effect of cryopreservation on goat semen characteristics related to sperm freezability.

PubMed

Dorado, J; Muñoz-Serrano, A; Hidalgo, M

2010-08-01

Seminal quality parameters were used to evaluate the effect of freeze-thawing procedure on goat sperm characteristics, and to relate possible changes in sperm parameters to cryopreservation success. Semen samples (n=110) were frozen with TRIS and milk-based extenders and thawed. Sperm quality parameters (motility, morphology and acrosome) were compared between fresh and frozen-thawed samples. Sperm freezability was judged by classifying the semen samples as "suitable" or "not suitable" according to the sperm quality parameters assessed before and after thawing. Fertility data was obtained after cervical insemination with frozen semen doses. The ejaculates were grouped into two categories according to their fertility results. In experiment 1, significant differences were found between semen extenders (P<0.001), bucks (P<0.05) and ejaculates within the same male (P<0.05) in terms of sperm quality. There was no seasonal effect (P>0.05) on the majority of the sperm parameters assessed after thawing. Moreover, significant differences (P<0.001) in semen parameters assessed in fresh semen and frozen-thawed samples were found between groups. The effect of the freeze-thawing procedure on sperm quality parameters was also different (P<0.05) between extenders within the same group. The number of sperm quality parameters that had changed after cryopreservation was lower in "suitable" semen samples before and after thawing. In experiment 2, no differences (P>0.05) in semen parameters assessed in fresh semen and frozen-thawed samples were found between groups. The effect of freezing and thawing on sperm quality parameters were different (P<0.05) between extenders within the same group. Only mean beat cross frequency (BCF) values were significantly higher (P<0.05) in TRIS diluted samples that led to successful pregnancies after artificial insemination. In conclusion, CASA-derived motility parameters, together with traditional semen assessment methods, give valuable information on sperm quality before and after freezing. Therefore, the identification of ejaculates as "good" or "bad" based on fresh and post-thaw semen parameters studied in the present experiment were good indicators of goat semen freezability, although the fertilizing capacity of frozen-thawed goat spermatozoa are not revealed by this quality study. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Bioimpedance spectroscopy can precisely discriminate human breast carcinoma from benign tumors.

PubMed

Du, Zhenggui; Wan, Hangyu; Chen, Yu; Pu, Yang; Wang, Xiaodong

2017-01-01

Intraoperative frozen pathology is critical when a breast tumor is not diagnosed before surgery. However, frozen tumor tissues always present various microscopic morphologies, leading to a high misdiagnose rate from frozen section examination. Thus, we aimed to identify breast tumors using bioimpedance spectroscopy (BIS), a technology that measures the tissues' impedance. We collected and measured 976 specimens from breast patients during surgery, including 581 breast cancers, 190 benign tumors, and 205 normal mammary gland tissues. After measurement, Cole-Cole curves were generated by a bioimpedance analyzer and parameters R0/R∞, fc, and α were calculated from the curve. The Cole-Cole curves showed a trend to differentiate mammary gland, benign tumors, and cancer. However, there were some curves overlapped with other groups, showing that it is not an ideal model. Subsequent univariate analysis of R0/R∞, fc, and α showed significant differences between benign tumor and cancer. However, receiver operating characteristic (ROC) analysis indicated the diagnostic value of fc and R0/R∞ were not superior to frozen sections (area under curve [AUC] = 0.836 and 0.849, respectively), and α was useless in diagnosis (AUC = 0.596). After further research, we found a scatter diagram that showed a synergistic effect of the R0/R∞ and fc, in discriminating cancer from benign tumors. Thus, we used multivariate analysis, which revealed that these two parameters were independent predictors, to combine them. A simplified equation, RF = 0.2fc + 3.6R0/R∞, based on multivariate analysis was developed. The ROC curve for RF' showed an AUC = 0.939, and the sensitivity and specificity were 82.62% and 95.79%, respectively. To match a clinical setting, the diagnostic criteria were set at 6.91 and 12.9 for negative and positive diagnosis, respectively. In conclusion, RF' derived from BIS can discriminate benign tumor and cancers, and integrated criteria were developed for diagnosis.

Ventricular, but not atrial, M2-muscarinic receptors increase in the canine pacing-overdrive model of heart failure.

PubMed

Wilkinson, M; Giles, A; Armour, J A; Cardinal, R

1996-01-01

To investigate the effects of heart failure induced by chronic rapid ventricular pacing (six weeks) on canine atrial and ventricular muscarinic receptors. Dogs (n = 4) were fitted with a bipolar pacing electrode connected to a Medtronic pacemaker set at 240 stimuli/min. Pacing was maintained for six weeks. Tissue samples obtained from the left atrium and ventral wall of the left ventricle were frozen at -70 degrees C. Control tissue was obtained from normal dogs (n = 6) following anesthesia and thoracotomy. M2-muscarinic receptors were characterized and quantified in tissue micropunches using the hydrophilic ligand [3H] N-methyl-scopolamine (NMS). Cardiac tissue bound [3H] NMS with the specificity of an M2 subtype. Tachycardia-induced heart failure did not affect atrial muscarinic receptors but signify left ventricular myocytes (control 160.0 +/- 10.0 fmol/mg protein versus heart failure 245.0 +/- 25.0 fmol/mg protein; P < 0.01). Canine ventricular muscarinic receptors display a specificity for the M2 subtype. In contrast to previous work, tachycardia-induced heart failure was accompanied by an increase (+ 53%) in ventricular, but not atrial, M2 receptors compared with normal dogs.

Boric acid-enhanced embedding medium for cryomicrotomy.

PubMed

Lim, Jin Ik; Park, Hun-Kuk

2012-05-01

A polyvinyl alcohol (PVA)/polyethylene glycol (PEG)-based resin is commonly used as a cryoembedding medium for the histological analysis of frozen tissue sections. However, it is not easy to obtain sufficient numbers of satisfactory reproducible sections owing to the differences between the mechanical properties of the medium and embedded tissue and the low cohesive force of the medium. We describe a modified PVA-based cryoembedding medium, composed of PVA (10wt% and 15wt%) with the addition of boric acid (from 0 to 5wt%), that can improve the sectioning properties and efficiency of frozen tissue for histological analysis. The amount of load under the same compressive displacement as well as cohesive force increased with increasing boric acid and PVA contents. 15wt% PVA and 3wt% boric acid was determined as an optimal composition for cryoembedding material based on the sectioning efficiency measured by the numbers of unimpaired sectioned slices and the amount of load under the same compressive displacement test. On the basis of the results of routine hematoxylin and eosin staining of cryosections of tissue embedded in a medium with 3wt% boric acid and PVA, it was concluded that the modified PVA cryoembedding medium can improve the efficiency of cryosectioning for subsequent histological or histochemical analysis of various tissues. Copyright © 2011 Elsevier GmbH. All rights reserved.

Understanding the response of winter cereals to freezing stress through freeze-fixation and 3d reconstruction of ice formation in crowns

USDA-ARS?s Scientific Manuscript database

One of the most difficult aspects of understanding mechanisms involved in winterhardiness is knowing where ice is formed and how it interacts with tissues in the frozen state. Many tissues recover and change shape during thawing which prevents a clear picture of ice formation and how individual cel...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundaramurthi, Prakash; Patapoff, Thomas W.; Suryanarayanan, Raj

To study the crystallization of trehalose in frozen solutions and to understand the phase transitions during the entire freeze-drying cycle. Aqueous trehalose solution was cooled to -40 C in a custom-designed sample holder. The frozen solution was warmed to -18 C and annealed, and then dried in the sample chamber of the diffractometer. XRD patterns were continuously collected during cooling, annealing and drying. After cooling, hexagonal ice was the only crystalline phase observed. However, upon annealing, crystallization of trehalose dihydrate was evident. Seeding the frozen solution accelerated the solute crystallization. Thus, phase separation of the lyoprotectant was observed in frozenmore » solutions. During drying, dehydration of trehalose dihydrate yielded a substantially amorphous anhydrous trehalose. Crystallization of trehalose, as trehalose dihydrate, was observed in frozen solutions. The dehydration of the crystalline trehalose dihydrate to substantially amorphous anhydrate occurred during drying. Therefore, analyzing the final lyophile will not reveal crystallization of the lyoprotectant during freeze-drying. The lyoprotectant crystallization can only become evident by continuous monitoring of the system during the entire freeze-drying cycle. In light of the phase separation of trehalose in frozen solutions, its ability to serve as a lyoprotectant warrants further investigation.« less

MicroRNAs are suitable for assessment as biomarkers from formalin-fixed paraffin-embedded tissue, and miR-24 represents an appropriate reference microRNA for diffuse large B-cell lymphoma studies.

PubMed

Culpin, Rachel Emily; Sieniawski, Michal; Proctor, Stephen John; Menon, Geetha; Mainou-Fowler, Tryfonia

2013-03-01

Tissue biopsy specimens in the form of formalin-fixed paraffin-embedded tissue (FFPET) represent a valuable resource for biomarker identification and validation. However, to date, they remain an underused asset due to uncertainty regarding RNA extraction and the reliability of downstream techniques, including quantitative RT-PCR. Recently, much interest has emerged in the study of microRNAs; small single-stranded RNAs with a role in transcriptional regulation, that are thought to be well preserved in FFPET. In this study, we show that microRNA expression is comparable between FFPET and matched fresh-frozen samples (miR-17-5p: p=0.01, miR-92: p=0.003), and demonstrate that no significant deterioration in expression occurs over prolonged FFPET storage (p=0.06). Furthermore, microRNA expression is equivalent dependant on RNA extraction method (p<0.001) or DNAse treatment of total RNA (p<0.001). Finally, we validate miR-24 as a suitable reference microRNA for diffuse large B-cell lymphoma (DLBCL) FFPET studies.

The effect of Arabic gum on frozen dough properties and the sensory assessments of the bread produced.

PubMed

Tavakoli, Hamid Reza; Jonaidi Jafari, Nematollah; Hamedi, Hassan

2017-04-01

The use of hydrocolloids in frozen dough has become frequent as bread improvers due to their anti-staling effect. Nevertheless, the impact of both different frozen storage and Arabic gum level in non-prefermented flat dough with following thawing procedure have not been studied. This work intended to study the effect of three different ratio of Arabic gum on rheological properties of 1, 7, and 30 days of frozen storage and the quality of the bread made from. In order to gain the least detrimental effects on gluten network, we used rapid rate freezing and microwave heating in thawing stage. Rheological results showed that the unfrozen samples to which Arabic gum had been added rendered the highest resistance to extension. The resistance of gum fortified samples were less than fresh dough, however the decline was not significant in 3.0% Arabic gum dough kept in a month storage (p > .05). The similar findings were obtained for extensibility and adhesiveness; in which the maximum incorporation of Arabic gum lessen the destructive impact of long freezing storage. Addition of 3% gum could be able to retard staling through an increment in hydrophilic bonds between water molecules and amylose during thawing (p < .05). The overall rating of Arabic gum enriched samples was similar with bread made from non-frozen dough, even after 30 days of storage as indicated by the sensory evaluation of breads. Producing a chapatti-like fermented bread without long fermentation period. Formulation a frozen dough without using chemical additives. Introducing a proper use of a new defrosting method with the aim of achieving a better texture. Improvement in retarding staling by the use of Gum Arabic after 7 days. © 2016 Wiley Periodicals, Inc.

Effect of different concentrations of egg yolk and virgin coconut oil in Tris-based extenders on chilled and frozen-thawed bull semen.

PubMed

Tarig, A A; Wahid, H; Rosnina, Y; Yimer, N; Goh, Y M; Baiee, F H; Khumran, A M; Salman, H; Ebrahimi, M

2017-07-01

The aim of this study was to evaluate the effects of 8% virgin coconut oil (VCO) combined with different percentages of egg yolk in Tris extender on the quality of chilled and frozen-thawed bull semen. A total of 24 ejaculates from four bulls were collected using an electroejaculator. Semen samples were diluted with 8% VCO in Tris extender which contained different concentrations 0% (control), 4%, 8%, 12%, 16% and 20% egg yolk. The diluted semen samples were divided into two fractions: one was chilled and stored at 4°C until evaluation after 24, 72, and 144h; the second fraction was processed by chilling for 3h at 4°C to equilibrate, then packaged in 0.25ml straws and frozen and stored in liquid nitrogen at -196°C until evaluation after 7 and 14 days. Both chilled and frozen semen samples were then thawed at 37°C and assessed for general motility using computer-assisted semen analysis (CASA), viability, acrosome integrity, and morphology (eosin-nigrosin), membrane integrity (hypo-osmotic swelling test) and lipid peroxidation (thiobarbituric acid-reactive substances (TBARS)). The results indicate treatments with 8%, 12%, 16% and 20% egg yolk with 8% VCO had greater sperm quality (P<0.05) as compared with the control. The treatment with 20% egg yolk had the greatest sperm quality (P<0.05) among the treated groups for both chilled and frozen-thawed semen. In conclusion, the use of 8% VCO combined with 20% egg yolk in a Tris-based extender enhanced the values for chilled and frozen-thawed quality variables of bull sperm. Copyright © 2017 Elsevier B.V. All rights reserved.

Immunoreactive LH in long-term frozen human urine samples.

PubMed

Singh, Gurmeet Kaur Surindar; Jimenez, Mark; Newman, Ron; Handelsman, David J

2014-04-01

Urine provides a convenient non-invasive alternative to blood sampling for measurement of certain hormones. Urinary luteinizing hormone (LH) measurements have been used for endocrinology research and anti-doping testing. However, the commercially available LH immunoassays are developed and validated for human blood samples but not urine so that LH assays intended for use with urine samples need thorough validation. Therefore, the present study evaluated the measurement of urinary LH immunoreactivity using previously validated immunofluorometric (IF) and immunochemiluminometric (ICL) LH assays after prolonged frozen storage. LH was measured in serial urine samples following administration of a single injection of one of two doses of recombinant human chorionic hormone (rhCG) with assays run at the end of study (2008) and again after four years of frozen (-20 °C) storage where samples were stored without adding preservatives. The ICL assay showed quantitatively reproducible LH measurements after prolonged -20 °C storage. However, the IF immunoassay gave consistently lower LH levels relative to ICL (2008) with a further proportionate reduction after four years of sample storage (2012). Yet, both the assays displayed similar patterns of the time-course of urine LH measurement both before and after four years of frozen storage. In conclusion, we found that both immunoassays are suitable for urinary LH measurements with ICL assay being more robust for quantitative urinary LH measurement such as for anti-doping purposes, whereas the IF could be applicable for research studies where urine LH levels are compared within-study but not in absolute terms. Copyright © 2013 John Wiley & Sons, Ltd.

Significant impact of amount of PCR input templates on various PCR-based DNA methylation analysis and countermeasure.

PubMed

Liu, Zhaojun; Zhou, Jing; Gu, Liankun; Deng, Dajun

2016-08-30

Methylation changes of CpG islands can be determined using PCR-based assays. However, the exact impact of the amount of input templates (TAIT) on DNA methylation analysis has not been previously recognized. Using COL2A1 gene as an input reference, TAIT difference between human tissues with methylation-positive and -negative detection was calculated for two representative genes GFRA1 and P16. Results revealed that TAIT in GFRA1 methylation-positive frozen samples (n = 332) was significantly higher than the methylation-negative ones (n = 44) (P < 0.001). Similar difference was found in P16 methylation analysis. The TAIT-related effect was also observed in methylation-specific PCR (MSP) and denatured high performance liquid chromatography (DHPLC) analysis. Further study showed that the minimum TAIT for a successful MethyLight PCR reaction should be ≥ 9.4 ng (CtCOL2A1 ≤ 29.3), when the cutoff value of the methylated-GFRA1 proportion for methylation-positive detection was set at 1.6%. After TAIT of the methylation non-informative frozen samples (n = 94; CtCOL2A1 > 29.3) was increased above the minimum TAIT, the methylation-positive rate increased from 72.3% to 95.7% for GFRA1 and 26.6% to 54.3% for P16, respectively (Ps < 0.001). Similar results were observed in the FFPE samples. In conclusion, TAIT critically affects results of various PCR-based DNA methylation analyses. Characterization of the minimum TAIT for target CpG islands is essential to avoid false-negative results.

Development of new method and protocol for cryopreservation related to embryo and oocytes freezing in terms of fertilization rate: A comparative study including review of literature.

PubMed

Barik, Mayadhar; Bajpai, Minu; Patnaik, Santosh; Mishra, Pravash; Behera, Priyamadhaba; Dwivedi, Sada Nanda

2016-01-01

Cryopreservation is basically related to meritorious thin samples or small clumps of cells that are cooled quickly without loss. Our main objective is to establish and formulate an innovative method and protocol development for cryopreservation as a gold standard for clinical uses in laboratory practice and treatment. The knowledge regarding usefulness of cryopreservation in clinical practice is essential to carry forward the clinical practice and research. We are trying to compare different methods of cryopreservation (in two dozen of cells) at the same time we compare the embryo and oocyte freezing interms of fertilization rate according to the International standard protocol. The combination of cryoprotectants and regimes of rapid cooling and rinsing during warming often allows successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include semen, blood, tissue samples like tumors, histological cross-sections, human eggs and human embryos. Although presently many studies have reported that the children born from frozen embryos or "frosties," show consistently positive results with no increase in birth defects or development abnormalities is quite good enough and similar to our study (50-85%). We ensure that cryopreservation technology provided useful cell survivability, tissue and organ preservation in a proper way. Although it varies according to different laboratory conditions, it is certainly beneficial for patient's treatment and research. Further studies are needed for standardization and development of new protocol.

Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

PubMed Central

2013-01-01

Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. Conclusions The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms. PMID:24160452

Research ethics in Canada: experience of a group operating a human embryo and fetal tissue bank.

PubMed

Milos, N; Bamforth, S; Bagnall, K

1999-04-01

A Canadian research group is establishing a human embryo and fetal tissue bank. Its purpose is to provide researchers with frozen or fixed tissue specimens for use in protein and gene expression studies. Several legal and ethical issues have arisen, including questions about consent, use of these rare tissues, cost recovery, and profit-making. These issues are discussed here in light of the present lack of legislation in Canada. We make recommendations in these areas, and suggest that the bank's operations could legally fall under the jurisdiction of the Human Tissue Gift Act.

Is sperm cryopreservation at -150 degree C a feasible alternative?

PubMed

Medrano, A; Cabrera, F; González, F; Batista, M; Gracia, A

2002-01-01

A series of experiments was carried out to validate a -150 degree C ultra-low temperature freezer for its possible use to properly freeze and store semen. In the first part, crude sample handling was simulated to see whether temperature of stored samples was maintained within a safe range; also, the freezing point and latent heat of fusion plateau of a semen extender were monitored. In the second part, buck semen was (i) frozen in liquid nitrogen and stored in the ultra-low freezer, (ii) frozen and stored in the ultra-low freezer, and (iii) frozen and stored in liquid nitrogen, to compare sperm cryosurvival between freezing methods. Both, frequent removal of samples and long opening of the freezer door did not negatively affect stored sample temperature; latent heat of fusion plateau was 5 minutes long. Semen stored either at -150 degree C or at -196 degree C cryosurvived similarly after 2 days and after 2 months of cryopreservation.

Wide-field optical coherence elastography for intraoperative assessment of tumour margins in breast cancer (Conference Presentation)

NASA Astrophysics Data System (ADS)

Allen, Wes M.; Chin, Lixin; Sampson, David D.; Kennedy, Brendan F.

2016-03-01

Incomplete excision of tumour margins is a major issue in breast-conserving surgery. Currently 20 - 60% of cases require a second surgical procedure required as a result of cancer recurrence. A number of techniques have been proposed to assess margin status, including frozen section analysis and imprint cytology. However, the recurrence rate after using these techniques remains very high. Over the last several years, our group has been developing optical coherence elastography (OCE) as a tool for the intraoperative assessment of tumour margins in breast cancer. We have reported a feasibility study on 65 ex vivo samples from patients undergoing mastectomy or wide local excision demonstrates the potential of OCE in differentiating benign from malignant tissue. In this study, malignant tissue was readily distinguished from surrounding relative tissue by a distinctive heterogeneous pattern in micro-elastograms. To date the largest field of view for a micro-elastogram is 20 x 20mm, however, lumpectomy samples are typically ~50 x 50 x 30mm. For OCE to progress as a useful clinical tool, elastograms must be acquired over larger areas to allow a greater portion of the surface area of lumpectomies to be assessed. Here, we propose a wide-field OCE scanner that utilizes a piezoelectric transducer with an internal diameter of 65mm. In this approach partially overlapped elastograms are stitched together forming a mosaic with overall dimensions of 50 x 50mm in a total acquisition time of 15 - 30 minutes. We present results using this approach on both tissue-mimicking phantoms and tissue, and discuss prospects for shorter acquisitions times.

Characterization of canine mitochondrial protein expression in natural and induced forms of idiopathic dilated cardiomyopathy.

PubMed

Lopes, Rosana; Solter, Philip F; Sisson, D David; Oyama, Mark A; Prosek, Robert

2006-06-01

To map canine mitochondrial proteins and identify qualitative and quantitative differences in heart mitochondrial protein expression between healthy dogs and dogs with naturally occurring and induced dilated cardiomyopathy (DCM). Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with induced DCM. Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by >or= 2-fold between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. Within narrow pH gradients of control canine heart mitochondrial samples, a total of 1,528 protein spots were revealed. Forty subunits of heart mitochondrial proteins that differ significantly from control tissues were altered in tissue specimens from dogs with naturally occurring and induced forms of DCM. The most affected heart mitochondrial proteins in both groups were those of oxidative phosphorylation (55%). Upregulation of manganese superoxide dismutase was suggestive of heart oxidative injury in tissue specimens from dogs with both forms of DCM. Evidence of apoptosis was associated with overexpression of the heart mitochondrial voltage-dependent anion channel-2 protein and endonuclease G in tissue specimens from dogs with induced DCM. Alterations of heart mitochondrial proteins related to oxidative phosphorylation dysfunction were more prevalent in tissue specimens from dogs with induced or naturally occurring DCM, compared with those of control dogs.

Attachment sites of the coracoclavicular ligaments are characterized by fibrocartilage differentiation: a study on human cadaveric tissue.

PubMed

Ockert, B; Braunstein, V; Sprecher, C; Shinohara, Y; Kirchhoff, C; Milz, S

2012-02-01

We analyzed the immunohistochemical labeling patterns of the extracellular matrix of the coracoclavicular ligaments (CCL) in order to relate the molecular composition of the attachment sites to their mechanical environment. Ligaments were exposed from 12 fresh-frozen human cadaveric samples (four males, mean age: 48.6 ± 12.1 years). Cryosection of methanol-fixed and decalcified tissue was cut and sections were labeled with a panel of monoclonal antibodies directed against collagens, proteoglycans and proteins of vascular components. Attachment sites of both ligaments showed characteristic fibrocartilaginous labeling of collagen type II, aggrecan and link protein in all samples. Labeling for type II collagen was most conspicuous at the insertion of the coracoid process. Morphometry of adjacent samples revealed a fibrocartilage zone of 10-15% in relationship with the ligament proper, where labeling for type II collagen, aggrecan and link protein was negative. The presence of fibrocartilage at both entheses of the trapezoid and conoid ligament suggests that the CCL complex is subject to shear/compression forces. A variable fibrocartilage differentiation at the entheses of both ligaments may be related to the marked change in loading and insertion angle that the ligaments undergo during shoulder movement. © 2010 John Wiley & Sons A/S.

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

PubMed

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.

Effect of osmotic dehydration pretreatment and glassy state storage on the quality attributes of frozen mangoes under long-term storage.

PubMed

Zhao, Jin-Hong; Xiao, Hong-Wei; Ding, Yang; Nie, Ying; Zhang, Yu; Zhu, Zhen; Tang, Xuan-Ming

2017-05-01

Changes in the quality of frozen mango cuboids were investigated during long-term glassy state storage with and without osmotic dehydration pretreatment. The mango cuboids were dehydrated in mixed solutions (sucrose: glucose: fructose in a ratio of 3.6:1:3) of different concentrations (30, 40, and 50% (wt/wt)) prior to freezing and then stored at -55 °C (in the glassy state) for 6 months. The results revealed that compared with the untreated samples, osmotic pretreatment decreased total color difference (reduced by 15.6-62.3%), drip loss (reduced by 8.2-29.5%) and titration acidity (reduced by 1.3-9.4%), while increasing hardness (increased by 48.8-82.3%), vitamin C content (increased by 72.5-120.6%) and total soluble solids (increased by 21.8-53.7%) of frozen mangoes after 6 months. Dehydration with a sugar concentration of 40% was considered as the optimal pretreatment condition. In addition, a storage temperature of -55 °C provided better retention of quality than rubbery state storage at -18 °C. With prolonged storage time, the quality of frozen mangoes continued to change, even in the glassy state. However, the changes in quality of the osmotic-dehydrated samples were less than those of the untreated samples. The current work indicates that osmotic pretreatment and glassy state storage significantly improved the quality of frozen mangoes during long-term storage.

Biophysical interpretation and ex-vivo characterization of scattered light from tumor-associated breast stroma

NASA Astrophysics Data System (ADS)

Laughney, Ashley; Krishnaswamy, Venkat; Schwab, Mary; Wells, Wendy A.; Paulsen, Keith D.; Pogue, Brian W.

2009-02-01

The purpose of this study was to extract scatter parameters related to tissue ultra-structures from freshly excised breast tissue and to assess whether evident changes in scatter across diagnostic categories is primarily influenced by variation in the composition of each tissues subtypes or by physical remodeling of the extra-cellular environment. Pathologists easily distinguish between epithelium, stroma and adipose tissues, so this classification was adopted for macroscopic subtype classification. Micro-sampling reflectance spectroscopy was used to characterize single-backscattered photons from fresh, excised tumors and normal reduction specimens with sub-millimeter resolution. Phase contrast microscopy (sub-micron resolution) was used to characterize forward-scattered light through frozen tissue from the DHMC Tissue Bank, representing normal, benign and malignant breast tissue, sectioned at 10 microns. The packing density and orientation of collagen fibers in the extracellular matrix (ECM) associated with invasive, normal and benign epithelium was evaluated using transmission electron microscopy (TEM). Regions of interest (ROIs) in the H&E stained tissues were identified for analysis, as outlined by a pathologist as the gold standard. We conclude that the scatter parameters associated with tumor specimens (Npatients=6, Nspecimens=13) significantly differs from that of normal reductions (Npatients=6, Nspecimens=10). Further, tissue subtypes may be identified by their scatter spectra at sub-micron resolution. Stromal tissue scatters significantly more than the epithelial cells embedded in its ECM and adipose tissue scatters much less. However, the scatter signature of the stroma at the sub-micron level is not particularly differentiating in terms of a diagnosis.

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

NASA Technical Reports Server (NTRS)

Choi, S. Y.; Lai, S.; Klotz, R.; Popova, Y.; Chakravarty, K.; Beegle, J. E.; Wigley, C. L.; Globus, R. K.

2014-01-01

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development; Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science. Mouse livers and spleens were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection operations including storage at -80 C for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RIN(is) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNAlater) and liver (fast-freezing) at various time points post-euthanasia (from 5 min up to 105 min). The RNA recovered was of high quality (spleen, RIN (is) greater than 8; liver, RIN (is) greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at the latest time (105 min). Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) Many additional tissues for gene expression analysis can be obtained by dissection following prolonged storage of the tissue in situ at -80 C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to all future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

An efficient protocol for tissue sampling and DNA isolation from the stem bark of Leguminosae trees.

PubMed

Novaes, R M L; Rodrigues, J G; Lovato, M B

2009-02-03

Traditionally, molecular studies of plant species have used leaves as the source of DNA. However, sampling leaves from tall tree species can be quite difficult and expensive. We developed a sequence of procedures for using stem bark as a source of DNA from Leguminosae trees of the Atlantic Forest and the Cerrado. Leguminosae is an important species-rich family in these two highly diverse and endangered biomes. A modified CTAB protocol for DNA isolation is described, and details of the procedures for sampling and storage of the bark are given. The procedures were initially developed for three species, and then their applicability for 15 other species was evaluated. DNA of satisfactory quality was obtained from the bark of all species. The amounts of DNA obtained from leaves were slightly higher than from bark samples, while its purity was the same. Storing the bark frozen or by drying in silica gel yielded similar results. Polymerase chain reaction amplification worked for both plastid and nuclear genomes. This alternative for isolating DNA from bark samples of trees facilitates field work with these tree species.

Environmental Factors Related to a Semiarid Climate Influence the Freezability of Sperm from Collared Peccaries (Pecari tajacu Linnaeus, 1758).

PubMed

Maia, Keilla M; Souza, Ana L P; Praxedes, Erica C G; Bezerra, Luana G P; Silva, Andreia M; Campos, Livia B; Moreira, Samara S J; Apolinário, Carlos A C; Souza, João B F; Silva, Alexandre R

2018-04-30

The influence of environmental factors in a semiarid climate on characteristics of fresh and frozen/thawed sperm collected from collared peccaries (Pecari tajacu) was assessed. Semen from 11 male collared peccaries was collected by electroejaculation during the peaks of the dry and rainy periods while rainfall indices, air temperatures, relative humidity levels, and wind speeds were measured. The number, motility, morphology, osmotic response, and membrane integrity of sperm in the collected ejaculates were assessed. Samples were then frozen in liquid nitrogen, thawed, and reassessed. The rainfall index of the rainy period (73.2 mm) was significantly higher than that of the dry period (13.6 mm) and the relative humidity was significantly higher during the rainy period (74.6%) than it was during the dry period (66.8%). Air temperature and wind speed did not differ between the two periods. Characteristics of sperm in the fresh samples were not affected by environmental parameters. In contrast, computerized analysis revealed that sperm in samples frozen during the rainy period exhibited better post-thaw membrane integrity (28.6 ± 6%), motility (29.5 ± 7.7%), and rapid sperm population (13.7 ± 6.2%) than did sperm in samples frozen during the dry period (23.4 ± 3% membrane integrity, 14.6 ± 4.1% motility, and 4.1 ± 1.2% rapid sperm; p < 0.05). Other characteristics of the frozen/thawed sperm did not differ depending on the period in which they were collected. We demonstrated that environmental parameters did not affect the quality of fresh sperm, but could influence the freezability of sperm collected from collared peccaries raised under a semiarid climate.

Sealing Penetrating Eye Injuries Using Photoactivated Bonding

DTIC Science & Technology

2010-09-30

on  corneal tissue. To evaluate inflammation all 15 rabbits will be euthanized with  phenobarbital . The  10 corneoscleral button of tissue will be...will be euthanized with  phenobarbital . The  corneoscleral button of tissue will be removed and divided into portions for paraffin and frozen sections

Antigen Masking During Fixation and Embedding, Dissected

PubMed Central

Scalia, Carla Rossana; Boi, Giovanna; Bolognesi, Maddalena Maria; Riva, Lorella; Manzoni, Marco; DeSmedt, Linde; Bosisio, Francesca Maria; Ronchi, Susanna; Leone, Biagio Eugenio; Cattoretti, Giorgio

2016-01-01

Antigen masking in routinely processed tissue is a poorly understood process caused by multiple factors. We sought to dissect the effect on antigenicity of each step of processing by using frozen sections as proxies of the whole tissue. An equivalent extent of antigen masking occurs across variable fixation times at room temperature. Most antigens benefit from longer fixation times (>24 hr) for optimal detection after antigen retrieval (AR; for example, Ki-67, bcl-2, ER). The transfer to a graded alcohol series results in an enhanced staining effect, reproduced by treating the sections with detergents, possibly because of a better access of the polymeric immunohistochemical detection system to tissue structures. A second round of masking occurs upon entering the clearing agent, mostly at the paraffin embedding step. This may depend on the non-freezable water removal. AR fully reverses the masking due both to the fixation time and the paraffin embedding. AR itself destroys some epitopes which do not survive routine processing. Processed frozen sections are a tool to investigate fixation and processing requirements for antigens in routine specimens. PMID:27798289

Characterization of neuroendocrine tumors of the pancreas by real-time quantitative polymerase chain reaction. A methodological approach.

PubMed

Annaratone, Laura; Volante, Marco; Asioli, Sofia; Rangel, Nelson; Bussolati, Gianni

2013-06-01

The aim of this study was to assess the suitability of using real-time quantitative PCR (RT-qPCR) to characterize neuroendocrine (NE) tumors of the pancreas. For a series of tumors, we evaluated several genes of interest, and the data were matched with the "classical" immunohistochemical (IHC) features. In 21 cases, we extracted RNA from formalin-fixed paraffin-embedded (FFPE) blocks, and in nine cases, we also extracted RNA from fresh-frozen tissue. The RT-qPCR procedure was performed using two sets of customized arrays. The test using the first set, covering 96 genes of interest, was focused on assessing the feasibility of the procedure, and the results were used to select 18 genes indicative of NE differentiation, clinical behavior, and therapeutic responsiveness for use in the second set of arrays. Threshold cycle (Ct) values were used to calculate the fold-changes in gene expression using the 2-∆∆Ct method. Statistical procedures were used to analyze the results, which were matched with the IHC and follow-up data. Material from fresh-frozen samples performed better in terms of the level of amplification, but acceptable and concordant results were also obtained from FFPE samples. In addition, high concordance was observed between the mRNA and protein expression levels of somatostatin receptor type 2A (R = 0.52, p = 0.016). Genes associated with NE differentiation, as well as the gastrin-releasing peptide receptor and O-6-methylguanine-DNA methyltransferase genes, were underexpressed, whereas angiogenesis-associated markers (CDH13 and SLIT2) were overexpressed in tissues with malignant behavior. The RT-qPCR procedure is practical and feasible in economic terms for the characterization of NE tumors of the pancreas and can complement morphological and IHC-based evaluations. Thus, the results of the RT-qPCR procedure might offer an objective basis for therapeutic choices.

Sperm Cryopreservation in Live-Bearing Xiphophorus Fishes: Offspring Production from Xiphophorus variatus and Strategies for Establishment of Sperm Repositories

PubMed Central

Cuevas-Uribe, Rafael; Savage, Markita G.; Walter, Ronald B.; Tiersch, Terrence R.

2012-01-01

Abstract Cryopreservation of sperm from Xiphophorus fishes has produced live young in three species: X. hellerii, X. couchianus, and X. maculatus. In this study, the goal was to establish protocols for sperm cryopreservation and artificial insemination to produce live young in X. variatus, and to identify needs for repository development. The objectives were to: 1) collect basic biological characteristics of males; 2) cryopreserve sperm from X. variatus, 3) harvest live young from cryopreserved sperm, and 4) discuss the requirements for establishment of sperm repositories. The 35 males used in this study had a body weight of 0.298±0.096 g (mean±SD), body length of 2.5±0.2 cm, and testis weight of 6.4±3.4 mg. The sperm production per gram of testis was 2.33±1.32×109 cells. After freezing, the post-thaw motility decreased significantly to 37%±17% (ranging from 5% to 70%) (p=0.000) from 57%±14% (40%–80%) of fresh sperm (N=20). Artificial insemination of post-thaw sperm produced confirmed offspring from females of X. hellerii and X. variatus. This research, taken together with previous studies, provides a foundation for development of strategies for sperm repositories of Xiphophorus fishes. This includes: 1) the need for breeding strategies for regeneration of target populations, 2) identification of minimum fertilization capacity of frozen samples, 3) identification of fish numbers necessary for sampling and their genetic relationships, 4) selection of packaging containers for labeling and biosecurity, 5) assurance of quality control and standardization of procedures, 6) information systems that can manage the data associated with cryopreserved samples, including the genetic data, 7) biological data of sampled fish, 8) inventory data associated with frozen samples, and 9) data linking germplasm samples with other related materials such as body tissues or cells saved for DNA and RNA analyses. PMID:22924335

Sperm cryopreservation in live-bearing Xiphophorus fishes: offspring production from Xiphophorus variatus and strategies for establishment of sperm repositories.

PubMed

Yang, Huiping; Cuevas-Uribe, Rafael; Savage, Markita G; Walter, Ronald B; Tiersch, Terrence R

2012-09-01

Cryopreservation of sperm from Xiphophorus fishes has produced live young in three species: X. hellerii, X. couchianus, and X. maculatus. In this study, the goal was to establish protocols for sperm cryopreservation and artificial insemination to produce live young in X. variatus, and to identify needs for repository development. The objectives were to: 1) collect basic biological characteristics of males; 2) cryopreserve sperm from X. variatus, 3) harvest live young from cryopreserved sperm, and 4) discuss the requirements for establishment of sperm repositories. The 35 males used in this study had a body weight of 0.298±0.096 g (mean±SD), body length of 2.5±0.2 cm, and testis weight of 6.4±3.4 mg. The sperm production per gram of testis was 2.33±1.32×10(9) cells. After freezing, the post-thaw motility decreased significantly to 37%±17% (ranging from 5% to 70%) (p=0.000) from 57%±14% (40%-80%) of fresh sperm (N=20). Artificial insemination of post-thaw sperm produced confirmed offspring from females of X. hellerii and X. variatus. This research, taken together with previous studies, provides a foundation for development of strategies for sperm repositories of Xiphophorus fishes. This includes: 1) the need for breeding strategies for regeneration of target populations, 2) identification of minimum fertilization capacity of frozen samples, 3) identification of fish numbers necessary for sampling and their genetic relationships, 4) selection of packaging containers for labeling and biosecurity, 5) assurance of quality control and standardization of procedures, 6) information systems that can manage the data associated with cryopreserved samples, including the genetic data, 7) biological data of sampled fish, 8) inventory data associated with frozen samples, and 9) data linking germplasm samples with other related materials such as body tissues or cells saved for DNA and RNA analyses.

A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue.

PubMed

Sarkar, F H; Valdivieso, M; Borders, J; Yao, K L; Raval, M M; Madan, S K; Sreepathi, P; Shimoyama, R; Steiger, Z; Visscher, D W

1995-12-01

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.

Disseminated phaeohyphomycosis in weedy seadragons (Phyllopteryx taeniolatus) and leafy seadragons (Phycodurus eques) caused by species of Exophiala, including a novel species.

PubMed

Nyaoke, Akinyi; Weber, E Scott; Innis, Charles; Stremme, Donald; Dowd, Cynthia; Hinckley, Lynn; Gorton, Timothy; Wickes, Brian; Sutton, Deanna; de Hoog, Sybren; Frasca, Salvatore

2009-01-01

During the period from January 2002 to March 2007, infections by melanized fungi were identified with greater frequency in aquarium-maintained leafy seadragons (Phycodurus eques) and weedy seadragons (Phyllopteryx taeniolatus), pivotal species to the educational and environmental concerns of the aquarium industry and conservation groups. The objective of this study was to characterize the pathology and identify fungi associated with phaeohyphomycotic lesions in these species. Samples from 14 weedy and 6 leafy seadragons were received from 2 institutions and included fresh, frozen, and formalin-fixed tissues from necropsy and biopsy specimens. Fresh and frozen tissues were cultured for fungi on Sabouraud dextrose agar only or both Sabouraud dextrose agar and inhibitory mold agar with gentamicin and chloramphenicol at 30 degrees C. Isolates were processed for morphologic identification and molecular sequence analysis of the internal transcribed spacer region and D1/D2 domains of the large subunit ribosomal RNA gene. Lesions were extensive and consisted of parenchymal and vascular necrosis with fungal invasion of gill (11/20), kidney (14/20), and other coelomic viscera with or without cutaneous ulceration (13/20). Exophiala sp. isolates were obtained from 4 weedy and 3 leafy seadragons and were identified to species level in 6 of 7 instances, namely Exophiala angulospora (1) and a novel species of Exophiala (5), based on nucleotide sequence comparisons and phylogenetic analyses. Disseminated phaeohyphomycosis represents an important pathologic condition of both weedy and leafy seadragons for which 2 species of Exophiala, 1 a novel species, have been isolated.

An audit of parents'/guardians' wishes recorded after coronial autopsies in cases of sudden unexpected death in infancy: issues raised and future directions.

PubMed

Cohen, M C; Blakey, S; Donn, T; McGovern, S; Parry, L

2009-07-01

In the U.K., cases of sudden unexpected death in infancy are under the jurisdiction of the Coroner and consent for a post-mortem is not required. Prior to the Human Tissue Act 2006 (HTA) there was also no requirement to request retention of tissue (blocks and slides). The HTA stipulates that parental/ guardian consent is mandatory to retain or dispose of all tissues after the Coroners' purposes have been fulfilled. In 2007, in order to avoid confusion with the consent needed for hospital post-mortems, a new form was introduced by Sheffield Children's Hospital NHS Foundation Trust (SCH) called Record of parents'/guardians'wishes regarding samples taken at a Coroner's post mortem. This version specifically asks if blocks and slides may be retained as part of the medical record, or are to be disposed of, and for parental agreement (or not) for the frozen tissue, blocks and slides to be used for education, audit, quality control and medical research. One hundred and nineteen Coroners' postmortems covering the years 2006-2007 were reviewed. All parents/guardians (P/G) were contacted and the outcomes of P/G wishes recorded by SCH staff, Coroners' Officers (CO) and Police Family Liaison Officers (PFLO) were analysed and compared (44% from CO were outstanding at the time of audit). Any delay in recording P/G wishes by these three groups was also compared. In 2006, parental agreement to the use of blocks and slides for education, audit, quality control and medical research was 94%, 77% and 75% for SCH, CO and PFLO, respectively. In 2007 it was 84%, 37% and 100% for the same groups. Permission for the retention of frozen tissue given to SCH, CO and PFLO was 90%, 62% and 100% in 2006 and 90%, 44% and 100% in 2007, respectively. Cases where parents did not wish for the retention or use of tissue (including blocks and slides) were 3%, 15% and 0% in 2006 for SCH, CO and PFLO respectively, and 0% for all groups in 2007. Training of staff in all aspects of post-mortem and bereavement care is essential for ascertaining parental wishes. Families should be provided with the knowledge that allows them to make informed choices. The analysis of the results of the audit supports this view.

Homogenization of tissues via picosecond-infrared laser (PIRL) ablation: Giving a closer view on the in-vivo composition of protein species as compared to mechanical homogenization.

PubMed

Kwiatkowski, M; Wurlitzer, M; Krutilin, A; Kiani, P; Nimer, R; Omidi, M; Mannaa, A; Bussmann, T; Bartkowiak, K; Kruber, S; Uschold, S; Steffen, P; Lübberstedt, J; Küpker, N; Petersen, H; Knecht, R; Hansen, N O; Zarrine-Afsar, A; Robertson, W D; Miller, R J D; Schlüter, H

2016-02-16

Posttranslational modifications and proteolytic processing regulate almost all physiological processes. Dysregulation can potentially result in pathologic protein species causing diseases. Thus, tissue species proteomes of diseased individuals provide diagnostic information. Since the composition of tissue proteomes can rapidly change during tissue homogenization by the action of enzymes released from their compartments, disease specific protein species patterns can vanish. Recently, we described a novel, ultrafast and soft method for cold vaporization of tissue via desorption by impulsive vibrational excitation (DIVE) using a picosecond-infrared-laser (PIRL). Given that DIVE extraction may provide improved access to the original composition of protein species in tissues, we compared the proteome composition of tissue protein homogenates after DIVE homogenization with conventional homogenizations. A higher number of intact protein species was observed in DIVE homogenates. Due to the ultrafast transfer of proteins from tissues via gas phase into frozen condensates of the aerosols, intact protein species were exposed to a lesser extent to enzymatic degradation reactions compared with conventional protein extraction. In addition, total yield of the number of proteins is higher in DIVE homogenates, because they are very homogenous and contain almost no insoluble particles, allowing direct analysis with subsequent analytical methods without the necessity of centrifugation. Enzymatic protein modifications during tissue homogenization are responsible for changes of the in-vivo protein species composition. Cold vaporization of tissues by PIRL-DIVE is comparable with taking a snapshot at the time of the laser irradiation of the dynamic changes that occur continuously under in-vivo conditions. At that time point all biomolecules are transferred into an aerosol, which is immediately frozen. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Homogenization of tissues via picosecond-infrared laser (PIRL) ablation: Giving a closer view on the in-vivo composition of protein species as compared to mechanical homogenization

PubMed Central

Kwiatkowski, M.; Wurlitzer, M.; Krutilin, A.; Kiani, P.; Nimer, R.; Omidi, M.; Mannaa, A.; Bussmann, T.; Bartkowiak, K.; Kruber, S.; Uschold, S.; Steffen, P.; Lübberstedt, J.; Küpker, N.; Petersen, H.; Knecht, R.; Hansen, N.O.; Zarrine-Afsar, A.; Robertson, W.D.; Miller, R.J.D.; Schlüter, H.

2016-01-01

Posttranslational modifications and proteolytic processing regulate almost all physiological processes. Dysregulation can potentially result in pathologic protein species causing diseases. Thus, tissue species proteomes of diseased individuals provide diagnostic information. Since the composition of tissue proteomes can rapidly change during tissue homogenization by the action of enzymes released from their compartments, disease specific protein species patterns can vanish. Recently, we described a novel, ultrafast and soft method for cold vaporization of tissue via desorption by impulsive vibrational excitation (DIVE) using a picosecond-infrared-laser (PIRL). Given that DIVE extraction may provide improved access to the original composition of protein species in tissues, we compared the proteome composition of tissue protein homogenates after DIVE homogenization with conventional homogenizations. A higher number of intact protein species was observed in DIVE homogenates. Due to the ultrafast transfer of proteins from tissues via gas phase into frozen condensates of the aerosols, intact protein species were exposed to a lesser extent to enzymatic degradation reactions compared with conventional protein extraction. In addition, total yield of the number of proteins is higher in DIVE homogenates, because they are very homogenous and contain almost no insoluble particles, allowing direct analysis with subsequent analytical methods without the necessity of centrifugation. Biological significance Enzymatic protein modifications during tissue homogenization are responsible for changes of the in-vivo protein species composition. Cold vaporization of tissues by PIRL-DIVE is comparable with taking a snapshot at the time of the laser irradiation of the dynamic changes that occur continuously under in-vivo conditions. At that time point all biomolecules are transferred into an aerosol, which is immediately frozen. PMID:26778141

Tissue Quality Assessment Using a Novel Direct Elasticity Assessment Device (The E-Finger): A Cadaveric Study of Prostatectomy Dissection

PubMed Central

Good, Daniel W.; Khan, Ashfaq; Hammer, Steven; Scanlan, Paul; Shu, Wenmiao; Phipps, Simon; Parson, Simon H.; Stewart, Grant D.; Reuben, Robert; McNeill, S. Alan

2014-01-01

Introduction Minimally invasive radical prostatectomy (RP) (robotic and laparoscopic), have brought improvements in the outcomes of RP due to improved views and increased degrees of freedom of surgical devices. Robotic and laparoscopic surgeries do not incorporate haptic feedback, which may result in complications secondary to inadequate tissue dissection (causing positive surgical margins, rhabdosphincter damage, etc). We developed a micro-engineered device (6 mm2 sized) [E-finger]) capable of quantitative elasticity assessment, with amplitude ratio, mean ratio and phase lag representing this. The aim was to assess the utility of the device in differentiating peri-prostatic tissue types in order to guide prostate dissection. Material and Methods Two embalmed and 2 fresh frozen cadavers were used in the study. Baseline elasticity values were assessed in bladder, prostate and rhabdosphincter of pre-dissected embalmed cadavers using the micro-engineered device. A measurement grid was created to span from the bladder, across the prostate and onto the rhabdosphincter of fresh frozen cadavers to enable a systematic quantitative elasticity assessment of the entire area by 2 independent assessors. Tissue was sectioned along each row of elasticity measurement points, and stained with haematoxylin and eosin (H&E). Image analysis was performed with Image Pro Premier to determine the histology at each measurement point. Results Statistically significant differences in elasticity were identified between bladder, prostate and sphincter in both embalmed and fresh frozen cadavers (p = <0.001). Intra-class correlation (ICC) reliability tests showed good reliability (average ICC = 0.851). Sensitivity and specificity for tissue identification was 77% and 70% respectively to a resolution of 6 mm2. Conclusions This cadaveric study has evaluated the ability of our elasticity assessment device to differentiate bladder, prostate and rhabdosphincter to a resolution of 6 mm2. The results provide useful data for which to continue to examine the use of elasticity assessment devices for tissue quality assessment with the aim of giving haptic feedback to surgeons performing complex surgery. PMID:25384014

Conventional freezing plus high pressure-low temperature treatment: Physical properties, microbial quality and storage stability of beef meat.

PubMed

Fernández, Pedro P; Sanz, Pedro D; Molina-García, Antonio D; Otero, Laura; Guignon, Bérengère; Vaudagna, Sergio R

2007-12-01

Meat high-hydrostatic pressure treatment causes severe decolouration, preventing its commercialisation due to consumer rejection. Novel procedures involving product freezing plus low-temperature pressure processing are here investigated. Room temperature (20°C) pressurisation (650MPa/10min) and air blast freezing (-30°C) are compared to air blast freezing plus high pressure at subzero temperature (-35°C) in terms of drip loss, expressible moisture, shear force, colour, microbial quality and storage stability of fresh and salt-added beef samples (Longissimus dorsi muscle). The latter treatment induced solid water transitions among ice phases. Fresh beef high pressure treatment (650MPa/20°C/10min) increased significantly expressible moisture while it decreased in pressurised (650MPa/-35°C/10min) frozen beef. Salt addition reduced high pressure-induced water loss. Treatments studied did not change fresh or salt-added samples shear force. Frozen beef pressurised at low temperature showed L, a and b values after thawing close to fresh samples. However, these samples in frozen state, presented chromatic parameters similar to unfrozen beef pressurised at room temperature. Apparently, freezing protects meat against pressure colour deterioration, fresh colour being recovered after thawing. High pressure processing (20°C or -35°C) was very effective reducing aerobic total (2-log(10) cycles) and lactic acid bacteria counts (2.4-log(10) cycles), in fresh and salt-added samples. Frozen+pressurised beef stored at -18°C during 45 days recovered its original colour after thawing, similarly to just-treated samples while their counts remain below detection limits during storage.

Handling, storage, and preparation of human tissues.

PubMed

Dressler, L G; Visscher, D

2001-05-01

Human tissue for flow cytometry must be prepared as an adequate single-cell suspension. The appropriate methods for tissue collection, transport, storage, and dissociation depend on the cell parameters being measured and the localization of the markers. This unit includes a general method for collecting and transporting human tissue and preparing a tissue imprint. Protocols are supplied for tissue disaggregation by either mechanical or enzymatic means and for preparation of single-cell suspensions of whole cells from fine-needle aspirates, pleural effusions, abdominal fluids, or other body fluids. Other protocols detail preparation of intact nuclei from fresh, frozen, or paraffin-embedded tissue. Support protocols cover fixation, cryospin preparation, cryopreservation, and removal of debris.

Microgravity

NASA Image and Video Library

1996-03-24

Astronaut Michael Clifford places a liquid nitrogen Dewar containing frozen protein solutions aboard Russia's space station Mir during a visit by the Space Shuttle (STS-76). The protein samples were flash-frozen on Earth and will be allowed to thaw and crystallize in the microgravity environment on Mir Space Station. A later crew will return the Dewar to Earth for sample analysis. Dr. Alexander McPherson of the University of California at Riverside is the principal investigator. Photo credit: NASA/Johnson Space Center.

Microgravity

NASA Image and Video Library

1996-09-20

Astronaut Tom Akers places a liquid nitrogen Dewar containing frozen protein solutions aboard Russia's space Station Mir during a visit by the Space Shuttle (STS-79). The protein samples were flash-frozen on Earth and will be allowed to thaw and crystallize in the microgravity environment on Mir Space Station. A later crew will return the Dewar to Earth for sample analysis. Dr. Alexander McPherson of the University of California at Riverside is the principal investigator. Photo credit: NASA/Johnson Space Center.

Respiratory analysis of coupled mitochondria in cryopreserved liver biopsies.

PubMed

García-Roche, Mercedes; Casal, Alberto; Carriquiry, Mariana; Radi, Rafael; Quijano, Celia; Cassina, Adriana

2018-07-01

The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at -80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

A cryopreservation method for Pasteurella multocida from wetland samples

USGS Publications Warehouse

Moore, Melody K.; Shadduck, D.J.; Goldberg, Diana R.; Samuel, M.D.

1998-01-01

A cryopreservation method and improved isolation techniques for detection of Pasteurella multocida from wetland samples were developed. Wetland water samples were collected in the field, diluted in dimethyl sulfoxide (DMSO, final concentration 10%), and frozen at -180 C in a liquid nitrogen vapor shipper. Frozen samples were transported to the laboratory where they were subsequently thawed and processed in Pasteurella multocida selective broth (PMSB) to isolate P. multocida. This method allowed for consistent isolation of 2 to 18 organisms/ml from water seeded with known concentrations of P. multocida. The method compared favorably with the standard mouse inoculation method and allowed for preservation of the samples until they could be processed in the laboratory.

Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality.

PubMed

Dictor, Michael; Skogvall, Ingela; Warenholt, Janina; Rambech, Eva

2007-01-01

Two-colour flow cytometry was compared with multiplex PCR with capillary electrophoresis for clonality determination in specific categories of B-cell lymphoma. FTA cards were evaluated for preserving DNA from node imprints and expediting molecular analysis. A single-tube multiplex PCR targeted IGH and lymphoma-specific translocations in DNA extracted from 180 frozen lymphoid tissues and DNA bound to FTA cards from 192 fresh tissues and 137 aspirates. PCR results were compared with flow cytometry in the extracted and aspirated samples. Overall, single-tube multiplex PCR sensitivity was equivalent in the sample groups (intergroup range 79%-91%). False negatives were associated with tumour origin in the follicle centre. Multiplex PCR and flow cytometry were equally sensitive and together detected 98% of B-cell lymphomas. Additional two-tube targeting of IGK suggested an overall molecular sensitivity >90%. False positive (pseudoclonal) single-tube multiplex PCR was associated with necrosis and sparse lymphocytes. Multiplex PCR using template DNA bound to an FTA card effectively detects B-lymphocyte clonality, obviates DNA extraction and refrigeration, and can be used without diminished sensitivity in fine needle aspirates or node imprints as a replacement for or complement to flow cytometry at any point in the diagnostic work-up.

Laser capture microdissection tailored to type 1 diabetes mellitus research.

PubMed

Szulawski, Robert; Nakazawa, Masato; McCall, Kelly D; James, Calvin B L; Schwartz, Frank L

2016-01-01

RNA isolation from pancreatic islets poses unique challenges. Here, we present a reproducible means of obtaining high-quality RNA from juvenile rodent islets in sufficient quantities for use in ex vivo expression studies. Tissue was extracted from female non-obese diabetic (NOD) toll-like receptor 3 (TLR3)(+/+) and (TLR3)(-/-) mice in the pre-diabetic stage. Samples were frozen in liquid nitrogen, sectioned, fixed in a highly alcoholic solution, and stained with an alcoholic cresyl violet (CV) solution. Rehydration of the fixed sections was minimized. Islets were identified visually and isolated with the Leica LMD6000 laser capture microdissection (LCM) system to yield samples highly enriched in islet RNA. Real time qPCR was performed on the islet cDNA using probes for CXC chemokine ligand 10 (CXCL10), an inflammatory marker that plays a critical role in the pathogenesis of type 1 diabetes mellitus (TIDM). This method represents an improvement over currently described LCM techniques for rodent pancreatic islets and makes feasible expression studies using small amounts of starting tissue without the need for RNA pre-amplification. This has immediate implications for ongoing TIDM studies using the NOD mouse.

Hamstring autograft versus soft-tissue allograft in anterior cruciate ligament reconstruction: a systematic review and meta-analysis of randomized controlled trials.

PubMed

Cvetanovich, Gregory L; Mascarenhas, Randy; Saccomanno, Maristella F; Verma, Nikhil N; Cole, Brian J; Bush-Joseph, Charles A; Bach, Bernard R

2014-12-01

To compare outcomes of anterior cruciate ligament (ACL) reconstruction with hamstring autograft versus soft-tissue allograft by systematic review and meta-analysis. A systematic review of randomized controlled studies comparing hamstring autograft with soft-tissue allograft in ACL reconstruction was performed. Studies were identified by strict inclusion and exclusion criteria. Descriptive statistics were reported. Where possible, the data were pooled and a meta-analysis was performed using RevMan software (The Nordic Cochrane Centre, The Cochrane Collaboration, Copenhagen, Denmark). Dichotomous data were reported as risk ratios, whereas continuous data were reported as standardized mean differences and 95% confidence intervals. Heterogeneity was assessed by use of I(2) for each meta-analysis. Study methodologic quality was analyzed with the Modified Coleman Methodology Score and Jadad scale. Five studies with 504 combined patients (251 autograft and 253 allograft; 374 male and 130 female patients) with a mean age of 29.9 ± 2.2 years were included. The allografts used were fresh-frozen hamstring, irradiated hamstring, mixture of fresh-frozen and cryopreserved hamstring, fresh-frozen tibialis anterior, and fresh-frozen Achilles tendon grafts without bone blocks. The mean follow-up period was 47.4 ± 26.9 months, with a mean follow-up rate of 83.3% ± 8.6%. Two studies found a longer operative time with autograft than with allograft (77.1 ± 2.0 minutes v 59.9 ± 0.9 minutes, P = .008). Meta-analysis showed no statistically significant differences between autografts and allografts for any outcome measures (P > .05 for all tests). One study found significantly greater laxity for irradiated allograft than for autograft. The methodologic quality of the 5 studies was poor, with a mean Modified Coleman Methodology Score of 54.4 ± 6.9 and mean Jadad score of 1.6 ± 1.5. On the basis of this systematic review and meta-analysis of 5 randomized controlled trials, there is no statistically significant difference in outcome between patients undergoing ACL reconstruction with hamstring autograft and those undergoing ACL reconstruction with soft-tissue allograft. These results may not extrapolate to younger patient populations. The methodology of the available randomized controlled trials comparing hamstring autograft and soft-tissue allograft is poor. Level II, systematic review of Level I and II studies. Copyright © 2014 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Ex vivo fluorescence confocal microscopy for fast evaluation of tumour margins during Mohs surgery.

PubMed

Bennàssar, A; Vilata, A; Puig, S; Malvehy, J

2014-02-01

Ex vivo fluorescence confocal microscopy (FCM) enables real-time imaging of skin morphology directly in freshly excised tissue. FCM displays wide field-of-view mosaics with cellular resolution, thus enabling a rapid bedside pathology. An application of interest is rapid detection of residual basal cell carcinoma (BCC) in skin excisions during Mohs surgery. We sought to evaluate the sensitivity and specificity of ex vivo imaging with FCM for the detection of residual BCC in Mohs tissue excisions, and to calculate the time invested up to the diagnosis for both FCM and frozen sections. Eighty consecutive BCCs were prospectively collected and the margins scanned with ex vivo FCM, including excisions with and without residual BCC of all major subtypes. Each mosaic was divided into two or four, resulting in 480 submosaics for study. Every confocal submosaic was assessed for the presence or absence of BCC and compared with standard frozen sections as the gold standard. Furthermore, the time spent for each technique was calculated and compared. The overall sensitivity and specificity of detecting residual BCC were 88% and 99%, respectively. Moreover, the new technique reduced by almost two-thirds the time invested when compared with the processing of a frozen section (P < 0·001). The results demonstrate the feasibility of confocal mosaicing microscopy in fresh tissue for rapid surgical pathology, potentially to expedite and guide Mohs surgery with high accuracy. This observation is an important step towards the goal of using real-time surgical pathology for skin tumours. © 2013 British Association of Dermatologists.

Skin cancer margin analysis within minutes with full-field OCT (Conference Presentation)

NASA Astrophysics Data System (ADS)

Dalimier, Eugénie; Ogrich, Lauren; Morales, Diego; Cusack, Carrie Ann; Abdelmalek, Mark; Boccara, Claude; Durkin, John

2017-02-01

Non-melanoma skin cancer (NMSC) is the most common cancer. Treatment consists of surgical removal of the skin cancer. Traditional excision involves the removal of the visible skin cancer with a significant margin of normal skin. On cosmetically sensitive areas, Mohs micrographic tissue is the standard of care. Mohs uses intraoperative microscopic margin assessment which minimizes the surgical defect and can help reduce the recurrence rate by a factor of 3. The current Mohs technique relies on frozen section tissue slide preparation which significantly lengthens operative time and requires on-site trained histotechnicians. Full-Field Optical Coherence Tomography (FFOCT) is a novel optical imaging technique which provides a quick and efficient method to visualize cancerous areas in minutes, without any preparation or destruction of the tissue. This study aimed to evaluate the potential of FFOCT for the analysis of skin cancer margins during Mohs surgery. Over 150 images of Mohs specimens were acquired intraoperatively with FFOCT before frozen section analysis. The imaging procedure took less than 5 minutes for each specimen. No artifacts on histological preparation were found arising from FFOCT manipulation; however frozen section artifact was readily seen on FFOCT. An atlas was established with FFOCT images and corresponding histological slides to reveal FFOCT reading criteria of normal and cancerous structures. Blind analysis showed high concordance between FFOCT and histology. FFOCT can potentially reduce recurrence rates while maintaining short surgery times, optimize clinical workflow, and decrease healthcare costs. For the patient, this translates into smaller infection risk, decreased stress, and better comfort.

Evaluation of motility, membrane status and DNA integrity of frozen-thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation.

PubMed

Montano, G A; Kraemer, D C; Love, C C; Robeck, T R; O'Brien, J K

2012-06-01

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

Frozen section evaluation via dynamic real-time non-robotic Telepathology system in a university Cancer center by resident / faculty cooperation team.

PubMed

Vosoughi, Aram; Smith, Paul Taylor; Zeitouni, Joseph A; Sodeman, Gregori M; Jorda, Merce; Gomez-Fernandez, Carmen; Garcia-Buitrago, Monica; Petito, Carol K; Chapman, Jennifer R; Campuzano-Zuluaga, German; Rosenberg, Andrew E; Kryvenko, Oleksandr N

2018-04-30

Frozen section telepathology interpretation experience has been largely limited to practices with locations significantly distant from one another with sporadic need for frozen section diagnosis. In 2010 we established a real-time non-robotic telepathology system in a very active cancer center for daily frozen section service. Herein, we evaluate its accuracy compared to direct microscopic interpretation performed in the main hospital by the same faculty and its cost-efficiency over a 1-year period. From 643 (1416 parts) cases requiring intraoperative consultation, 333 cases (690 parts) were examined by telepathology and 310 cases (726 parts) by direct microscopy. Corresponding discrepancy rates were 2.6% (18 cases: 6 (0.9%) sampling and 12 (1.7%) diagnostic errors) and 3.2% (23 cases: 8 (1.1%) sampling and 15 (2.1%) diagnostic errors), P=.63. The sensitivity and specificity of intraoperative frozen diagnosis were 0.92 and 0.99, respectively, in telepathology, and 0.90 and 0.99, respectively, in direct microscopy. There was no correlation of error incidence with post graduate year level of residents involved in the telepathology service. Cost analysis indicated that the time saved by telepathology was $19691 over one year of the study period while the capital cost for establishing the system was $8924. Thus, real-time non-robotic telepathology is a reliable and easy to use tool for frozen section evaluation in busy clinical settings, especially when frozen section service involves more than one hospital, and it is cost efficient when travel is a component of the service. Copyright © 2018. Published by Elsevier Inc.

The effect of refrigerated and frozen storage on butter flavor and texture.

PubMed

Krause, A J; Miracle, R E; Sanders, T H; Dean, L L; Drake, M A

2008-02-01

Butter is often stored for extended periods of time; therefore, it is important for manufacturers to know the refrigerated and frozen shelf life. The objectives of this study were to characterize the effect of refrigerated and frozen storage on the sensory and physical characteristics of butter. Fresh butter was obtained on 2 occasions from 2 facilities in 113-g sticks and 4-kg bulk blocks (2 facilities, 2 package forms). Butters were placed into both frozen (-20 degrees C) and refrigerated storage (5 degrees C). Frozen butters were sampled after 0, 6, 12, 15, and 24 mo; refrigerated butters were sampled after 0, 3, 6, 9, 12, 15, and 18 mo. Every 3 mo, oxidative stability index (OSI) and descriptive sensory analysis (texture, flavor, and color) were conducted. Every 6 mo, peroxide value (PV), free fatty acid value (FFV), fatty acid profiling, vane, instrumental color, and oil turbidity were examined. A mixed-model ANOVA was conducted to characterize the effects of storage time, temperature, and package type. Storage time, temperature, and package type affected butter flavor, OSI, PV, and FFV. Refrigerated butter quarters exhibited refrigerator/stale off-flavors concurrent with increased levels of oxidation (lower oxidative stability and higher PV and FFV) within 6 mo of refrigerated storage, and similar trends were observed for refrigerated bulk butter after 9 mo. Off-flavors were not evident in frozen butters until 12 or 18 mo for quarters and bulk butters, respectively. Off-flavors in frozen butters were not correlated with instrumental oxidation measurements. Because butter is such a desirable fat source in terms of flavor and textural properties, it is important that manufacturers understand how long their product can be stored before negative attributes develop.

Presentation of frozen shoulder among diabetic and non-diabetic patients.

PubMed

Uddin, Mohammad Moin; Khan, Aminuddin A; Haig, Andrew J; Uddin, Mohammad Kafil

2014-12-01

The literature is inconsistent regarding the level of pain and disability in frozen shoulder patients with or without diabetes mellitus. The aim of this study is to evaluate some demographic features of frozen shoulder patients and to look into the disparity of information by comparing the level of pain and disability due to frozen shoulder between diabetic and non-diabetic people. This is a prospective comparative study. People with frozen shoulder attending an outpatient department were selected by consecutive sampling. Disability levels were assessed by the Shoulder Pain & Disability Index (SPADI). Means of pain and disability scores were compared using unpaired t-test. Among 140 persons with shoulder pain 99 (71.4%) had frozen shoulder. From the participating 40 frozen shoulder patients, 26 (65%) were males and 14 (35%) were females. Seventeen participants (42.5%) were diabetic, two (5%) had impaired glucose tolerance and 21 (52.5%) patients were non-diabetic. Mean disability scores (SPADI) were 51 ± 15.5 in diabetic and 57 ± 16 in non-diabetic persons. The differences in pain and disability level were not statistically significance (respectively, p = 0.24 and p = 0.13 at 95% confidence interval). No difference was found in level of pain and disability level between frozen shoulder patients with and without diabetes.

Incorporation of nisin into a meat binding system to inhibit bacteria on beef surfaces.

PubMed

Cutter, C N; Siragusa, G R

1998-07-01

In two separate experiments, the bacteriocin, nisin, was incorporated into a commercially available meat binding system (Fibrimex) and applied to meat surfaces as a way of inhibiting the meat spoilage organism, Brochothrix thermosphacta during extended refrigerated storage. In experiment 1, pre-rigor lean beef carcass tissue (BCT) was inoculated with B. thermosphacta, left untreated (U), treated with 10 micrograms ml-1 nisin (N), Fibrimex (F) or Fibrimex containing 10 micrograms ml-1 nisin (FN), held aerobically at 4 degrees C for up to 7 d, and populations of B. thermosphacta and nisin activity determined. Experiment 2 determined the effects of the same treatments but on post-rigor, frozen and thawed lean BCT that was inoculated, vacuum-packaged, and stored at 4 degrees C for up to 14 d. In both experiments, N- and FN-treated tissues exhibited significantly lower populations of B. thermosphacta compared to U- and F-treated tissues, for the duration of refrigerated storage. Nisin activity was detected up to 7 d in N- and FN-treated samples from experiment 1. However, activity was detected only to days 0 and 2 in FN- and N-treated samples, respectively, from experiment 2. These studies indicate that the addition of a bacteriocin to a meat binding system and application to meat surfaces may be useful in reducing undesirable bacteria in restructured meat products.

High-resolution scanning electron microscopy of frozen-hydrated cells.

PubMed

Walther, P; Chen, Y; Pech, L L; Pawley, J B

1992-11-01

Cryo-fixed yeast Paramecia and sea urchin embryos were investigated with an in-lens type field-emission SEM using a cold stage. The goal was to further develop and investigate the processing of frozen samples for the low-temperature scanning electron microscope (LTSEM). Uncoated frozen-hydrated samples were imaged with the low-voltage backscattered electron signal (BSE). Resolution and contrast were sufficient to visualize cross-fractured membranes, nuclear pores and small vesicles in the cytoplasm. It is assumed that the resolution of this approach is limited by the extraction depth of the BSE which depends upon the accelerating voltage of the primary beam (V0). In this study, the lowest possible V0 was 2.6 kV because below this value the sensitivity of the BSE detector is insufficient. It is concluded that the resolution of the uncoated specimen could be improved if equipment were available for high-resolution BSE imaging at 0.5-2 kV. Higher resolution was obtained with platinum cryo-coated samples, on which intramembranous particles were easily imaged. These images even show the ring-like appearance of the hexagonally arranged intramembranous particles known from high-resolution replica studies. On fully hydrated samples at high magnification, the observation time for a particular area is limited by mass loss caused by electron irradiation. Other potential sources of artefacts are the deposition of water vapour contamination and shrinkage caused by the sublimation of ice. Imaging of partially dehydrated (partially freeze-dried) samples, e.g. high-pressure frozen Paramecium and sea urchin embryos, will probably become the main application in cell biology. In spite of possible shrinkage problems, this approach has a number of advantages compared with any other electron microscopy preparation method: no chemical fixation is necessary, eliminating this source of artefacts; due to partial removal of the water additional structures in the cytoplasm can be investigated; and finally, the mass loss due to electron beam irradiation is greatly reduced compared to fully frozen-hydrated specimens.

Photoacoustic characterization of radiofrequency ablation lesions

NASA Astrophysics Data System (ADS)

Bouchard, Richard; Dana, Nicholas; Di Biase, Luigi; Natale, Andrea; Emelianov, Stanislav

2012-02-01

Radiofrequency ablation (RFA) procedures are used to destroy abnormal electrical pathways in the heart that can cause cardiac arrhythmias. Current methods relying on fluoroscopy, echocardiography and electrical conduction mapping are unable to accurately assess ablation lesion size. In an effort to better visualize RFA lesions, photoacoustic (PA) and ultrasonic (US) imaging were utilized to obtain co-registered images of ablated porcine cardiac tissue. The left ventricular free wall of fresh (i.e., never frozen) porcine hearts was harvested within 24 hours of the animals' sacrifice. A THERMOCOOLR Ablation System (Biosense Webster, Inc.) operating at 40 W for 30-60 s was used to induce lesions through the endocardial and epicardial walls of the cardiac samples. Following lesion creation, the ablated tissue samples were placed in 25 °C saline to allow for multi-wavelength PA imaging. Samples were imaged with a VevoR 2100 ultrasound system (VisualSonics, Inc.) using a modified 20-MHz array that could provide laser irradiation to the sample from a pulsed tunable laser (Newport Corp.) to allow for co-registered photoacoustic-ultrasound (PAUS) imaging. PA imaging was conducted from 750-1064 nm, with a surface fluence of approximately 15 mJ/cm2 maintained during imaging. In this preliminary study with PA imaging, the ablated region could be well visualized on the surface of the sample, with contrasts of 6-10 dB achieved at 750 nm. Although imaging penetration depth is a concern, PA imaging shows promise in being able to reliably visualize RF ablation lesions.

Short communication: The effects of frozen storage on the survival of probiotic microorganisms found in traditionally and commercially manufactured kefir.

PubMed

O'Brien, K V; Aryana, K J; Prinyawiwatkul, W; Ordonez, K M Carabante; Boeneke, C A

2016-09-01

Kefir is a fermented milk traditionally made from a unique starter culture, which consists of numerous bacteria and yeast species bound together in an exopolysaccharide matrix produced by certain lactic acid bacteria. Many health benefits are associated with traditionally produced kefir; however, bulging and leaking packaging, caused by secondary yeast fermentation during storage, has limited large-scale manufacture. Commercial kefir products have been designed to reduce these effects by using a pure starter culture consisting of a mixture of bacteria and yeast species that give a flavor similar to traditional kefir, but some health benefits may be lost in commercial production due to reduced microbial diversity and lack of beneficial exopolysaccharides. In this study, traditional and commercial kefir was frozen to study the effects of frozen storage on the viability of probiotic bacteria over time. Traditional kefir was prepared by inoculating 1L of pasteurized whole goat milk with approximately 30g of kefir grains. Commercial kefir was prepared by inoculating 1L of full-fat, pasteurized goat milk with a commercial kefir starter. The milk was allowed to ferment at room temperature (24-28°C) until pH 4.6 was reached. Samples were frozen (-8 to -14°C) immediately following the completion of fermentation and were thawed and plated for lactobacilli, lactococci, and yeasts on d 0, 7, 14, and 30 of frozen storage. Lactobacilli, lactococci, and yeasts were significantly reduced in number during frozen storage; however, the traditionally produced kefir was shown to have significantly higher counts of bacteria and yeast at each sampling. We concluded that frozen storage and the development of frozen kefir products could eliminate most packaging concerns associated with the large-scale manufacture of traditionally produced kefir, resulting in increased production and marketability of this healthful product. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Filter Paper-based Nucleic Acid Storage in High-throughput Solid Tumor Genotyping.

PubMed

Stachler, Matthew; Jia, Yonghui; Sharaf, Nematullah; Wade, Jacqueline; Longtine, Janina; Garcia, Elizabeth; Sholl, Lynette M

2015-01-01

Molecular testing of tumors from formalin-fixed paraffin-embedded (FFPE) tissue blocks is central to clinical practice; however, it requires histology support and increases test turnaround time. Prospective fresh frozen tissue collection requires special handling, additional storage space, and may not be feasible for small specimens. Filter paper-based collection of tumor DNA reduces the need for histology support, requires little storage space, and preserves high-quality nucleic acid. We investigated the performance of tumor smears on filter paper in solid tumor genotyping, as compared with paired FFPE samples. Whatman FTA Micro Card (FTA preps) smears were prepared from 21 fresh tumor samples. A corresponding cytology smear was used to assess tumor cellularity and necrosis. DNA was isolated from FTA preps and FFPE core samples using automated methods and quantified using SYBR green dsDNA detection. Samples were genotyped for 471 mutations on a mass spectrophotometry-based platform (Sequenom). DNA concentrations from FTA preps and FFPE correlated for untreated carcinomas but not for mesenchymal tumors (Spearman σ=0.39 and σ=-0.1, respectively). Average DNA concentrations were lower from FTA preps as compared with FFPE, but DNA quality was higher with less fragmentation. Seventy-six percent of FTA preps and 86% of FFPE samples generated adequate DNA for genotyping. FTA preps tended to perform poorly for collection of DNA from pretreated carcinomas and mesenchymal neoplasms. Of the 16 paired DNA samples that were genotyped, 15 (94%) gave entirely concordant results. Filter paper-based sample preservation is a feasible alternative to FFPE for use in automated, high-throughput genotyping of carcinomas.

Impact of Frozen Storage on the Anthocyanin and Polyphenol Content of American Elderberry Fruit Juice

PubMed Central

Johnson, Mitch C.; Thomas, Andrew L.; Greenlief, C. Michael

2015-01-01

The effects of frozen storage on the anthocyanin and polyphenol content of elderberry fruit juice are investigated. Juice from three genotypes of American elderberry (Adams II, Bob Gordon, and Wyldewood) was screened for total phenolic (TP) and total monomeric anthocyanin (TMA) content with spectrophotometric methods. The individual anthocyanin content (IAC) of the juice was tested by coupling solid phase extraction with ultra-performance liquid chromatography/tandem mass spectrometry. Juice samples were tested initially upon harvest, then again after 3, 6, and 9 months of frozen storage. Juice from the three different genotypes had significantly different TP, TMA, and IAC profiles initially (p<0.05). The TP,, TMA, and IAC content of the juice from different genotypes were significantly affected (p<0.05) by the frozen storage time, suggesting that both genotype and length of frozen storage time can affect the anthocyanin content of elderberry fruit juice. PMID:26028422

Effects of ionizing radiation on sensorial, chemical, and microbiological quality of frozen corn and peas.

PubMed

Fan, Xuetong; Sokorai, Kimberly J B

2007-08-01

The effects of irradiation (0, 1.8, and 4.5 kGy) on the quality of frozen corn and peas were investigated during a 12month period of postirradiation storage at -18 degrees C. Irradiation of frozen corn and peas caused a reduction in ascorbic acid content of both vegetables and a loss of texture in peas but had no significant effects on instrumental color parameters (L*, a*, and b*), carotenoid and chlorophyll content, or antioxidant capacity of corn and peas. Irradiation reduced microbial loads of frozen peas and increased display life at 23 degrees C of thawed peas by preserving the green color, apparently because of slower increases in the population of acid-producing microorganisms in the irradiated samples. Overall, irradiation significantly reduced the microbial load and increased the display life of peas and had minimal detrimental effects on the quality of frozen corn and peas.

Distinctive Glycerophospholipid Profiles of Human Seminoma and Adjacent Normal Tissues by Desorption Electrospray Ionization Imaging Mass Spectrometry

NASA Astrophysics Data System (ADS)

Masterson, Timothy A.; Dill, Allison L.; Eberlin, Livia S.; Mattarozzi, Monica; Cheng, Liang; Beck, Stephen D. W.; Bianchi, Federica; Cooks, R. Graham

2011-08-01

Desorption electrospray ionization mass spectrometry (DESI-MS) has been successfully used to discriminate between normal and cancerous human tissue from different anatomical sites. On the basis of this, DESI-MS imaging was used to characterize human seminoma and adjacent normal tissue. Seminoma and adjacent normal paired human tissue sections (40 tissues) from 15 patients undergoing radical orchiectomy were flash frozen in liquid nitrogen and sectioned to 15 μm thickness and thaw mounted to glass slides. The entire sample was two-dimensionally analyzed by the charged solvent spray to form a molecular image of the biological tissue. DESI-MS images were compared with formalin-fixed, hematoxylin and eosin (H&E) stained slides of the same material. Increased signal intensity was detected for two seminolipids [seminolipid (16:0/16:0) and seminolipid (30:0)] in the normal tubule testis tissue; these compounds were undetectable in seminoma tissue, as well as from the surrounding fat, muscle, and blood vessels. A glycerophosphoinositol [PI(18:0/20:4)] was also found at increased intensity in the normal testes tubule tissue when compared with seminoma tissue. Ascorbic acid (i.e., vitamin C) was found at increased amounts in seminoma tissue when compared with normal tissue. DESI-MS analysis was successfully used to visualize the location of several types of molecules across human seminoma and normal tissues. Discrimination between seminoma and adjacent normal testes tubules was achieved on the basis of the spatial distributions and varying intensities of particular lipid species as well as ascorbic acid. The increased presence of ascorbic acid within seminoma compared with normal seminiferous tubules was previously unknown.

Freeze substitution in 3 hours or less.

PubMed

McDonald, K L; Webb, R I

2011-09-01

Freeze substitution is a process for low temperature dehydration and fixation of rapidly frozen cells that usually takes days to complete. New methods for freeze substitution have been developed that require only basic laboratory tools: a platform shaker, liquid nitrogen, a metal block with holes for cryotubes and an insulated container such as an ice bucket. With this equipment, excellent freeze substitution results can be obtained in as little as 90 min for cells of small volume such as bacteria and tissue culture cells. For cells of greater volume or that have significant diffusion barriers such as cuticles or thick cell walls, one can extend the time to 3 h or more with dry ice. The 3-h method works well for all manner of specimens, including plants and Caenorhabditis elegans as well as smaller samples. Here, we present the basics of the techniques and some results from Nicotiana leaves, C. elegans adult worms, Escherichia coli and baby hamster kidney tissue culture cells. © 2011 The Authors Journal of Microscopy © 2011 Royal Microscopical Society.

Adult celiac disease with acetylcholine receptor antibody positive myasthenia gravis

PubMed Central

Freeman, Hugh J; Gillett, Helen R; Gillett, Peter M; Oger, Joel

2009-01-01

Celiac disease has been associated with some autoimmune disorders. A 40-year-old competitive strongman with celiac disease responded to a gluten-free diet, but developed profound and generalized motor weakness with acetylcholine receptor antibody positive myasthenia gravis, a disorder reported to occur in about 1 in 5000. This possible relationship between myasthenia gravis and celiac disease was further explored in serological studies. Frozen stored serum samples from 23 acetylcholine receptor antibody positive myasthenia gravis patients with no intestinal symptoms were used to screen for celiac disease. Both endomysial and tissue transglutaminase antibodies were examined. One of 23 (or, about 4.3%) was positive for both IgA-endomysial and IgA tissue transglutaminase antibodies. Endoscopic studies subsequently showed duodenal mucosal scalloping and biopsies confirmed the histopathological changes of celiac disease. Celiac disease and myasthenia gravis may occur together more often than is currently appreciated. The presence of motor weakness in celiac disease may be a clue to occult myasthenia gravis, even in the absence of intestinal symptoms. PMID:19824105

Rapid Intraoperative Molecular Characterization of Glioma

PubMed Central

Shankar, Ganesh M.; Francis, Joshua M.; Rinne, Mikael L.; Ramkissoon, Shakti H.; Huang, Franklin W.; Venteicher, Andrew S.; Akama-Garren, Elliot H.; Kang, Yun Jee; Lelic, Nina; Kim, James C.; Brown, Loreal E.; Charbonneau, Sarah K.; Golby, Alexandra J.; Pedamallu, Chandra Sekhar; Hoang, Mai P.; Sullivan, Ryan J.; Cherniack, Andrew D.; Garraway, Levi A.; Stemmer-Rachamimov, Anat; Reardon, David A.; Wen, Patrick Y.; Brastianos, Priscilla K.; Curry, William T.; Barker, Fred G.; Hahn, William C.; Nahed, Brian V.; Ligon, Keith L.; Louis, David N.; Cahill, Daniel P.; Meyerson, Matthew

2016-01-01

IMPORTANCE Conclusive intraoperative pathologic confirmation of diffuse infiltrative glioma guides the decision to pursue definitive neurosurgical resection. Establishing the intraoperative diagnosis by histologic analysis can be difficult in low-cellularity infiltrative gliomas. Therefore, we developed a rapid and sensitive genotyping assay to detect somatic single-nucleotide variants in the telomerase reverse transcriptase (TERT) promoter and isocitrate dehydrogenase 1 (IDH1). OBSERVATIONS This assay was applied to tissue samples from 190 patients with diffuse gliomas, including archived fixed and frozen specimens and tissue obtained intraoperatively. Results demonstrated 96% sensitivity (95% CI, 90%–99%) and 100% specificity (95% CI, 95%–100%) for World Health Organization grades II and III gliomas. In a series of live cases, glioma-defining mutations could be identified within 60 minutes, which could facilitate the diagnosis in an intraoperative timeframe. CONCLUSIONS AND RELEVANCE The genotyping method described herein can establish the diagnosis of low-cellularity tumors like glioma and could be adapted to the point-of-care diagnosis of other lesions that are similarly defined by highly recurrent somatic mutations. PMID:26181761

Survival of human pre-antral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix.

PubMed

Amorim, Christiani A; Van Langendonckt, Anne; David, Anu; Dolmans, Marie-Madeleine; Donnez, Jacques

2009-01-01

Ovarian tissue cryopreservation is a promising technique to safeguard fertility in cancer patients. However, in some types of cancer, there is a risk of transmitting malignant cells present in the cryopreserved tissue. To avoid such a risk, pre-antral follicles could be isolated from ovarian tissue and grown in vitro. On the basis of this assumption, the aim of our study was to investigate in vitro survival and growth of pre-antral follicles after cryopreservation of ovarian tissue and follicular isolation, followed by encapsulation in alginate beads. Ovarian biopsies from four patients were frozen and thawed. Pre-antral follicles were then isolated and embedded in an alginate matrix before in vitro culture for 7 days. Small pre-antral follicles (42.98 +/- 9.06 microm) from frozen-thawed tissue can survive and develop after enzymatic isolation and in vitro culture. A total of 159 follicles were incubated in a three-dimensional system (alginate hydrogel) and, after 7 days, all of them showed an increase in size (final size 56.73 +/- 13.10 microm). The survival rate of the follicles was 90% (oocyte and all granulosa cells viable). Our preliminary results indicate that alginate hydrogels may be a suitable system for in vitro culture of isolated human pre-antral follicles. However, more studies are required to establish whether follicular morphology and functionality can be maintained using this matrix.

Slow Freezing, but Not Vitrification Supports Complete Spermatogenesis in Cryopreserved, Neonatal Sheep Testicular Xenografts

PubMed Central

Pukazhenthi, Budhan S.; Nagashima, Jennifer; Travis, Alexander J.; Costa, Guilherme M.; Escobar, Enrique N.; França, Luiz R.; Wildt, David E.

2015-01-01

The ability to spur growth of early stage gametic cells recovered from neonates could lead to significant advances in rescuing the genomes of rare genotypes or endangered species that die unexpectedly. The purpose of this study was to determine, for the first time, the ability of two substantially different cryopreservation approaches, slow freezing versus vitrification, to preserve testicular tissue of the neonatal sheep and subsequently allow initiation of spermatogenesis post-xenografting. Testis tissue from four lambs (3-5 wk old) was processed and then untreated or subjected to slow freezing or vitrification. Tissue pieces (fresh, n = 214; slow freezing, then thawing, n = 196; vitrification, then warming, n = 139) were placed subcutaneously under the dorsal skin of SCID mice and then grafts recovered and evaluated 17 wk later. Grafts from fresh and slow frozen tissue contained the most advanced stages of spermatogenesis, including normal tubule architecture with elongating spermatids in ~1% (fresh) and ~10% (slow frozen) of tubules. Fewer than 2% of seminiferous tubules advanced to the primary spermatocyte stage in xenografts derived from vitrified tissue. Results demonstrate that slow freezing of neonatal lamb testes was far superior to vitrification in preserving cellular integrity and function after xenografting, including allowing ~10% of tubules to retain the capacity to resume spermatogenesis and yield mature spermatozoa. Although a first for any ruminant species, findings also illustrate the importance of preemptive studies that examine cryo-sensitivity of testicular tissue before attempting this type of male fertility preservation on a large scale. PMID:25923660

A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories

PubMed Central

Lou, Jerry J; Mirsadraei, Leili; Sanchez, Desiree E; Wilson, Ryan W; Shabihkhani, Maryam; Lucey, Gregory M; Wei, Bowen; Singer, Elyse J; Mareninov, Sergey; Yong, William H

2014-01-01

Frozen biospecimens are crucial for translational research and contain well preserved nucleic acids and protein. However, the risk for catastrophic freezer failure as well as space, cost, and environmental concerns argue for evaluating long-term room temperature storage alternatives. Formalin-fixed paraffin embedded (FFPE) tissues have great value but their use is limited by cross-linking and fragmentation of nucleic acids, as well as loss of enzymatic activity. Stabilization solutions can now robustly preserve fresh tissue for up to 7 days at room temperature. For longer term storage, commercial vendors of chemical matrices claim real time stability of nucleic acids of over 2 years and their accelerated aging studies to date suggest stability for 12 years for RNA and 60 years for DNA. However, anatomic pathology biorepositories store mostly frozen tissue rather than nucleic acids. Small quantities of tissue can be directly placed on some chemical matrices to stabilize DNA, however RNA and proteins are not preserved. Current lyophilization approaches can preserve histomorphology, DNA, RNA, and proteins though RNA shows moderate degradation after 1–2 years. Formalin free fixatives show improved but varying abilities to preserve nucleic acids and face validation as well as cost barriers in replacing FFPE specimens. The paraffin embedding process can degrade RNA. Development of robust long-term room temperature biospecimen tissue storage technology can potentially reduce costs for the biomedical community in the face of growing targeted therapy needs and decreasing budgets. PMID:24362270

Effects of type of freeze straw and thaw temperature on the fertilizing capacity of frozen chicken semen.

PubMed

Duplaix, M; Sexton, T J

1984-04-01

Two experiments were conducted to determine the relationship between thawing temperature and the type of straw in which chicken semen was frozen. In Experiment 1, semen was frozen in three different types of plastic straws: US (.5-ml capacity), French (.5-ml capacity), and French mini (.25-ml capacity). Experiment 1 was divided into two trials to compare semen packaged in the different straws and thawed at 15 (Trial 1) or .5 C (Trial 2). Although there were distinguishable features of the freeze and thaw curves between samples frozen in the different straws, the type of freeze straw had no effect on the fertilizing capacity of frozen semen when thaw temperature was held constant: fertility, Days 2 to 4 after artificial insemination, ranged from 16 to 27% for Trial 1 and 45 to 47% for Trial 2. In Experiment 2, semen was frozen in US straws and thawed at either .5 or 15 C to assess the effect of the thaw temperature. Fertility of frozen semen, Days 2 to 4 after artificial insemination, was significantly higher when semen was thawed at .5 than at 15 C (62 vs. 20%).

Effect of Chitin Isolated from Crustaceans and Cephalopods on Denaturation of Fish Myofibrillar Protein and the State of Water during Frozen Storage

NASA Astrophysics Data System (ADS)

Yamashita, Yasumitsu; Zhang, Nong; Nozaki, Yukinori

The effect of chitin hydrolysate made from shell of crustaceans and cartilage of cephalopods on the denaturation and the state of water of lizard fish myofibrillar protein (Mf) during frozen storage at -250°C for 120 days were investigated. 5.0% chitin hydrolysate (dried matter) added Mf samples were frozen at -25°C and storage for 120 days, and the change of inactivation of Mf Ca-ATPase and the change of unfrozen water in Mf were examined during the frozen storage. The decrease of Mf Ca-ATPase during frozen storage were slow by addition of chitin hydrolysate. The amount of unfrozen water in Mf were increased by addition of chitin hydrolysate, and decreaced gradually during frozen storage. On the other hand, the amount of unfrozen water of the control were decreaced suddenly during frozen storage. Above results suggest that chitin hydrolysate has cryoprotective effect on Mf. It is guessed that the mechanism of cryoprotective effect of chitin hydrolysate may be caused by an interaction between hydration sphere of Mf and equatorial OH groups in the molecular structure of chitin hydrolysate.

Effect of sucrose and pectin addition on physical, chemical, thermal and rheological properties of frozen/thawed pineapple pulps

NASA Astrophysics Data System (ADS)

Conceição, Márcia Cavalcante; Fernandes, Tatiana Nunes; Prado, Mônica Elisabeth Torres; de Resende, Jaime Vilela

2012-09-01

Pectin (0-1.0 g/100 mL) and sucrose (0-20 g/100 mL) were added to pineapple pulp to improve their rheological properties, thermal properties and stability after freezing and thawing processes. The properties of the mixes were characterized before and after freezing and thawing. Samples were frozen at -20°C, and the freeze concentration was evaluated every 60 min. The thawing rate was evaluated at 19°C and quantified by photographic editing and image analysis software. The thawing rates and values for the freeze concentration were leveled out at pectin concentrations above 0.5 g/100 mL pectin, which indicated that pectin functions to maintain structural homogeneity during freezing. In the thawed samples, the plastic viscosity values were leveled out from pectin concentrations (0.25-0.75 g/100 mL) as the sucrose concentration increased when compared to unfrozen samples. The differences between the rheological parameters of the unfrozen and frozen/thawed pulps, the higher yield stress values after thawing were attributed to the size of suspended particles in the pulp. Applications can specify formulations of frozen products containing pectin, where these properties can be handled after thawing the product.

Concentration of Nitrate near the Surface of Frozen Salt Solutions

NASA Astrophysics Data System (ADS)

Michelsen, R. R. H.; Marrocco, H. A.

2017-12-01

The photolysis of nitrate near the surface of snow and ice in Earth's environment results in the emission of nitrogen oxides (NO, NO2 and, in acidic snow, HONO) and OH radicals. As a result, nitrate photolysis affects the composition and oxidative capacity of the overlying atmosphere. Photolysis yields depend in part on how much nitrate is close enough to the surface to be photolyzed. These concentrations are assumed to be higher than the concentrations of nitrate that are measured in melted snow and ice samples. However, near-surface concentrations of nitrate have not been directly measured. In this work, laboratory studies of the concentration of nitrate in frozen aqueous solutions are described. Individual aqueous solutions of nitric acid, sodium nitrate, and magnesium nitrate were mixed. Attenuated total reflection infrared spectroscopy was utilized to measure the nitrate and liquid water signals within 200 - 400 nm of the lower surface of frozen samples. Temperature was varied from -18°C to -2°C. In addition to the amount of nitrate observed, changes to the frozen samples' morphology with annealing are discussed. Nitrate concentrations near the lower surface of these frozen solutions are high: close to 1 M at warmer temperatures and almost 4 M at the coldest temperature. Known freezing point depression data describe the observed concentrations better than ideal solution thermodynamics, which overestimate concentration significantly at colder temperatures. The implications for modeling the chemistry of snow are discussed. Extending and relating this work to the interaction of gas-phase nitric acid with the surfaces of vapor-deposited ice will also be explored.

Progressive Consent and Specimen Accrual Models to Address Sustainability: A Decade's Experience at an Oregon Biorepository.

PubMed

Ost, John A; Newton, Paul W; Neilson, Duncan R; Cioffi, Joseph A; Wackym, P Ashley; Perkins, R Serene

2017-02-01

The Legacy Biorepository is a College of American Pathologists-accredited biorepository operating within a seven-hospital healthcare system, with a decade's experience in specimen accrual, storage, and distribution. While standardization of our practices through accreditation remains a priority, we along with others face challenges with regard to sustainability. Purposeful changes in our consent process, which we term "progressive consent," are expected to improve sustainability and operational flexibility while increasing our scientific impact. Until 2015, informed consent was performed primarily by biorepository staff at an estimated time of 1 hour per case. After a process improvement exercise, we successfully changed our informed consent process to a modified front-door model, with use of material and data for research as an opt-in or opt-out selection on the institutional patient informed consent form provided to surgery patients in the healthcare system. Successful implementation of this change required the engagement and participation of multiple stakeholders in healthcare system leadership, hospital administration, research, legal, regulatory, and patient care levels. A modified front-door consent enabled us to collect an additional 38 specimens in the first two quarters of 2016, with a time commitment of 15.75 hours, a time savings per specimen increasing in Q2 over Q1. We estimate a potential savings of 43 hours in 2016. This progressive model allowed us to maintain our frozen sample collection while increasing the availability of paraffin-embedded tissue and bodily fluids. Augmenting our tissue collection added little expense per case (approximately half that of each frozen tissue aliquot) and increased the range of biospecimens collected. Biorepository financial sustainability is a critical issue. Thorough evaluation and modification of existing procedures and collection models, as well as cost recovery initiatives, can translate into savings. Sustainability, process improvement, and scientific impact broadly overlap and continue to require operational critique and implementation of strategic changes.

76 FR 31575 - United States Standards for Grades of Frozen Onions

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-01

... processors of frozen onions in the United States. The petition provided information on style, sample size... change in the style designations for minced style, and a correction to the text. The members agreed with the proposed section concerning requirements for Styles, Type I, Whole onions and Type II, Pearl...

Meiotic activity in orthotopic xenografts derived from human postpubertal testicular tissue.

PubMed

Van Saen, D; Goossens, E; Bourgain, C; Ferster, A; Tournaye, H

2011-02-01

Grafting of frozen-thawed testicular tissue has been suggested as a novel fertility preservation method for patients undergoing gonadotoxic treatments. However, this technique still needs further optimization before any clinical application. So far, grafting of human testicular tissue has only been performed to the back skin of nude mice and has shown spermatogonial stem-cell survival and occasionally differentiation up to primary spermatocytes. In this study, orthotopic grafting to mouse testes was evaluated as an alternative, and the effect of freezing and the donor's age was studied. Human testicular tissue was obtained from two prepubertal (aged 3 and 5) and two postpubertal (aged 12 and 13) boys. Both fresh and frozen-thawed testicular tissue was grafted to the testis of immuno-deficient nude mice. Four and nine months after transplantation, testes were analyzed by histology and immunohistochemistry. Four and nine months after transplantation, spermatogonial stem cells were observed in all tissue grafts. Germ cell survival was found to be higher in xenografts from the older boys when compared with that from younger donors. Furthermore, no differentiation was observed in the xenografts from younger patients, but the grafts of two older donors showed differentiation up to the primary spermatocyte level, with the presence of secondary spermatocytes in the oldest donor 9 months after transplantation. This xenografting study shows that intratesticular grafting results in high germ cell survival. In grafts derived from the older boys, meiotic activity was maintained in the xenografts for at least 9 months. Although difficult to conduct due to the scarcity of the tissue, more comparative research is needed to elucidate an optimal grafting strategy.

Effect of Chitin Isolated from Crustaceans and Cephalopods on Denaturation of Fish Myofibrillar Protein and the State of Water during Frozen Storage

NASA Astrophysics Data System (ADS)

Arredondo Romero, Eduardo; Nakamura, Yukio; Yamashita, Yasumitsu; Ichikawa, Hisashi; Goto, Shingi; Osatomi, Kiyoshi; Nozaki, Yukinori

From the point of view of utilization of shells as a waste product of fishery industry, the cryoprotective effect of chitin made from shell of crustaceans (Japanese fan lobster and Japanese swimming crab) and cartilage of cephalopods (spear squid) are studied. Chitin from the shells and cartilage were added to lizard fish myofibrils, and the changes of unfrozen water in myofibrils and ATPase activity of myofibrillar protein were observed during frozen storage at -250°C for 120days. The amount of unfrozen water were increased by addition of three kinds of chitin, and decreased moderately during forzen storage. Whereas, in the chitin free sample, the amount of unfrozen water were decreased rapidly during frozen storage. Changes of ATPase activity of samples showed similar tendency to that of the amount of unfrozen water. The present moderate cryoprotective effect of chitin and data of unfrozen water and ATPase activity of myofibrillar protein suggest the importance of the amount of unfrozen water in frozen matrix.

Optimization of preservation and storage time of sponge tissues to obtain quality mRNA for next-generation sequencing.

PubMed

Riesgo, Ana; Pérez-Porro, Alicia R; Carmona, Susana; Leys, Sally P; Giribet, Gonzalo

2012-03-01

Transcriptome sequencing with next-generation sequencing technologies has the potential for addressing many long-standing questions about the biology of sponges. Transcriptome sequence quality depends on good cDNA libraries, which requires high-quality mRNA. Standard protocols for preserving and isolating mRNA often require optimization for unusual tissue types. Our aim was assessing the efficiency of two preservation modes, (i) flash freezing with liquid nitrogen (LN₂) and (ii) immersion in RNAlater, for the recovery of high-quality mRNA from sponge tissues. We also tested whether the long-term storage of samples at -80 °C affects the quantity and quality of mRNA. We extracted mRNA from nine sponge species and analysed the quantity and quality (A260/230 and A260/280 ratios) of mRNA according to preservation method, storage time, and taxonomy. The quantity and quality of mRNA depended significantly on the preservation method used (LN₂) outperforming RNAlater), the sponge species, and the interaction between them. When the preservation was analysed in combination with either storage time or species, the quantity and A260/230 ratio were both significantly higher for LN₂-preserved samples. Interestingly, individual comparisons for each preservation method over time indicated that both methods performed equally efficiently during the first month, but RNAlater lost efficiency in storage times longer than 2 months compared with flash-frozen samples. In summary, we find that for long-term preservation of samples, flash freezing is the preferred method. If LN₂ is not available, RNAlater can be used, but mRNA extraction during the first month of storage is advised. © 2011 Blackwell Publishing Ltd.

Ductal carcinoma of breast: nuclear grade as a predictor of S-phase fraction.

PubMed

Dabbs, D J

1993-06-01

Nuclear grade (NG) and S-phase fraction (SPF) are established independent prognostic variables for ductal breast carcinomas. Nuclear grade can be assigned by a pathologist in a simple fashion during histopathologic evaluation of the tumor, while SPF requires flow cytometric evaluation of tumor samples. This prospective study was undertaken to determine whether elevated SPF could be predicted from NG alone and how NG and SPF correlate with c-erbB-2 expression. Eighty-two breast carcinomas of ductal type were assigned an NG of low (grade 1 or grade 2) or high (grade 3). S-phase fraction was recorded initially from fresh-frozen tissue samples and was designated as either low SPF (below the value designated as the cutoff for elevated SPF) or high SPF (a value at or greater than the cutoff value). On fresh tissue the NG predicted the range of SPF (low or high) in 89% of cases. Four percent of the cases that did not correlate could definitely be attributed to sample error. The remaining 7% that did not correlate could have been due to sample error, specimen quality, or tumor heterogeneity, as demonstrated by reversal of SPF range as performed on paraffin blocks of tumor. Eighty-eight percent of the tumors positive for c-erbB-2 were NG 3 and 12% were NG 2. All c-erbB-2 tumors were aneuploid. This study demonstrates the importance of carefully assigning NGs on tissue and indicates the importance of reviewing flow cytometric data side by side with histopathologic parameters to detect discrepancies between these two modalities. Careful nuclear grading assignment can accurately predict the range of SPF.

Novel biomarker identification using metabolomic profiling to differentiate radiation necrosis and recurrent tumor following Gamma Knife radiosurgery.

PubMed

Lu, Alex Y; Turban, Jack L; Damisah, Eyiyemisi C; Li, Jie; Alomari, Ahmed K; Eid, Tore; Vortmeyer, Alexander O; Chiang, Veronica L

2017-08-01

OBJECTIVE Following an initial response of brain metastases to Gamma Knife radiosurgery, regrowth of the enhancing lesion as detected on MRI may represent either radiation necrosis (a treatment-related inflammatory change) or recurrent tumor. Differentiation of radiation necrosis from tumor is vital for management decision making but remains difficult by imaging alone. In this study, gas chromatography with time-of-flight mass spectrometry (GC-TOF) was used to identify differential metabolite profiles of the 2 tissue types obtained by surgical biopsy to find potential targets for noninvasive imaging. METHODS Specimens of pure radiation necrosis and pure tumor obtained from patient brain biopsies were flash-frozen and validated histologically. These formalin-free tissue samples were then analyzed using GC-TOF. The metabolite profiles of radiation necrosis and tumor samples were compared using multivariate and univariate statistical analysis. Statistical significance was defined as p ≤ 0.05. RESULTS For the metabolic profiling, GC-TOF was performed on 7 samples of radiation necrosis and 7 samples of tumor. Of the 141 metabolites identified, 17 (12.1%) were found to be statistically significantly different between comparison groups. Of these metabolites, 6 were increased in tumor, and 11 were increased in radiation necrosis. An unsupervised hierarchical clustering analysis found that tumor had elevated levels of metabolites associated with energy metabolism, whereas radiation necrosis had elevated levels of metabolites that were fatty acids and antioxidants/cofactors. CONCLUSIONS To the authors' knowledge, this is the first tissue-based metabolomics study of radiation necrosis and tumor. Radiation necrosis and recurrent tumor following Gamma Knife radiosurgery for brain metastases have unique metabolite profiles that may be targeted in the future to develop noninvasive metabolic imaging techniques.

Effects of frozen storage on survival of Staphylococcus aureus and enterotoxin production in precooked tuna meat.

PubMed

Wu, Xulei; Su, Yi-Cheng

2014-08-01

This study investigated the survival of Staphylococcus aureus in precooked tuna meat for producing canned products during frozen storage (-20 ± 2 °C) as well as its growth and enterotoxin production at 35 to 37 °C after the storage. Samples (50 ± 5 g) of precooked albacore (loin, chunk, and flake) and skipjack (chunk and flake) tuna were inoculated with 5 enterotoxin-producing strains of S. aureus at a level of approximately 3.5 log CFU/g and individually packed in a vacuum bag after 3 h incubation at 35 to 37 °C. Vacuum-packed samples were stored in a freezer (-20 ± 2 °C) for 4 wk. The frozen samples were then thawed in 37 °C circulating water for 2 h and incubated at 35 to 37 °C for 22 h. Populations of S. aureus in all precooked tuna samples decreased slightly (<0.7 log CFU/g) after 4 wk of storage at -20 ± 2 °C, but increased rapidly once the samples were thawed and held at 35 to 37 °C. Total S. aureus counts in albacore and skipjack samples increased by greater than 3 log CFU/g after 6 and 8 h of exposure to 35 to 37 °C, respectively. All samples became spoiled after 10 h of exposure to 35 to 37 °C, while no enterotoxin was detected in any samples. However, enterotoxins were detected in albacore loin and other samples after 12 and 24 h of incubation at 35 to 37 °C, respectively. Frozen precooked tuna meat should be used for producing canned tuna within 6 to 8 h of thawing to avoid product spoilage and potential enterotoxin production by S. aureus in contaminated precooked tuna meat. © 2014 Institute of Food Technologists®

Applications of Mass Spectrometry Imaging for Safety Evaluation.

PubMed

Bonnel, David; Stauber, Jonathan

2017-01-01

Mass spectrometry imaging (MSI) was first derived from techniques used in physics, which were then incorporated into chemistry followed by application in biology. Developed over 50 years ago, and with different principles to detect and map compounds on a sample surface, MSI supports modern biology questions by detecting biological compounds within tissue sections. MALDI (matrix-assisted laser desorption/ionization) imaging trend analysis in this field shows an important increase in the number of publications since 2005, especially with the development of the MALDI imaging technique and its applications in biomarker discovery and drug distribution. With recent improvements of statistical tools, absolute and relative quantification protocols, as well as quality and reproducibility evaluations, MALDI imaging has become one of the most reliable MSI techniques to support drug discovery and development phases. MSI allows to potentially address important questions in drug development such as "What is the localization of the drug and its metabolites in the tissues?", "What is the pharmacological effect of the drug in this particular region of interest?", or "Is the drug and its metabolites related to an atypical finding?" However, prior to addressing these questions using MSI techniques, expertise needs to be developed to become proficient at histological procedures (tissue preparation with frozen of fixed tissues), analytical chemistry, matrix application, instrumentation, informatics, and mathematics for data analysis and interpretation.

Male fertility preservation before gonadotoxic therapies

PubMed Central

Wyns, C.

2010-01-01

Background: Recent advances in cancer therapy have resulted in an increased number of long-term cancer survivors. Unfortunately, aggressive chemotherapy, radiotherapy and preparative regimens for bone marrow transplantation can severely affect male germ cells, including spermatogonial stem cells (SSCs), and lead to permanent loss of fertility. Different options for fertility preservation are dependent on the pubertal state of the patient. Methods: Relevant studies were identified by an extensive Medline search of English and French language articles. Results: Sperm cryopreservation prior to gonadotoxic treatment is a well established method after puberty. In case of ejaculation failure by masturbation, assisted ejaculation methods or testicular tissue sampling should be considered. Although no effective gonadoprotective drug is yet available for in vivo spermatogonial stem cell (SSC) protection in humans, current evidence supports the feasibility of immature testicular tissue (ITT) cryopreservation. The different cryopreservation protocols and available fertility restoration options from frozen tissue, i.e. cell suspension transplantation, tissue grafting and in vitro maturation, are presented. Results obtained in humans are discussed in the light of lessons learned from animal studies. Conclusion: Advances in reproductive technology have made fertility preservation a real possibility in young patients whose gonadal function is threatened by gonadotoxic therapies. The putative indications for such techniques, as well as their limitations according to disease, are outlined. PMID:25302103

Male fertility preservation before gonadotoxic therapies.

PubMed

Wyns, C

2010-01-01

Recent advances in cancer therapy have resulted in an increased number of long-term cancer survivors. Unfortunately, aggressive chemotherapy, radiotherapy and preparative regimens for bone marrow transplantation can severely affect male germ cells, including spermatogonial stem cells (SSCs), and lead to permanent loss of fertility. Different options for fertility preservation are dependent on the pubertal state of the patient. Relevant studies were identified by an extensive Medline search of English and French language articles. Sperm cryopreservation prior to gonadotoxic treatment is a well established method after puberty. In case of ejaculation failure by masturbation, assisted ejaculation methods or testicular tissue sampling should be considered. Although no effective gonadoprotective drug is yet available for in vivo spermatogonial stem cell (SSC) protection in humans, current evidence supports the feasibility of immature testicular tissue (ITT) cryopreservation. The different cryopreservation protocols and available fertility restoration options from frozen tissue, i.e. cell suspension transplantation, tissue grafting and in vitro maturation, are presented. RESULTS obtained in humans are discussed in the light of lessons learned from animal studies. Advances in reproductive technology have made fertility preservation a real possibility in young patients whose gonadal function is threatened by gonadotoxic therapies. The putative indications for such techniques, as well as their limitations according to disease, are outlined.

Downregulated SASH1 expression indicates poor clinical prognosis in gastric cancer.

PubMed

Zhou, Nan; Liu, Can; Wang, Xudong; Mao, Qinsheng; Jin, Qin; Li, Peng

2018-04-01

SASH1 (SAM- and SH3-domain containing 1), a novel candidate tumor suppressor, has attracted attention due to its role in intracellular signal transduction and its tumor prognostic value in diverse cancers. Reports have demonstrated that reduced SASH1 expression correlates with tumor proliferation, invasion, and metastasis. However, the expression and prognostic significance of SASH1 in gastric cancer (GC) remain unclear. In this study, 8 paired fresh-frozen GC tissues and corresponding gastric mucosal tissues were examined by Western blot to analyze the protein expression of SASH1. Seven hundred twenty-six formalin-fixed, paraffin-embedded (FFPE) gastric tissue samples were evaluated by immunohistochemical (IHC) to determine the correlations of SASH1 expression with clinicopathological factors and prognosis. Compared with adjacent noncancerous tissues, SASH1 was significantly downregulated in GC specimens. Analysis using the χ 2 test revealed that low SASH1 expression was significantly associated with advanced TNM stage (P < .001) in GC. Cox regression multivariable analyses demonstrated that SASH1 expression (P < .001), TNM stage (P < .001), preoperative CEA level (P = .003) and preoperative CA19-9 level (P = .002) were independent prognostic factors. Our clinical findings suggest that downregulated SASH1 expression could be used as an independent biomarker for poor prognosis in GC. Copyright © 2018. Published by Elsevier Inc.

Single-layer centrifugation through PureSperm® 80 selects improved quality spermatozoa from frozen-thawed dog semen.

PubMed

Dorado, J; Alcaraz, L; Gálvez, M J; Acha, D; Ortiz, I; Urbano, M; Hidalgo, M

2013-08-01

The aim of this study was to investigate whether single-layer centrifugation (SLC) with PureSperm® 80 could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were divided into two aliquots: one of them was used as control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, frozen-thawed control and frozen-thawed SLC treated samples. A multivariate clustering procedure separated 26,051 motile spermatozoa into three subpopulations (sP): sP1 consisting of highly active but non-progressive spermatozoa (40.3%), sP2 consisting of spermatozoa with high velocity and progressive motility (30.0%), and sP3 consisting of poorly active and non-progressive spermatozoa (29.7%). SLC with PureSperm® 80 yielded sperm suspensions with improved motility, morphology, viability and acrosome integrity (P<0.001). The frozen-thawed SLC treated samples were enriched in sP2, reaching a proportion of 44.1% of the present spermatozoa. From these results, we concluded that SLC with PureSperm® 80 may be an alternative and successful method for improving the quality of frozen-thawed dog spermatozoa. Moreover, sP2 (high-speed and progressive spermatozoa) was more frequently observed after SLC. Finally, this study also demonstrated that the general motile sperm structure present in dogs remained constant despite the effect caused by either cryopreservation or separation by SLC through PureSperm® 80. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of new method and protocol for cryopreservation related to embryo and oocytes freezing in terms of fertilization rate: A comparative study including review of literature

PubMed Central

Barik, Mayadhar; Bajpai, Minu; Patnaik, Santosh; Mishra, Pravash; Behera, Priyamadhaba; Dwivedi, Sada Nanda

2016-01-01

Background: Cryopreservation is basically related to meritorious thin samples or small clumps of cells that are cooled quickly without loss. Our main objective is to establish and formulate an innovative method and protocol development for cryopreservation as a gold standard for clinical uses in laboratory practice and treatment. The knowledge regarding usefulness of cryopreservation in clinical practice is essential to carry forward the clinical practice and research. Materials and Methods: We are trying to compare different methods of cryopreservation (in two dozen of cells) at the same time we compare the embryo and oocyte freezing interms of fertilization rate according to the International standard protocol. Results: The combination of cryoprotectants and regimes of rapid cooling and rinsing during warming often allows successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include semen, blood, tissue samples like tumors, histological cross-sections, human eggs and human embryos. Although presently many studies have reported that the children born from frozen embryos or “frosties,” show consistently positive results with no increase in birth defects or development abnormalities is quite good enough and similar to our study (50–85%). Conclusions: We ensure that cryopreservation technology provided useful cell survivability, tissue and organ preservation in a proper way. Although it varies according to different laboratory conditions, it is certainly beneficial for patient's treatment and research. Further studies are needed for standardization and development of new protocol. PMID:27512686

Substance P expression in the gingival tissue after upper third molar extraction: effect of ketoprofen, a preliminary study.

PubMed

Abbate, G M; Mangano, A; Sacerdote, P; Amodeo, G; Moschetti, G; Levrini, L

2017-01-01

The aim of this study was to evaluate substance P (SP) levels and the effect of a non-steroidal anti-inflammatory drug (NSAID), ketoprofen, on SP in the pericoronal gingival tissue after extraction of upper third molars. A sample of 20 young non-smoking systemically healthy adults of both sexes, with a healthy upper third molar to extract for orthodontic purposes, was selected. After extraction, a sample of the gingival tissue of the pericoronal region was collected with a sterile scalpel, placed into test tubes and kept frozen at -20°C until the SP determination. SP levels were determined by using a commercially available enzyme immunoassay (ELISA) kit. The subjects were randomly divided into two groups: group 1 received a single dose of ketoprofen 30 minutes prior to the experimental procedure. The subjects of group 2 did not receive any kind of drug administration before extraction. The patients were asked to complete a diary on the postoperative pain. A relevant amount of SP was measured in all the gingival samples. No statistically significant difference could be detected in SP expression between the two groups. In group 1 pain appearance was significantly delayed (6.2±0.13 hours) in comparison with group 2 (3.95±0.2 hours). In this small selected group of subjects and limited study design, preventive administration of ketoprofen did not significantly affect the gingival levels of SP, the clinical recommendation emerging is that of NSAID administration postoperatively but before pain appearance in order to optimize the management of pain of the patient.

The impact of milk handling procedures on Ostertagia ostertagi antibody ELISA test results.

PubMed

Vanderstichel, Raphaël; Dohoo, Ian; Stryhn, Henrik

2010-04-19

The impact of various milk handling stressors were analyzed using a commercially available enzyme-linked immunosorbent assay (ELISA) test measuring Ostertagia ostertagi antibodies in milk from dairy cattle (Svanovir). An indirect ELISA has the ability to determine the amount of milk production losses related to intestinal parasitism. The ELISA test recommends fresh defatted milk, however, milk collected from Dairy Herd Improvement (DHI) programs in North America undergo many stressors, including, heating, freezing and are not defatted. Normalized optical density ratios (ODRs) were compared between fresh defatted milk and milk subjected to one or more stressors with a linear mixed model accounting for differences in variation between the fresh and the frozen samples. Concordance correlation coefficients were also analyzed for comparisons to other similar studies. After accounting for random cow and container effects, the treatment factors interacted with each other (p<0.001). Biologically interesting contrasts were created to explain the interaction. The estimated difference in ODR between the milk samples handled according to recommendations of the manufacturers of Svanovir and the whole milk samples that were subjected to the most extreme treatment (heated, frozen, thawed, and re-frozen for 4 weeks) was 0.062 (p<0.001). This difference represented less than 5% of the range, and was thus considered biologically negligible. Frozen whole milk processed by DHI programs, the most likely method of collecting on-farm samples in North America, will likely yield reliable results for the indirect ELISA tests, particularly, Svanovir.

dbMDEGA: a database for meta-analysis of differentially expressed genes in autism spectrum disorder.

PubMed

Zhang, Shuyun; Deng, Libin; Jia, Qiyue; Huang, Shaoting; Gu, Junwang; Zhou, Fankun; Gao, Meng; Sun, Xinyi; Feng, Chang; Fan, Guangqin

2017-11-16

Autism spectrum disorders (ASD) are hereditary, heterogeneous and biologically complex neurodevelopmental disorders. Individual studies on gene expression in ASD cannot provide clear consensus conclusions. Therefore, a systematic review to synthesize the current findings from brain tissues and a search tool to share the meta-analysis results are urgently needed. Here, we conducted a meta-analysis of brain gene expression profiles in the current reported human ASD expression datasets (with 84 frozen male cortex samples, 17 female cortex samples, 32 cerebellum samples and 4 formalin fixed samples) and knock-out mouse ASD model expression datasets (with 80 collective brain samples). Then, we applied R language software and developed an interactive shared and updated database (dbMDEGA) displaying the results of meta-analysis of data from ASD studies regarding differentially expressed genes (DEGs) in the brain. This database, dbMDEGA ( https://dbmdega.shinyapps.io/dbMDEGA/ ), is a publicly available web-portal for manual annotation and visualization of DEGs in the brain from data from ASD studies. This database uniquely presents meta-analysis values and homologous forest plots of DEGs in brain tissues. Gene entries are annotated with meta-values, statistical values and forest plots of DEGs in brain samples. This database aims to provide searchable meta-analysis results based on the current reported brain gene expression datasets of ASD to help detect candidate genes underlying this disorder. This new analytical tool may provide valuable assistance in the discovery of DEGs and the elucidation of the molecular pathogenicity of ASD. This database model may be replicated to study other disorders.

A new method for determining dose rate distribution from radioimmuno-therapy using radiochromic media

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mayer, R.; Dillehay, L.E.; Shao, Y.

The purpose of this study is to describe and evaluate a new, simple, inexpensive method for directly measuring the radiation dose and its spatial distribution generated from explanted tissues of animals previously injected with radiolabeled immunoconjugates or other agents. This technique uses the newly developed radiochromic dye medium (Gafchromic[trademark]) which responds reproducibly for therapeutic dose exposures, has high spatial resolution, does not require film processing, and is relatively insensitive to ambient light. The authors have evaluated the dose distribution from LS174T tumors and selected normal tissues in nude mice previously injected with [sup 90]Y labeled anti-carcinoembryonic antigen antibodies. Individual tissuesmore » from sacrificed animals are halved and the flat section of the tissue is placed onto the dosimetry media and then frozen. The dosimetry medium is exposed to beta and Bremsstrahlung radiation originating from the frozen tissues. The relative darkening of the dosimetry medium depends on the dose deposited in the film. The dosimetry medium is scanned with a commercial flatbed scanner and the image intensity is digitally stored and quantitatively analyzed. Isodose curves are generated and compared to the actual tissue outline. The absorbed dose distribution due to [sup 90]Y exposure show only slight gradients in the interior of the tissue, with a markedly decreasing dose near the edges of the tissue. In addition, the isodose curves follow the tissue outline except in regions having radii of curvature smaller than the range of the beta-particle (R90 = 5 mm). These results suggest that the shape of the tumor, and its curvature, are important in determining the minimum dose delivered to the tumor by radiation from [sup 90]Y monoclonal antibodies, and hence in evaluating the tumor response to the radiation. 28 refs., 8 figs.« less

Use of Digitally Stained Multimodal Confocal Mosaic Images to Screen for Nonmelanoma Skin Cancer

PubMed Central

Mu, Euphemia W.; Lewin, Jesse M.; Stevenson, Mary L.; Meehan, Shane A.; Carucci, John A.; Gareau, Daniel S.

2017-01-01

IMPORTANCE Confocal microscopy has the potential to provide rapid bedside pathologic analysis, but clinical adoption has been limited in part by the need for physician retraining to interpret grayscale images. Digitally stained confocal mosaics (DSCMs) mimic the colors of routine histologic specimens and may increase adaptability of this technology. OBJECTIVE To evaluate the accuracy and precision of 3 physicians using DSCMs before and after training to detect basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) in Mohs micrographic surgery fresh-tissue specimens. DESIGN This retrospective study used 133 DSCMs from 64 Mohs tissue excisions, which included clear margins, residual BCC, or residual SCC. Discarded tissue from Mohs surgical excisions from the dermatologic surgery units at Memorial Sloan Kettering Cancer Center and Oregon Health & Science University were collected for confocal imaging from 2006 to 2011. Final data analysis and interpretation took place between 2014 and 2016. Two Mohs surgeons and a Mohs fellow, who were blinded to the correlating gold standard frozen section diagnoses, independently reviewed the DSCMs for residual nonmelanoma skin cancer (NMSC) before and after a brief training session (about 5 minutes). The 2 assessments were separated by a 6-month washout period. MAIN OUTCOMES AND MEASURES Diagnostic accuracy was characterized by sensitivity and specificity of detecting NMSC using DSCMs vs standard frozen histopathologic specimens. The diagnostic precision was calculated based on interobserver agreement and κ scores. Paired 2-sample t tests were used for comparative means analyses before and after training. RESULTS The average respective sensitivities and specificities of detecting NMSC were 90% (95% CI, 89%-91%) and 79% (95% CI, 52%-100%) before training and 99% (95% CI, 99%-99%) (P = .001) and 93% (95% CI, 90%-96%) (P = .18) after training; for BCC, they were 83% (95% CI, 59%-100%) and 92% (95% CI, 81%-100%) before training and 98% (95% CI, 98%-98%) (P = .18) and 97% (95% CI, 95%-100%) (P = .15) after training; for SCC, they were 73% (95% CI, 65%-81%) and 89% (95% CI, 72%-100%) before training and 100% (P = .004) and 98% (95% CI, 95%-100%) (P = .21) after training. The pretraining interobserver agreement was 72% (κ = 0.58), and the posttraining interobserver agreement was 98% (κ = 0.97) (P = .04). CONCLUSIONS AND RELEVANCE Diagnostic use of DSCMs shows promising correlation to frozen histologic analysis, but image quality was affected by variations in image contrast and mosaic-stitching artifact. With training, physicians were able to read DSCMs with significantly improved accuracy and precision to detect NMSC. PMID:27603676

Elevated expression of FABP3 and FABP4 cooperatively correlates with poor prognosis in non-small cell lung cancer (NSCLC).

PubMed

Tang, Zhiyuan; Shen, Qin; Xie, Hao; Zhou, Xiaoyu; Li, Jun; Feng, Jian; Liu, Hua; Wang, Wei; Zhang, Shu; Ni, Songshi

2016-07-19

Fatty acid binding proteins (FABPs) are intracellular lipid-binding proteins that are involved in a variety of biological cellular processes, including tumorigenesis. In this study, we explored the expression pattern of FABP3 and FABP4 in non-small cell lung cancer (NSCLC) as well as their roles in prognosis. We determined mRNA expression of FABP3 and FABP4 in matched pairs of cancerous and non-cancerous fresh frozen tissues from 30 NSCLC patients. Tissue microarray immunohistochemical analysis (TMA-IHC) was applied to determine the protein expression of FABP3 and FABP4 in 281 cancerous and 121 matched adjacent non-cancerous tissue samples. Our results showed that both mRNA and protein expression of FABP3 and FABP4 were significantly higher in cancerous tissues when compared to non-cancerous tissues. Furthermore, high expression of FABP3 or FABP4 in NSCLC was significantly associated with advanced tumor node metastasis (TNM) stage and had a negative impact on the overall survival of NSCLC patients. Concurrent high expression of FABP3 and FABP4 was significantly related to TNM stage. In conclusion, our research demonstrated that high FABP3 or FABP4 expression had strong prognostic value for overall survival in NSCLC. Detection of FABP3 and FABP4 cooperatively was helpful to predict the prognosis of NSCLC.

Elevated expression of FABP3 and FABP4 cooperatively correlates with poor prognosis in non-small cell lung cancer (NSCLC)

PubMed Central

Tang, Zhiyuan; Shen, Qin; Xie, Hao; Zhou, Xiaoyu; Li, Jun; Feng, Jian; Liu, Hua; Wang, Wei; Zhang, Shu; Ni, Songshi

2016-01-01

Fatty acid binding proteins (FABPs) are intracellular lipid-binding proteins that are involved in a variety of biological cellular processes, including tumorigenesis. In this study, we explored the expression pattern of FABP3 and FABP4 in non-small cell lung cancer (NSCLC) as well as their roles in prognosis. We determined mRNA expression of FABP3 and FABP4 in matched pairs of cancerous and non-cancerous fresh frozen tissues from 30 NSCLC patients. Tissue microarray immunohistochemical analysis (TMA-IHC) was applied to determine the protein expression of FABP3 and FABP4 in 281 cancerous and 121 matched adjacent non-cancerous tissue samples. Our results showed that both mRNA and protein expression of FABP3 and FABP4 were significantly higher in cancerous tissues when compared to non-cancerous tissues. Furthermore, high expression of FABP3 or FABP4 in NSCLC was significantly associated with advanced tumor node metastasis (TNM) stage and had a negative impact on the overall survival of NSCLC patients. Concurrent high expression of FABP3 and FABP4 was significantly related to TNM stage. In conclusion, our research demonstrated that high FABP3 or FABP4 expression had strong prognostic value for overall survival in NSCLC. Detection of FABP3 and FABP4 cooperatively was helpful to predict the prognosis of NSCLC. PMID:27323829

Presentation of frozen shoulder among diabetic and non-diabetic patients☆

PubMed Central

Uddin, Mohammad Moin; Khan, Aminuddin A.; Haig, Andrew J.; Uddin, Mohammad Kafil

2014-01-01

Objective The literature is inconsistent regarding the level of pain and disability in frozen shoulder patients with or without diabetes mellitus. The aim of this study is to evaluate some demographic features of frozen shoulder patients and to look into the disparity of information by comparing the level of pain and disability due to frozen shoulder between diabetic and non-diabetic people. Design This is a prospective comparative study. People with frozen shoulder attending an outpatient department were selected by consecutive sampling. Disability levels were assessed by the Shoulder Pain & Disability Index (SPADI). Means of pain and disability scores were compared using unpaired t-test. Results Among 140 persons with shoulder pain 99 (71.4%) had frozen shoulder. From the participating 40 frozen shoulder patients, 26 (65%) were males and 14 (35%) were females. Seventeen participants (42.5%) were diabetic, two (5%) had impaired glucose tolerance and 21 (52.5%) patients were non-diabetic. Mean disability scores (SPADI) were 51 ± 15.5 in diabetic and 57 ± 16 in non-diabetic persons. The differences in pain and disability level were not statistically significance (respectively, p = 0.24 and p = 0.13 at 95% confidence interval). Conclusions No difference was found in level of pain and disability level between frozen shoulder patients with and without diabetes. PMID:25983497

Assessment of sperm nuclear quality after in vitro maturation of fresh or frozen/thawed mouse pre-pubertal testes.

PubMed

Oblette, A; Rives, N; Dumont, L; Rives, A; Verhaeghe, F; Jumeau, F; Rondanino, C

2017-10-01

Is nuclear quality of in vitro generated spermatozoa from fresh or frozen/thawed pre-pubertal mouse testes similar to that of their in vivo counterparts? The production of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was not significantly increased in organotypic cultures compared to in vivo controls. Although murine spermatozoa have been produced in vitro from pre-pubertal testes, their nuclear DNA integrity has never been investigated. Fresh and frozen/thawed testicular fragments from 6 to 7 days postpartum (dpp) mice were cultured for 30 days. Testicular tissues were frozen by controlled slow freezing (CSF) or solid surface vitrification (SSV). In total, 30 fresh, 30 CSF, 30 SSV testes were used for in vitro maturation and 6 testes from 36 to 37 dpp mice were used as in vivo controls. Murine spermatozoa were extracted from pooled in vitro cultured testicular fragments and from in vivo controls. Sperm aneuploidy was analyzed by fluorescence in situ hybridization (FISH), DNA fragmentation by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling, chromatin condensation by aniline blue staining, telomere length and number by quantitative FISH, DNA oxidation by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Because of the low spermatogenic yield in cultures, a hundred spermatozoa extracted from pooled tissues were examined and compared to their in vivo counterparts. Most of spermatozoa generated in vitro and in vivo were haploid, contained unfragmented DNA and normally condensed chromatin. A similar proportion of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was found in cultures and in vivo. No significant difference in telomere length was found within the nuclei of in vitro and in vivo generated spermatozoa. However, the number of telomere spots was lower in gametes obtained from cultures of fresh, CSF and SSV testes than in their natural counterparts (P < 0.01). Moreover, the proportion of spermatozoa containing 8-OHdG was significantly increased in frozen/thawed tissues in comparison to fresh tissues and in vivo controls (P < 0.05). None. Further studies will be needed to enhance the production of spermatozoa in organotypic cultures while preserving their quality, to investigate epigenetic modifications and embryonic development. This is the first study comparing the nuclear quality of in vitro and in vivo generated murine spermatozoa. The organotypic culture system will have to be adapted for human tissue and extensive analyses of human gamete quality will have to be performed before potential clinical applications can be envisaged. This work was supported by Rouen University Hospital, Ligue contre le Cancer, Agence de la Biomédecine, Association Laurette Fugain, France Lymphome Espoir, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Régional Development Fund (ERDF). The authors declare that they have no conflict of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:journals.permissions@oup.com

Enhancement of the spectral selectivity of complex samples by measuring them in a frozen state at low temperatures in order to improve accuracy for quantitative analysis. Part II. Determination of viscosity for lube base oils using Raman spectroscopy.

PubMed

Kim, Mooeung; Chung, Hoeil

2013-03-07

The use of selectivity-enhanced Raman spectra of lube base oil (LBO) samples achieved by the spectral collection under frozen conditions at low temperatures was effective for improving accuracy for the determination of the kinematic viscosity at 40 °C (KV@40). A collection of Raman spectra from samples cooled around -160 °C provided the most accurate measurement of KV@40. Components of the LBO samples were mainly long-chain hydrocarbons with molecular structures that were deformable when these were frozen, and the different structural deformabilities of the components enhanced spectral selectivity among the samples. To study the structural variation of components according to the change of sample temperature from cryogenic to ambient condition, n-heptadecane and pristane (2,6,10,14-tetramethylpentadecane) were selected as representative components of LBO samples, and their temperature-induced spectral features as well as the corresponding spectral loadings were investigated. A two-dimensional (2D) correlation analysis was also employed to explain the origin for the improved accuracy. The asynchronous 2D correlation pattern was simplest at the optimal temperature, indicating the occurrence of distinct and selective spectral variations, which enabled the variation of KV@40 of LBO samples to be more accurately assessed.

Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression.

PubMed

Press, M F; Hung, G; Godolphin, W; Slamon, D J

1994-05-15

HER-2/neu oncogene amplification and overexpression of breast cancer tissue has been correlated with poor prognosis in women with both node-positive and node-negative disease. However, several studies have not confirmed this association. Review of these studies reveals the presence of considerable methodological variability including differences in study size, follow-up time, techniques and reagents. The majority of papers with clinical follow-up information are immunohistochemical studies using archival, paraffin-embedded breast cancers, and a variety of HER-2/neu antibodies have been used in these studies. Very little information, however, is available about the ability of the antibodies to detect overexpression following tissue processing for paraffin-embedding. Therefore, a series of antibodies, reported in the literature or commercially available, were evaluated to assess their sensitivity and specificity as immunohistochemical reagents. Paraffin-embedded samples of 187 breast cancers, previously characterized as frozen specimens for HER-2/neu amplification by Southern blot and for overexpression by Northern blot, Western blot, and immunohistochemistry, were used. Two multitumor paraffin-embedded tissue blocks were prepared from the previously analyzed breast cancers as a panel of cases to test a series of previously studied and/or commercially available anti-HER-2/neu antibodies. Immunohistochemical staining results obtained with 7 polyclonal and 21 monoclonal antibodies in sections from paraffin-embedded blocks of these breast cancers were compared. The ability of these antibodies to detect overexpression was extremely variable, providing an important explantation for the variable overexpression rate reported in the literature.

Autofluorescence and non-specific immunofluorescent labeling in frozen bovine intestinal tissue sections: Solutions for multi-color immunofluorescence experiments

USDA-ARS?s Scientific Manuscript database

Autofluorescence and non-specific immunofluorescent labeling are common challenges associated with immunofluorescence experiments. Autofluorescence typically demonstrates a broad emission spectrum, increasing the potential for overlap with experiments that utilize multiple fluorophores. During immun...

Clinical anatomy of the elbow and shoulder.

PubMed

Villaseñor-Ovies, Pablo; Vargas, Angélica; Chiapas-Gasca, Karla; Canoso, Juan J; Hernández-Díaz, Cristina; Saavedra, Miguel Ángel; Navarro-Zarza, José Eduardo; Kalish, Robert A

The elbow patients herein discussed feature common soft tissue conditions such as tennis elbow, golfers' elbow and olecranon bursitis. Relevant anatomical structures for these conditions can easily be identified and demonstrated by cross examination by instructors and participants. Patients usually present rotator cuff tendinopathy, frozen shoulder, axillary neuropathy and suprascapular neuropathy. The structures involved in tendinopathy and frozen shoulder can be easily identified and demonstrated under normal conditions. The axillary and the suprascapular nerves have surface landmarks but cannot be palpated. In neuropathy however, physical findings in both neuropathies are pathognomonic and will be discussed. Copyright © 2012 Elsevier España, S.L. All rights reserved.

Simultaneously Synchrotron X-ray Fluorescence and Ptychographic Imaging of Frozen Biological Single Cells

DOE PAGES

Chen, S.; Deng, J.; Nashed, Y. S. G.; ...

2016-07-25

Bionanoprobe (BNP), a hard x-ray fluorescence sample-scanning nanoprobe at the Advanced Photon Source of Argonne National Laboratory, has been used to quantitatively study elemental distributions in biological cells with sub-100 nm spatial resolution and high sensitivity. Cryogenic conditions enable biological samples to be studied in their frozen-hydrated state with both ultrastructure and elemental distributions more faithfully preserved compared to conventional chemical fixation or dehydration methods. Furthermore, radiation damage is reduced in two ways: the diffusion rate of free radicals is decreased at low temperatures; and the sample is embedded in vitrified ice, which reduces mass loss.

Transplantation and Perfusion of Microvascular Fragments in a Rodent Model of Volumetric Muscle Loss Injury

DTIC Science & Technology

2014-07-01

solution. Tissue processing and histological procedures Tissues were excised, weighed, embedded in a talcum- based gel, and flash frozen in 2...the overall level of perfusion was low and was not distributed evenly throughout the defect. GFP MVF transplantation MVFs derived from GFP...support vasculogenesis. However, the levels of VEGF secreted by MVFs were significantly lower than that of ASCs under both normoxic and hypoxic

Influences of Nutrition and Physical Forces on Bone Structure/Function Properties

DTIC Science & Technology

2005-10-01

weeks old. The mice were humanely euthanized at 20 wks of age, the left femur and eighth caudal vertebrae were dissected free of soft tissue and...regime, mice were humanely euthanized and the right tibiae were removed and dissected free of soft tissue and frozen in LRS. The right tibiae...Feld MS (1998) Histopathology of human coronary artherosclerosis by quantifying its chemical composition with Raman spectr- oscopy. Circulation 97:878

Detection of Panulirus argus Virus 1 (PaV1) in exported frozen tails of subadult-adult Caribbean spiny lobsters Panulirus argus.

PubMed

Huchin-Mian, Juan Pablo; Briones-Fourzán, Patricia; Simá-Alvarez, Raúl; Cruz-Quintana, Yanis; Pérez-Vega, Juan Antonio; Lozano-Alvarez, Enrique; Pascual-Jiménez, Cristina; Rodríguez-Canul, Rossanna

2009-09-23

The Caribbean spiny lobster Panulirus argus is a valuable fishing resource and the trade in frozen lobster tails is an important industry. However, the presence of the pathogenic virus Panulirus argus Virus 1 (PaV1), which causes systemic infection in P. argus and is particularly lethal to juvenile individuals, has not been previously examined in imported/exported lobster products. We used PCR assays to determine the presence of PaV1 in abdominal muscle tissue of 22 frozen P. argus tails exported from Belize to Mexico. Based on their size, the tails belonged to subadult-adult lobsters. Using specific primers targeted for PaV1 resulted in 11 tails showing a specific 499 bp band. The sequence of positive amplified fragments showed a high similarity to PaV1 (95% identity with GenBank accession no. EF206313.1). Although the pathogenicity of PaV1 was not evaluated in the present study, our results provide the first evidence of PaV1 in frozen lobster tails exported in the seafood industry as well as the first molecular evidence of PaV1 in adult lobsters.

Conservation science in a terrorist age: the impact of airport security screening on the viability and DNA integrity of frozen felid spermatozoa.

PubMed

Gloor, Kayleen T; Winget, Doug; Swanson, William F

2006-09-01

In response to growing terrorism concerns, the Transportation Security Administration now requires that all checked baggage at U.S. airports be scanned through a cabinet x-ray system, which may increase risk of radiation damage to transported biologic samples and other sensitive genetic material. The objective of this study was to investigate the effect of these new airport security regulations on the viability and DNA integrity of frozen felid spermatozoa. Semen was collected from two domestic cats (Felis silvestris catus) and one fishing cat (Prionailurus viverrinus), cryopreserved in plastic freezing straws, and transferred into liquid nitrogen dry shippers for security screening. Treatment groups included frozen samples from each male scanned once or three times using a Transportation Security Administration-operated cabinet x-ray system, in addition to non-scanned samples (i.e., negative control) and samples previously scanned three times and exposed to five additional high-intensity x-ray bursts (i.e., positive control). Dosimeters placed in empty dry shippers were used to quantify radiation exposure. Following treatment, straws were thawed and spermatozoa analyzed for post-thaw motility (percentage motile and rate of progressive movement), acrosome status, and DNA integrity using single-cell gel electrophoresis (i.e., the comet assay). Dosimeter measurements determined that each airport screening procedure produced approximately 16 mrem of radiation exposure. Our results indicated that all levels of radiation exposure adversely affected (P < 0.05) post-thaw sperm motility, but the percentage of acrosome-intact spermatozoa did not differ (P > 0.05) among treatment groups. Results also showed that the amount of double-stranded DNA damage was greater (P < 0.05) in sperm samples from both cat species scanned three times compared to samples scanned once or negative controls. Findings suggest that new airport security measures may cause radiation-induced damage to frozen spermatozoa and other valuable biologic samples transported on passenger aircraft and that alternative modes of sample transportation should be used whenever possible.

The effect of Taro (Colocasia esculenta L.) and Lesser Yam flour (Dioscorea esculenta L.) as thickener agent on physical characteristics of frozen wheygurt

NASA Astrophysics Data System (ADS)

Nurhartadi, E.; Utami, R.; Widowati, E.; Karunawati, B. M.

2017-11-01

The results showed that the addition ratio of taro and lesser yam flour affected to the physical characteristics of frozen wheygurt. The addition of lesser yam flour increased total soluble solids until the addition ratio of 2:2 due to the higher ash content of lesser yam (2.87%) than taro (0.44%). Sample with addition ratio of 1:3 and 0:4 significantly different compared to other samples, due to the starch content difference between taro (70-80%) and lesser yam (51.34%). Addition ratio of taro and lesser yam flour do not have a significant effect on the viscosity of the frozen wheygurt, due to both starch have similar setback viscosity. Lesser yam setback viscosity was 684.8 cP, while taro was 838.3 cP. Setback viscosity showed a high tendency of retrogradation. The addition ratio of taro and lesser yam flour have a significant effect to the overrun of frozen wheygurt. Addition ratio of taro and lesser yam flour have a significant effect to melting rate of frozen wheygurt. This result was caused by higher peak viscosity of taro starch compared to lesser yam, thus produced thicker gel than lesser yam. This lead increased water contents in the mixtures entrapped and slows down water mobility, hence melting rate would decrease.

High pressure treatments combined with sodium lactate to inactivate Escherichia coli O157:H7 and spoilage microbiota in cured beef carpaccio.

PubMed

Masana, Marcelo Oscar; Barrio, Yanina Ximena; Palladino, Pablo Martín; Sancho, Ana Maria; Vaudagna, Sergio Ramón

2015-04-01

High-pressure treatments (400 and 600 MPa) combined with the addition of sodium lactate (1 and 3%) were tested to reduce Escherichia coli O157:H7 (STEC O157) and spoilage microbiota contamination in a manufactured cured beef carpaccio in fresh or frozen conditions. Counts of spoilage microorganisms and STEC O157 were also examined during the curing step to prepare the carpaccio. STEC O157 counts remained almost unchanged through the curing process performed at 1 ± 1 °C for 12 days, with a small decrease in samples with 3% of sodium lactate. High-pressure treatments at 600 MPa for 5 min achieved an immediate reduction of up to 2 logarithmic units of STEC O157 in frozen carpaccio, and up to 1.19 log in fresh condition. Counts of spoilage bacteria diminished below detection limits in fresh or frozen carpaccio added with sodium lactate by the application of 400 and 600 MPa. Maximum injury on STEC O157 cells was observed at 600 MPa in carpaccio in fresh condition without added sodium lactate. Lethality of high-pressure treatments on STEC O157 was enhanced in frozen carpaccio, while the addition of sodium lactate at 3% reduced the lethality on STEC O157 in frozen samples, and the degree of injury in fresh carpaccio. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of sample preparation method on sensory quality of cooked chicken breast fillets processed for food service

USDA-ARS?s Scientific Manuscript database

Chicken fillets (Pectoralis major) are one of popular items for food service. In the store, especially in fast food service stores, ready-to-cook meat products are commonly stored in freezers before use. The frozen meat can be cooked either directly from a frozen stage or after thawing. However, the...

Evaluation of the antioxidant activity of root extract of pepper fruit (Dennetia tripetala), and it's potential for the inhibition of lipid peroxidation.

PubMed

Okolie, Ngozi Paulinus; Falodun, Abiodun; Davids, Oluseyi

2014-01-01

The antioxidant properties of ethanolic root extract of pepper fruit (Donnetia tripetala), and its effect on lipid peroxidation of some fresh beef tissues during frozen storage were investigated. The antioxidant parameters were assessed using standard methods, while malondialdehyde levels of different fresh beef tissue sections treated with the extract prior to freezing, were estimated in a colorimetric reaction with thiobarbituric acid. The H2O2-scavenging ability of the extract was similar to that of ascorbic acid, with a maximum scavenging power of 55.61 ±4.98%, and an IC50 value of 86µg/ml. The extract exhibited a concentration-dependent ferric ion-reducing power, although this was significantly lower relative to that of the ascorbic acid (p < 0.05). The total phenolic content was 212.5 ± 0.002 mg/g, while the nitric oxide-scavenging ability was 64.33 ± 0.2% after 150 min. The capacity of the extract to inhibit lipid peroxidation in frozen heart muscle slices was significantly higher than that of vitamin C (p < 0 .05), but comparable to vitamins C and E in frozen testes and kidney slices. These results suggest that the root extract of D. tripetala is rich in antioxidants which can be applied to meat preservation during refrigerated storage.

Magnetic resonance imaging, computed tomography, and gross anatomy of the canine tarsus.

PubMed

Deruddere, Kirsten J; Milne, Marjorie E; Wilson, Kane M; Snelling, Sam R

2014-11-01

To describe the normal anatomy of the soft tissues of the canine tarsus as identified on computed tomography (CT) and magnetic resonance imaging (MRI) and to evaluate specific MRI sequences and planes for observing structures of diagnostic interest. Prospective descriptive study. Canine cadavers (n = 3). A frozen cadaver pelvic limb was used to trial multiple MRI sequences using a 1.5 T superconducting magnet and preferred sequences were selected. Radiographs of 6 canine cadaver pelvic limbs confirmed the tarsi were radiographically normal. A 16-slice CT scanner was used to obtain 1 mm contiguous slices through the tarsi. T1-weighted, proton density with fat suppression (PD FS) and T2-weighted MRI sequences were obtained in the sagittal plane, T1-weighted, and PD FS sequences in the dorsal plane and PD FS sequences in the transverse plane. The limbs were frozen for one month and sliced into 4-5 mm thick frozen sections. Anatomic sections were photographed and visually correlated to CT and MR images. Most soft tissue structures were easiest to identify on the transverse MRI sections with cross reference to either the sagittal or dorsal plane. Bony structures were easily identified on all CT, MR, and gross sections. The anatomy of the canine tarsus can be readily identified on MR imaging. © Copyright 2014 by The American College of Veterinary Surgeons.

Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions.

PubMed Central

Grober, J S; Bowen, B L; Ebling, H; Athey, B; Thompson, C B; Fox, D A; Stoolman, L M

1993-01-01

Blood monocytes are the principal reservoir for tissue macrophages in rheumatoid synovitis. Receptor-mediated adhesive interactions between circulating cells and the synovial venules initiate recruitment. These interactions have been studied primarily in cultured endothelial cells. Thus the functional activities of specific adhesion receptors, such as the endothelial selectins and the leukocytic integrins, have not been evaluated directly in diseased tissues. We therefore examined monocyte-microvascular interactions in rheumatoid synovitis by modifying the Stamper-Woodruff frozen section binding assay initially developed to study lymphocyte homing. Specific binding of monocytes to venules lined by low or high endothelium occurred at concentrations as low as 5 x 10(5) cells/ml. mAbs specific for P-selectin (CD62, GMP-140/PADGEM) blocked adhesion by > 90% in all synovitis specimens examined. In contrast, P-selectin-mediated adhesion to the microvasculature was either lower or absent in frozen sections of normal foreskin and placenta. mAbs specific for E-selectin (ELAM-1) blocked 20-50% of monocyte attachment in several RA synovial specimens but had no effect in others. mAbs specific for LFA-1, Mo1/Mac 1, the integrin beta 2-chain, and L-selectin individually inhibited 30-40% of adhesion. An mAb specific for the integrin beta 1-chain inhibited the attachment of elutriated monocytes up to 20%. We conclude that P-selectin associated with the synovial microvasculature initiates shear-resistant adhesion of monocytes in the Stamper-Woodruff assay and stabilizes bonds formed by other selectins and the integrins. Thus the frozen section binding assay permits direct evaluation of leukocyte-microvascular adhesive interactions in inflamed tissues and suggests a prominent role for P-selectin in monocyte recruitment in vivo. Images PMID:7685772

Gene expression profiling of histologically normal breast tissue in females with human epidermal growth factor receptor 2‑positive breast cancer.

PubMed

Zubor, Pavol; Hatok, Jozef; Moricova, Petra; Kapustova, Ivana; Kajo, Karol; Mendelova, Andrea; Sivonova, Monika Kmetova; Danko, Jan

2015-02-01

Gene expression profile‑based taxonomy of breast cancer (BC) has been described as a significant breakthrough in comprehending the differences in the origin and behavior of cancer to allow individually tailored therapeutic approaches. In line with this, we hypothesized that the gene expression profile of histologically normal epithelium (HNEpi) could harbor certain genetic abnormalities predisposing breast tissue cells to develop human epidermal growth factor receptor 2 (HER2)‑positive BC. Thus, the aim of the present study was to assess gene expression in normal and BC tissue (BCTis) from patients with BC in order to establish its value as a potential diagnostic marker for cancer development. An array study evaluating a panel of 84 pathway‑ and disease‑specific genes in HER2‑positive BC and tumor‑adjacent HNEpi was performed using quantitative polymerase chain reaction in 12 patients using microdissected samples from frozen tissue. Common prognostic and predictive parameters of BC were assessed by immunohistochemistry and in situ hybridization. In the BCTis and HNEpi samples of 12 HER2‑positive subjects with BC, the expression of 2,016 genes was assessed. A total of 39.3% of genes were deregulated at a minimal two‑fold deregulation rate and 10.7% at a five‑fold deregulation rate in samples of HNEpi or BCTis. Significant differences in gene expression between BCTis and HNEpi samples were revealed for BCL2L2, CD44, CTSD, EGFR, ERBB2, ITGA6, NGFB, RPL27, SCBG2A1 and SCGB1D2 genes (P<0.05), as well as GSN, KIT, KLK5, SERPINB5 and STC2 genes (P<0.01). Insignificant differences (P<0.07) were observed for CCNA1, CLU, DLC1, GABRP and IL6 genes. The ontological gene analyses revealed that the majority of the deregulated genes in the HNEpi samples were part of the functional gene group directly associated with BC origin and prognosis. Functional analysis showed that the most frequent gene deregulations occurred in genes associated with apoptosis and cell cycle regulation in BCTis samples, and with angiogenesis, regulation of the cell cycle and transcriptional activity in HNEpi samples. The molecular profiling of HNEpi breast tissue revealed gene expression abnormalities that may represent potential markers of increased risk for HER2‑positive malignant transformation of breast tissue, and may be able to be employed as predictors of prognosis.

Proximate composition, fatty acids, cholesterol, minerals in frozen red porgy.

PubMed

Miniadis-Meimaroglou, Sofia; Dimizas, Christos; Loukas, Vassilis; Moukas, Athanasios; Vlachos, Alexandra; Thomaidis, Nikolaos; Paraskevopoulou, Vassiliki; Dasenakis, Manolis

2007-04-01

The proximate composition of frozen red porgy (Pagrus pagrus) was determined. The moisture, ash, protein and total lipids (45.5+/-1.4% PL of which 90.4+/-2.0% PhL) were found to be 71.7+/-1.0%, 1.73+/-0.12%, 21.5+/-0.8% and 0.81+/-0.09% of the wet muscle tissue, respectively. 16:0 and 18:0 were the main SFA, 18:1 (omega-9 and omega-7) the main MUFA while DHA, EPA and arachidonic acid were the main polyunsaturated fatty acids (PUFA). The SFA/PUFA ratio was 1.5 and the omega-3/omega-6 ratio was 3.02. The cholesterol content was found to be 8.18+/-0.34 mg/100 g of the wet muscle tissue. Ni, Cr, Mn, Cu, Zn, Fe and Mg were determined in the muscles, skin, hepatopancreas and head of the fish. The covering percentage of the recommended daily allowance/intake (RDA/RDI) for each mineral, in the muscle tissue, has been calculated to 14.2% (males) and 7.89% (females) for Fe, 2.87% for Cu, 4.07% for Zn 0.4% for Mn, 13.9% for Ni, 20.2% for Cr and 10.4% for Mg.

Qualitative and quantitative assessment of DNA quality of frozen beef based on DNA yield, gel electrophoresis and PCR amplification and their correlations to beef quality.

PubMed

Zhao, Jing; Zhang, Ting; Liu, Yongfeng; Wang, Xingyu; Zhang, Lan; Ku, Ting; Quek, Siew Young

2018-09-15

Freezing is a practical method for meat preservation but the quality of frozen meat can deteriorate with storage time. This research investigated the effect of frozen storage time (up to 66 months) on changes in DNA yield, purity and integrity in beef, and further analyzed the correlation between beef quality (moisture content, protein content, TVB-N value and pH value) and DNA quality in an attempt to establish a reliable, high-throughput method for meat quality control. Results showed that frozen storage time influenced the yield and integrity of DNA significantly (p < 0.05). The DNA yield decreased as frozen storage time increased due to DNA degradation. The half-life (t 1/2  = ln2/0.015) was calculated as 46 months. The DNA quality degraded dramatically with the increased storage time based on gel electrophoresis results. Polymerase chain reaction (PCR) products from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) were observed in all frozen beef samples. Using real-time PCR for quantitative assessment of DNA and meat quality revealed that correlations could be established successfully with mathematical models to evaluate frozen beef quality. Copyright © 2018 Elsevier Ltd. All rights reserved.

RNA-seq transcriptome analysis of formalin fixed, paraffin-embedded canine meningioma

PubMed Central

Grenier, Jennifer K.; Foureman, Polly A.; Sloma, Erica A.

2017-01-01

Meningiomas are the most commonly reported primary intracranial tumor in dogs and humans and between the two species there are similarities in histology and biologic behavior. Due to these similarities, dogs have been proposed as models for meningioma pathobiology. However, little is known about specific pathways and individual genes that are involved in the development and progression of canine meningioma. In addition, studies are lacking that utilize RNAseq to characterize gene expression in clinical cases of canine meningioma. The primary objective of this study was to develop a technique for which high quality RNA can be extracted from formalin-fixed, paraffin embedded tissue and then used for transcriptome analysis to determine patterns of gene expression. RNA was extracted from thirteen canine meningiomas–eleven from formalin fixed and two flash-frozen. These represented six grade I and seven grade II meningiomas based on the World Health Organization classification system for human meningioma. RNA was also extracted from fresh frozen leptomeninges from three control dogs for comparison. RNAseq libraries made from formalin fixed tissue were of sufficient quality to successfully identify 125 significantly differentially expressed genes, the majority of which were related to oncogenic processes. Twelve genes (AQP1, BMPER, FBLN2, FRZB, MEDAG, MYC, PAMR1, PDGFRL, PDPN, PECAM1, PERP, ZC2HC1C) were validated using qPCR. Among the differentially expressed genes were oncogenes, tumor suppressors, transcription factors, VEGF-related genes, and members of the WNT pathway. Our work demonstrates that RNA of sufficient quality can be extracted from FFPE canine meningioma samples to provide biologically relevant transcriptome analyses using a next-generation sequencing technique, such as RNA-seq. PMID:29073243

Pathology and diagnosis of avian bornavirus infection in wild Canada geese (Branta canadensis), trumpeter swans (Cygnus buccinator) and mute swans (Cygnus olor) in Canada: a retrospective study.

PubMed

Delnatte, Pauline; Ojkic, Davor; Delay, Josepha; Campbell, Doug; Crawshaw, Graham; Smith, Dale A

2013-04-01

Nine hundred and fifty-five pathology cases collected in Ontario between 1992 and 2011 from wild free-ranging Canada geese, trumpeter swans and mute swans were retrospectively evaluated for the pathology associated with avian bornavirus (ABV) infection. Cases were selected based on the presence of upper gastrointestinal impaction, central nervous system histopathology or clinical history suggestive of ABV infection. The proportion of birds meeting at least one of these criteria was significantly higher at the Toronto Zoo (30/132) than elsewhere in Ontario (21/823). Central, peripheral and autonomic nervous tissues were examined for the presence of lymphocytes and plasma cells on histopathology. The presence of virus was assessed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) on frozen brains and on formalin-fixed paraffin-embedded tissues. Among selected cases, 86.3% (44/51) were considered positive on histopathology, 56.8% (29/51) were positive by immunohistochemistry, and RT-PCR was positive on 88.2% (15/17) of the frozen brains and 78.4% (40/51) of the formalin-fixed paraffin-embedded samples. Histopathological lesions included gliosis and lymphoplasmacytic perivascular cuffing in brain (97.7%), spinal cord (50%), peripheral nerves (55.5%) and myenteric ganglia or nerves (62.8%), resembling lesions described in parrots affected with proventricular dilatation disease. Partial amino acid sequences of the nucleocapsid gene from seven geese were 100% identical amongst themselves and 98.1 to 100% identical to the waterfowl sequences recently described in the USA. Although ABV has been identified in apparently healthy geese, our study confirmed that ABV can also be associated with significant disease in wild waterfowl species.

Tumor Touch Imprints as Source for Whole Genome Analysis of Neuroblastoma Tumors

PubMed Central

Brunner, Clemens; Brunner-Herglotz, Bettina; Ziegler, Andrea; Frech, Christian; Amann, Gabriele; Ladenstein, Ruth; Ambros, Inge M.; Ambros, Peter F.

2016-01-01

Introduction Tumor touch imprints (TTIs) are routinely used for the molecular diagnosis of neuroblastomas by interphase fluorescence in-situ hybridization (I-FISH). However, in order to facilitate a comprehensive, up-to-date molecular diagnosis of neuroblastomas and to identify new markers to refine risk and therapy stratification methods, whole genome approaches are needed. We examined the applicability of an ultra-high density SNP array platform that identifies copy number changes of varying sizes down to a few exons for the detection of genomic changes in tumor DNA extracted from TTIs. Material and Methods DNAs were extracted from TTIs of 46 neuroblastoma and 4 other pediatric tumors. The DNAs were analyzed on the Cytoscan HD SNP array platform to evaluate numerical and structural genomic aberrations. The quality of the data obtained from TTIs was compared to that from randomly chosen fresh or fresh frozen solid tumors (n = 212) and I-FISH validation was performed. Results SNP array profiles were obtained from 48 (out of 50) TTI DNAs of which 47 showed genomic aberrations. The high marker density allowed for single gene analysis, e.g. loss of nine exons in the ATRX gene and the visualization of chromothripsis. Data quality was comparable to fresh or fresh frozen tumor SNP profiles. SNP array results were confirmed by I-FISH. Conclusion TTIs are an excellent source for SNP array processing with the advantage of simple handling, distribution and storage of tumor tissue on glass slides. The minimal amount of tumor tissue needed to analyze whole genomes makes TTIs an economic surrogate source in the molecular diagnostic work up of tumor samples. PMID:27560999

Effect of different concentrations of soybean lecithin and virgin coconut oil in Tris-based extender on the quality of chilled and frozen-thawed bull semen

PubMed Central

Tarig, A. A.; Wahid, H.; Rosnina, Y.; Yimer, N.; Goh, Y. M.; Baiee, F. H.; Khumran, A. M.; Salman, H.; Assi, M. A.; Ebrahimi, M.

2017-01-01

Aim: The objective of this study was to evaluate the effects of different concentrations of soybean lecithin (SL) and virgin coconut oil (VCO) in Tris-based extender on chilled and frozen-thawed bull semen quality parameters. Materials and Methods: A total of 24 ejaculates were collected from four bulls via an electroejaculator. Semen samples were diluted with 2% VCO in Tris-based extender which consists of various concentrations of SL (1, 1.25, 1.5, and 1.75%). A 20% egg yolk in Tris used as a positive control (C+). The diluted semen samples were divided into two fractions; one for chilling which were stored at 4°C for 24, 72, and 144 h before evaluated for semen quality parameters. The second fraction used for freezing was chilled for 3 h at 4°C, packed into 0.25 mL straws and then cryopreserved in liquid nitrogen. The samples were then evaluated after 7 and 14 days. Chilled and frozen semen samples were thawed at 37°C and assessed for general motility using computer-assisted semen analysis, viability, acrosome integrity and morphology (eosin-nigrosin stain), membrane integrity, and lipid peroxidation using thiobarbituric acid reaction test. Results: The results showed that all the quality parameters assessed were significantly (p<0.05) improved at 1.5% SL concentration in chilled semen. Treatment groups of 1, 1.25, 1.5, and 1.75% SL were higher in quality parameters than the control group (C+) in chilled semen. However, all the quality parameters in frozen-thawed semen were significantly higher in the C+ than the treated groups. Conclusion: In conclusion, supplementation of 1.5% SL in 2% VCO Tris-based extender enhanced the chilled bull semen. However, there was no marked improvement in the frozen-thawed quality parameters after treatment. PMID:28717321

Effect of different concentrations of soybean lecithin and virgin coconut oil in Tris-based extender on the quality of chilled and frozen-thawed bull semen.

PubMed

Tarig, A A; Wahid, H; Rosnina, Y; Yimer, N; Goh, Y M; Baiee, F H; Khumran, A M; Salman, H; Assi, M A; Ebrahimi, M

2017-06-01

The objective of this study was to evaluate the effects of different concentrations of soybean lecithin (SL) and virgin coconut oil (VCO) in Tris-based extender on chilled and frozen-thawed bull semen quality parameters. A total of 24 ejaculates were collected from four bulls via an electroejaculator. Semen samples were diluted with 2% VCO in Tris-based extender which consists of various concentrations of SL (1, 1.25, 1.5, and 1.75%). A 20% egg yolk in Tris used as a positive control (C+). The diluted semen samples were divided into two fractions; one for chilling which were stored at 4°C for 24, 72, and 144 h before evaluated for semen quality parameters. The second fraction used for freezing was chilled for 3 h at 4°C, packed into 0.25 mL straws and then cryopreserved in liquid nitrogen. The samples were then evaluated after 7 and 14 days. Chilled and frozen semen samples were thawed at 37°C and assessed for general motility using computer-assisted semen analysis, viability, acrosome integrity and morphology (eosin-nigrosin stain), membrane integrity, and lipid peroxidation using thiobarbituric acid reaction test. The results showed that all the quality parameters assessed were significantly (p<0.05) improved at 1.5% SL concentration in chilled semen. Treatment groups of 1, 1.25, 1.5, and 1.75% SL were higher in quality parameters than the control group (C+) in chilled semen. However, all the quality parameters in frozen-thawed semen were significantly higher in the C+ than the treated groups. In conclusion, supplementation of 1.5% SL in 2% VCO Tris-based extender enhanced the chilled bull semen. However, there was no marked improvement in the frozen-thawed quality parameters after treatment.

Breed differences of bull frozen-thawed semen.

PubMed

Ntemka, A; Tsousis, G; Brozos, C; Kiossis, E; Boscos, C M; Tsakmakidis, I A

2016-12-01

The objective of this study was to investigate the quality of frozen-thawed semen from different bull breeds. Commercial frozen-thawed bull semen samples (26 per breed, 130 totally) of five breeds (Holstein [Η], Brown Swiss [BS], Limousin [L], Belgian Blue [BB], Blonde d' Aquitaine [BA]) were used. After thawing, each semen sample was subjected to thermal resistance test (TR) for 0.5 and 1 hr at 38°C and hypo-osmotic swelling test (HOST) for 1 hr at 150 mOsm at 37°C. Additionally, all samples were evaluated at times 0 hr (thawing), 0.5 hr (TR), 1 hr (TR) for kinetics by CASA [progressive, immotile, rapid, medium, slow moving spermatozoa, curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), linearity (LIN), straightness (STR), beat cross-frequency (BCF), amplitude of lateral head displacement (ALH), wobble (WOB)]. Moreover, directly after thawing, all semen samples were evaluated for morphometry, morphology, viability and DNA fragmentation. Statistical analysis was conducted using a mixed model for repeated measures. The results showed (a) higher VCL after thawing in H, L breeds compared to BB and BA, (b) higher VAP after thawing in L compared to BB, BA, (c) higher values of progressive spermatozoa after TR in H, BS compared to BB, BA, (d) higher values of rapid spermatozoa after thawing and 0.5 hr of TR in H, BS, L compared to BB, BA, (e) lower viability in BA after thawing compared to H, BS, BB, (f) lower morphological abnormalities in H compared to L, BB, (g) higher head length in Η compared to BB. No significant differences were observed in the results from HOST and DNA fragmentation between breeds. In conclusion, quality characteristics of frozen-thawed bull semen are dependent on the breed. Frozen semen from BB and BA breeds should be handled more carefully after thawing, as it is more sensitive to stress. © 2016 Blackwell Verlag GmbH.

On the compressibility and temperature boundary of warm frozen soils

NASA Astrophysics Data System (ADS)

Qi, Jilin; Dang, Boxiang; Guo, Xueluan; Sun, Xiaoyu; Yan, Xu

2017-04-01

A silty-clay obtained along the Qinghai-Tibetan railway and a standard Chinese sand were taken as study objects. Saturated frozen soil samples were prepared for testing. Step-load was used and confined compression was carried out on the soils under different temperatures. Compression index and pseudo-preconsolidation pressure (PPC) were obtained. Unlike unfrozen soils, PPC is not associated with stress history. However, it is still the boundary of elastic and plastic deformations. Different compression indexes can be obtained from an individual compression curve under pressures before and after PPC. The parameters at different thermal and stress conditions were analyzed. It is found that temperature plays a critical role in mechanical behaviours of frozen soils. Efforts were then made on the silty-clay in order to suggest a convincing temperature boundary in defining warm frozen soil. Three groups of ice-rich samples with different ice contents were prepared and tested under confined compression. The samples were compressed under a constant load and with 5 stepped temperatures. Strain rates at different temperatures were examined. It was found that the strain rate at around -0.6°C increased abruptly. Analysis of compression index was performed on the data both from our own testing program and from the literature, which showed that at about -1°C was a turning point in the curves for compression index against temperature. Based on both our work and taking into account the unfrozen water content vs. temperature, the range of -1°C to -0.5°C seems to be the temperature where the mechanical properties change greatly. For convenience, -1.0°C can be defined as the boundary for warm frozen soils.

Effect of cooling rate on sperm quality of cryopreserved Andalusian donkey spermatozoa.

PubMed

Demyda-Peyrás, S; Bottrel, M; Acha, D; Ortiz, I; Hidalgo, M; Carrasco, J J; Gómez-Arrones, V; Gósalvez, J; Dorado, J

2018-06-01

The aim of this study was to evaluate the effect of different cooling rates on post-thaw quality of cryopreserved donkey spermatozoa. Eighteen ejaculates from six adult Andalusian donkeys (three ejaculates per donkey) were collected using an artificial vagina. Pooled semen samples (two ejaculates per pool) were divided into three aliquots, and frozen in Gent freezing extender using three different cryopreservation protocols (P): P1 (conventional slow freezing, as control): semen pre-cooled in an Equitainer for 2 h and frozen in liquid nitrogen (LN 2 ) vapour; P2 (controlled pre-freeze cooling rate): semen pre-cooled at a controlled rate for 73 min and frozen in LN 2 vapour; and P3 (rapid freezing) semen frozen immediately in LN 2 vapour. After thawing at 37 °C for 30 s, semen samples were assessed for motility, morphology, acrosome and plasma membrane integrity; spermatozoa were also tested for DNA integrity. Significant (P < 0.01) differences were found between the cryopreservation protocols for all sperm parameters evaluated, except for DNA integrity. Semen samples frozen using P2 showed significantly (P < 0.01) higher values for sperm motility, morphology, sperm membrane integrity, and acrosome integrity. On the contrary, P3 reduced sperm motility (P < 0.01) and increased the percentage of spermatozoa with damaged plasma membrane (P < 0.001). In our study, we demonstrated that the sperm of Andalusian donkey is particularly sensitive to the cooling rate used before freezing. Furthermore, Andalusian donkey semen can be successfully cryopreserved using controlled cooling rates combined with freezing in LN 2 vapour. Copyright © 2018 Elsevier B.V. All rights reserved.

Spawn viability in edible mushrooms after freezing in liquid nitrogen without a cryoprotectant.

PubMed

Mata, Gerardo; Pérez-Merlo, Rosalía

2003-08-01

Five strains of edible mushrooms (Lentinula boryana, Lentinula edodes, Pleurotus djamor, Pleurotus pulmonarius, and Volvariella volvacea) were studied. Spawn were prepared from sorghum seeds and then incubated for 14 days under optimum conditions for each species. Once covered by mycelia, the sorghum seeds were placed in polycarbonate vials for freezing in liquid nitrogen. The effect of adding a cryoprotective solution before freezing (either 10% glycerol v/v or 5% dimethylsulfoxide v/v) was evaluated as a function of mycelial growth and percent viability. Three main treatments were undertaken: (1) freezing with a glycerol or dimethylsulfoxide cryoprotectant, (2) freezing with water and (3) freezing without cryoprotectant or water. Samples were maintained frozen for a week, after which time they were thawed (10 min at 30 degrees C) and the seeds placed in Petri dishes with a culture medium. A recovery rate of 96.8% was obtained for the total number of samples summed over all strains and treatments. In contrast, 99.2% of the samples frozen without cryoprotectant were recovered. The recovery of frozen mycelia was delayed with respect to a control group, which was not frozen. However, no difference was observed in percent recovery and mycelial diameter when a new series of spawn was prepared from mycelia that had been previously frozen. Results obtained from this experiment demonstrate that an adequate recovery of mycelia can be obtained without using a cryoprotectant. This capacity might enable large quantities of commercial mushroom strains to be handled at reduced production costs. It is suggested that the mycelia survived freezing without cryoprotectants because they were embedded and protected within the sorghum seeds used to elaborate the spawn.

Quality Evaluation of Pork with Various Freezing and Thawing Methods

PubMed Central

2014-01-01

In this study, the physicochemical and sensory quality characteristics due to the influence of various thawing methods on electro-magnetic and air blast frozen pork were examined. The packaged pork samples, which were frozen by air blast freezing at −45℃ or electro-magnetic freezing at −55℃, were thawed using 4 different methods: refrigeration (4±1℃), room temperature (RT, 25℃), cold water (15℃), and microwave (2450 MHz). Analyses were carried out to determine the drip and cooking loss, water holding capacity (WHC), moisture content and sensory evaluation. Frozen pork thawed in a microwave indicated relatively less thawing loss (0.63-1.24%) than the other thawing methods (0.68-1.38%). The cooking loss after electro-magnetic freezing indicated 37.4% by microwave thawing, compared with 32.9% by refrigeration, 36.5% by RT, and 37.2% by cold water in ham. The thawing of samples frozen by electro-magnetic freezing showed no significant differences between the methods used, while the moisture content was higher in belly thawed by microwave (62.0%) after electro-magnetic freezing than refrigeration (54.8%), RT (61.3%), and cold water (61.1%). The highest overall acceptability was shown for microwave thawing after electro-magnetic freezing but there were no significant differences compared to that of the other samples. PMID:26761493

Antigen detection systems

USDA-ARS?s Scientific Manuscript database

Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

Impact of water extractable arabinoxylan from rye bran on the frozen steamed bread dough quality.

PubMed

Wang, Pei; Tao, Han; Jin, Zhengyu; Xu, Xueming

2016-06-01

Impact of water extractable arabinoxylan from rye bran on frozen steamed bread dough quality was investigated in terms of the bread characteristics, ice crystallization, yeast activity as well as the gluten molecular weight distribution and glutenin macropolymer content in the present study. Results showed that water extractable arabinoxylan significantly improved bread characteristics during the 60-day frozen storage. Less water was crystallized in the water extractable arabinoxylan dough during storage, which could explain the alleviated yeast activity loss. For all the frozen dough samples, more soluble high molecular weight (Mw ≈ 91,000-688,000) and low molecular weight (Mw ≈ 91,000-16,000) proteins were derived from glutenin macropolymer depolymerization. Nevertheless, water extractable arabinoxylan dough developed higher glutenin macropolymer content with lowered level of soluble low molecular weight proteins throughout the storage. This study suggested water extractable arabinoxylan from rye bran had great potential to be served as an effective frozen steamed bread dough improver. Copyright © 2016 Elsevier Ltd. All rights reserved.

Intracytoplasmic sperm injection outcomes with cryopreserved testicular sperm aspiration samples.

PubMed

Roque, M; Valle, M; Marques, F; Sampaio, M; Geber, S

2016-04-01

Intracytoplasmic sperm injection (ICSI) may be performed with testicular frozen-thawed spermatozoa in patients with nonobstructive azoospermia (NOA). Sperm retrieval can be performed in advance of oocyte aspiration, as it may avoid the possibility of no recovery of spermatozoa on the day of oocyte pickup. There are few studies available in the literature concerning the use of frozen-thawed spermatozoa obtained from testicular sperm aspiration (TESA). To evaluate the effects and the outcomes of ICSI with frozen-thawed spermatozoa obtained by TESA, we performed a retrospective analysis of 43 ICSI cycles using frozen-thawed TESA. We obtained acceptable results with a fertilisation rate of 67.9%, an implantation rate (IR) of 17.1%, and clinical and ongoing pregnancy rates of 41.9% and 37.2% respectively. The results of this study suggest that performing ICSI using cryopreserved frozen-thawed testicular spermatozoa with TESA as a first option is a viable, safe, economic and effective method for patients with NOA. © 2015 Blackwell Verlag GmbH.

Effect of freezing of sputum samples on flow cytometric analysis of lymphocyte subsets.

PubMed

Jaksztat, E; Holz, O; Paasch, K; Kelly, M M; Hargreave, F E; Cox, G; Magnussen, H; Jörres, R A

2004-08-01

Sputum samples should be processed shortly after induction to prevent cell degradation. For intermediate storage, freezing of homogenised samples or immediate fixation have been shown to be suitable for cytospins. The aim of this study was to investigate whether freezing or immediate fixation of sputum affect the analysis of lymphocyte subsets by flow cytometry. Selected plugs from 24 sputum samples were homogenised. One aliquot was processed immediately and analysed by flow cytometry. A second aliquot was homogenised, frozen at -20 C after addition of dimethylsulfoxide and stored for a median time of 6 days. In six samples a third aliquot was fixed in formalin after induction and stored for up to 72 h before further processing. Compared to immediate processing, percentages of total lymphocytes and T-suppressor cells were elevated after being frozen, with a minor decrease in the T4/T8 ratio. Proportions of total lymphocytes, T-helper and T-suppressor cells correlated between native and frozen samples, intra-class correlation coefficients being 0.74, 0.85 and 0.70, respectively. The formalin-fixed aliquots could not be analysed with the antibodies used. In conclusion, freezing seems to be a suitable technique to store sputum samples for flow cytometry of CD3, CD4 and CD8 lymphocyte subsets. Its effects were minor compared to the variation between subjects.

A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues.

PubMed Central

Casey, T. T.; Cousar, J. B.; Collins, R. D.

1988-01-01

Routine fixation and paraffin embedding destroys many hematopoietic and lymphoid differentiation antigens detected by flow cytometry or frozen section immunohistochemistry. On the other hand, morphologic evaluation is difficult in flow cytometric or frozen section studies. A simplified three-step plastic embedding system using acetone-fixed tissues embedded in glycol-methacrylate (GMA) resin has been found to provide both excellent morphologic and antigenic preservation. With our system, a wide variety of antigens are detected in plastic sections without trypsinization or prolonged embedding procedures; pan-B (CD19, CD22), pan-T (CD7, CD5, CD3, CD2), T-subset (CD4, CD8, CD1, CD25) markers as well as surface immunoglobulin and markers for myeloid and mononuclear-phagocyte cells are preserved. In summary, modifications of plastic embedding techniques used in this study simplify the procedure, apparently achieve excellent antigenic preservation, and facilitate evaluation of morphologic details in relation to immunocytochemical markers. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:3282442

Identification of regions of normal grey matter and white matter from pathologic glioblastoma and necrosis in frozen sections using Raman imaging.

PubMed

Kast, Rachel; Auner, Gregory; Yurgelevic, Sally; Broadbent, Brandy; Raghunathan, Aditya; Poisson, Laila M; Mikkelsen, Tom; Rosenblum, Mark L; Kalkanis, Steven N

2015-11-01

In neurosurgical applications, a tool capable of distinguishing grey matter, white matter, and areas of tumor and/or necrosis in near-real time could greatly aid in tumor resection decision making. Raman spectroscopy is a non-destructive spectroscopic technique which provides molecular information about the tissue under examination based on the vibrational properties of the constituent molecules. With careful measurement and data processing, a spatial step and repeat acquisition of Raman spectra can be used to create Raman images. Forty frozen brain tissue sections were imaged in their entirety using a 300-µm-square measurement grid, and two or more regions of interest within each tissue were also imaged using a 25 µm-square step size. Molecular correlates for histologic features of interest were identified within the Raman spectra, and novel imaging algorithms were developed to compare molecular features across multiple tissues. In previous work, the relative concentration of individual biomolecules was imaged. Here, the relative concentrations of 1004, 1300:1344, and 1660 cm(-1), which correspond primarily to protein and lipid content, were simultaneously imaged across all tissues. This provided simple interpretation of boundaries between grey matter, white matter, and diseased tissue, and corresponded with findings from adjacent hematoxylin and eosin-stained sections. This novel, yet simple, multi-channel imaging technique allows clinically-relevant resolution with straightforward molecular interpretation of Raman images not possible by imaging any single peak. This method can be applied to either surgical or laboratory tools for rapid, non-destructive imaging of grey and white matter.

Confocal laser endomicroscopy for brain tumor surgery: a milestone journey from microscopy to cellular surgery (Conference Presentation)

NASA Astrophysics Data System (ADS)

Charalampaki, Cleopatra

2017-02-01

The aim in brain tumor surgery is maximal tumor resection with minimal damage of normal neuronal tissue. Today diagnosis of tumor and definition of tumor borders intraoperatively is based on various visualization methods as well as on the histopathologic examination of a limited number of biopsy specimens via frozen sections. Unfortunately, intraoperative histopathology bears several shortcomings, and many biopsies are inconclusive. Therefore, the desirable treatment could be to have the ability to identify intraoperative cellular structures, and differentiate tumor from normal functional brain tissue on a cellular level. To achieve this goal new technological equipment integrated with new surgical concepts is needed.Confocal Laser Endomicroscopy (CLE) is an imaging technique which provides microscopic information of tissue in real-time. We are able to use these technique to perform intraoperative "optical biopsies" in bringing the microscope inside to the patients brain through miniaturized fiber-optic probes, and allow real-time histopathology. In our knowledge we are worldwide the only one neurosurgical group using CLE intraoperative for brain tumor surgery. We can detect and characterize intraoperative tumor cells, providing immediate online diagnosis without the need for frozen sections. It also provides delineation of borders between tumor and normal tissue on a cellular level, making surgical margins more accurate than ever before. The applications of CLE-assisted neurosurgery help to accurate the therapy by extending the resection borders and protecting the functionality of normal brain tissue in critical eloquent areas.

Training effect of the exchange bias in sputter deposited Fe3O4 thin films with varying thickness

NASA Astrophysics Data System (ADS)

Muhammed Shameem, P. V.; Senthil Kumar, M.

2018-07-01

The training effect property of the exchange bias in the reactively sputtered polycrystalline Fe3O4 thin films of varying thicknesses in the range 25-200 nm are studied. Structural studies by X-ray diffraction, X-ray photoelectron spectroscopy and selected area electron diffraction confirm the formation of single phase Fe3O4. The scanning electron spectroscopy images show that the grains are uniformly distributed. All the samples show clear and consistent exchange bias training behaviour due to the dynamics of the spins at the interface of the ferrimagnetic core and the spin glass-like surface of the grains. The analysis of the training effect data of the exchange bias field HE measured at 2 K by using three different models show that the model based on the relaxation of the frozen and rotatable spin components at the interface gives the best description for all the samples. From this model, it is found that the reversible interface spins relax around 7 times faster than the frozen interface spins at 2 K for all the samples and that their relative relaxation rates are independent of the sample thickness. This constancy show that the relative relaxation rates of the interfacial frozen and rotatable spin components is a material dependent property. The frozen component of the interfacial spins of each sample is found to be dominated at the initial stage of the training. A direct equivalence between the HE and remanence asymmetry ME is observed. Above the spin freezing temperature, the training effect measurements at 75 K show that the HE decreases sharply with successive field cycling as compared to the measurements made at 2 K and the HE vanishes after first few cycles.

Comparison of cook loss, shear force, and sensory descriptive profiles of boneless skinless white meat cooked from a frozen or thawed state.

PubMed

Zhuang, Hong; Savage, Elizabeth M

2013-11-01

Four replications were conducted to compare quality measurements, cook loss, shear force, and sensory quality attributes of cooked boneless skinless white meat, broiler breast fillets (pectoralis major) prepared directly from a frozen state or prepared from a thawed state. In each replication, fresh broiler fillets (removed from carcasses 6-8 h postmortem) were procured from a local commercial processing plant and stored in a -20°C freezer until use. On the sensory evaluation date, fillets were cooked to an endpoint temperature of 78°C either directly from the frozen state (thawing during cooking) or after the frozen samples were thawed in a refrigerator (2°C) overnight (thawing before cooking). Cook loss and Warner-Bratzler (WB) shear force were used as indicators for instrumental quality measurements. Sensory quality measurements were conducted by trained descriptive panelists using 0 to 15 universal intensity scales for 8 texture and 10 flavor attributes. Results show that there were no differences (P > 0.05) in measurements for sensory descriptive flavor attributes of cooked fillets between the 2 sample thawing methods, indicating that the sensory flavor profiles of both methods were similar to each other. However, WB shear force (36.98 N), cook loss (21.2%), sensory texture attributes of cohesiveness (intensity score was 5.59), hardness (5.14), rate of breakdown (5.50), and chewiness (5.21) of the breast fillets cooked directly from the frozen state were significantly higher (P < 0.05) than those of the breast meat cooked after being thawed (30.56 N, 19.0%, 5.19, 4.78, 5.29, and 5.02, respectively). These results indicate that cookery directly from frozen boneless skinless white meat can result in different measurement values of cook loss, shear force, and sensory descriptive texture attributes compared with cookery after frozen fillets are thawed.

System for and method of freezing biological tissue

NASA Technical Reports Server (NTRS)

Williams, T. E.; Cygnarowicz, T. A. (Inventor)

1978-01-01

Biological tissue is frozen while a polyethylene bag placed in abutting relationship against opposed walls of a pair of heaters. The bag and tissue are cooled with refrigerating gas at a time programmed rate at least equal to the maximum cooling rate needed at any time during the freezing process. The temperature of the bag, and hence of the tissue, is compared with a time programmed desired value for the tissue temperature to derive an error indication. The heater is activated in response to the error indication so that the temperature of the tissue follows the desired value for the time programmed tissue temperature. The tissue is heated to compensate for excessive cooling of the tissue as a result of the cooling by the refrigerating gas. In response to the error signal, the heater is deactivated while the latent heat of fusion is being removed from the tissue while the tissue is changing phase from liquid to solid.

Determination of polychlorinated biphenyl congeners and chlorinated pesticides in a fish tissue standard reference material.

PubMed

Poster, Dianne L; Kucklick, John R; Schantz, Michele M; Porter, Barbara J; Leigh, Stefan D; Wise, Stephen A

2003-01-01

The concentrations of a wide range of polychlorinated biphenyl congeners (PCBs) and chlorinated pesticides in a fish tissue Standard Reference Material (SRM) have been determined using multiple methods of analysis. This material, SRM 1946, Lake Superior Fish Tissue, was recently issued by the National Institute of Standards and Technology (NIST) and complements a suite of marine environmental natural-matrix SRMs that are currently available from NIST for the determination of organic contaminants such as aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), PCBs, and chlorinated pesticides. SRM 1946 is a fresh tissue homogenate (frozen) prepared from filleted adult lake trout (Salvelinus namaycush namaycush) collected from the Apostle Islands region of Lake Superior. SRM 1946 has certified and reference concentrations for PCB congeners, including the three non- ortho PCB congeners, and chlorinated pesticides. Certified concentrations are available for 30 PCB congeners and 15 chlorinated pesticides. Reference concentrations are available for 12 PCB congeners and 2 chlorinated pesticides. In addition, SRM 1946 is characterized for additional chemical constituents and properties: fatty acids, extractable fat, methylmercury, total mercury, selected trace elements, proximates, and caloric content. The characterization of chlorinated compounds is described in this paper with an emphasis on the approach used for the certification of the concentrations of PCB congeners and chlorinated pesticides. The PCB congener and chlorinated pesticide data are also compared to concentrations in other marine natural-matrix reference materials available from NIST (fish oil, mussel tissue, whale blubber, and a second fresh frozen fish tissue homogenate prepared from filleted adult lake trout collected from Lake Michigan) and from other organizations such as the National Research Council Canada (ground whole carp), the International Atomic Energy Agency (fish homogenate), and the European Commission Joint Research Centre [fish oils (cod and mackerel) and mussel tissue].

Detection of Cryptosporidium oocysts in fresh and frozen cattle faeces: comparison of three methods.

PubMed

Brook, E J; Christley, R M; French, N P; Hart, C A

2008-01-01

The aim of this study was to compare the performance of three commonly used screening tests for Cryptosporidium oocysts in fresh and frozen cattle faeces. Twenty-nine freshly voided faecal samples were collected from calves from three farms in the northwest of England. Three diagnostic tests for Cryptosporidium were carried out on each sample both before and after freezing - the modified Ziehl-Neelsen (MZN) and auramine phenol (APh) stains and a commercial enzyme immunoassay (EIA) kit, the ProSpecT Cryptosporidium Microplate assay (Remel, Lenexa, KS). Twelve samples were deemed positive by the reference standard (polymerase chain reaction, PCR). There were some discrepancies between the results of the screening tests and the levels of agreement were quantified. The sensitivity and specificity of each method was determined, with PCR as the gold standard. Sensitivity and specificity of the MZN stain was optimized when samples with fewer than two oocyst-like bodies were classified as negative. All three screening methods used were effective in detecting Cryptosporidium infection in both fresh and frozen calf faeces. This study has highlighted the value of determining characteristics of tests used for diagnosis and epidemiological studies.

Influence of freeze-thawing on hyaluronic acid binding of human spermatozoa.

PubMed

Nijs, Martine; Creemers, Eva; Cox, Annemie; Janssen, Mia; Vanheusden, Elke; Castro-Sanchez, Yovanna; Thijs, Herbert; Ombelet, Willem

2009-08-01

Mature human spermatozoa have at least three specific hyaluronic acid (HA) binding proteins present on their sperm membrane. These receptors play a role in the acrosome reaction, hyaluronidase activity, hyaluronan-mediated motility and sperm-zona and sperm-oolemmal binding. Cryopreservation of spermatozoa can cause ultrastructural and even molecular damage. The aim of this study was to investigate if HA binding receptors of human spermatozoa remain functional after freeze-thawing. Forty patients were enrolled in the study. Semen samples were analysed before and after cryopreservation. Parameters analysed included concentration, motility, morphology and hyaluronan binding. Samples were frozen in CBS straws using a glycerol-glucose-based cryoprotectant. HA binding was studied using the sperm-hyaluronan binding assay. Freeze-thawing resulted in a significant decline in motility: the percentage of motile spermatozoa reduced from 50.6 to 30.3% (P < 0.001). HA binding properties of frozen-thawed spermatozoa remained unchanged after the freeze-thawing process: 68.5 +/- 17.1% spermatozoa of the neat sample were bound to HA, as were 71.3 +/- 20.4 of the frozen-thawed sample. This study indicates that freeze-thawing did not alter the functional hyaluronan binding sites of mature motile spermatozoa, and therefore will not alter their fertilizing potential.

An evaluation of Brix refractometry instruments for measurement of colostrum quality in dairy cattle.

PubMed

Bielmann, V; Gillan, J; Perkins, N R; Skidmore, A L; Godden, S; Leslie, K E

2010-08-01

Acquisition of high quality colostrum is an important factor influencing neonatal calf health. Many methods have been used to assess the Ig concentration of colostrum; however, improved, validated evaluation tools are needed. The aims of this study were to evaluate both optical and digital Brix refractometer instruments for the measurement of Ig concentration of colostrum as compared with the gold standard radial immunodiffusion assay laboratory assessment and to determine the correlation between Ig measurements taken from fresh and frozen colostrum samples for both Brix refractometer instruments. This research was completed using 288 colostrum samples from 3 different farms. It was concluded that the optical and digital Brix refractometers were highly correlated for both fresh and frozen samples (r=0.98 and r=0.97, respectively). Correlation between both refractometer instruments for fresh and frozen samples and the gold standard radial immunodiffusion assay were determined to be very similar, with a correlation coefficient between 0.71 and 0.74. Both instruments exhibited excellent test characteristics, indicating an appropriate cut-off point of 22% Brix score for the identification of good quality colostrum. Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The Change of Total Anthocyanins in Blueberries and Their Antioxidant Effect After Drying and Freezing

PubMed Central

Srzednicki, George

2004-01-01

This study examined the effects of freezing, storage, and cabinet drying on the anthocyanin content and antioxidant activity of blueberries (Vaccinium corymbosum L). Fresh samples were stored for two weeks at 5°C while frozen samples were kept for up to three months at −20°C. There were two drying treatments, one including osmotic pretreatment followed by cabinet drying and the other involving only cabinet drying. Total anthocyanins found in fresh blueberries were 7.2 ± 0.5 mg/g dry matter, expressed as cyanidin 3-rutinoside equivalents. In comparison with fresh samples, total anthocyanins in untreated and pretreated dried blueberries were significantly reduced to 4.3 ± 0.1 mg/g solid content, 41% loss, and 3.7 ± 0.2 mg/g solid content, 49% loss, respectively. Osmotic treatment followed by a thermal treatment had a greater effect on anthocyanin loss than the thermal treatment alone. In contrast, the frozen samples did not show any significant decrease in anthocyanin level during three months of storage. Measurement of the antioxidant activity of anthocyanin extracts from blueberries showed there was no significant difference between fresh, dried, and frozen blueberries. PMID:15577185

Biologically active antimicrobial and antioxidant substances in the Helianthus annuus L. bee pollen.

PubMed

Fatrcová-Šramková, Katarína; Nôžková, Janka; Máriássyová, Magda; Kačániová, Miroslava

2016-01-01

The objective of this study was to measure the content of flavonoids, polyphenols, and carotenoids in the Helianthus annuus L. bee pollen. It was also to evaluate the ability of the dried, frozen, and freeze-dried extracts of sunflower (H. annuus) pollen, its scavenged free radicals and reducing action. Another aim of this study was to investigate the antimicrobial in vitro action of the H. annuus pollen extracts against the Gram-positive and Gram-negative bacteria and fungi. All pollen extracts showed medium antiradical activity and reductive ability. The most effective was the freeze-dried extract in both evaluation systems. The evaluation of the protective effects of DNA using a biosensor showed an opposite trending-frozen ˃ dried ˃ freeze-dried pollen. For the evaluation of antiradical activity, the DPPH method was used, and reductive ability was assessed by means of phosphomolybdic complex formation. The comparison of the polyphenols content shows higher values in freeze-dried bee pollen than in the dried and frozen pollen. The highest content of flavonoids was found in the frozen samples and the most carotenoids were present in the dried samples. In our study, the best antibacterial effects of the dried sunflower bee pollen extracts were found against Paenibacillus larvae, Pseudomonas aeruginosa, and Enterococcus raffinosus. The best inhibitory properties of the frozen sunflower bee pollen extracts were found against Escherichia coli, Pseudomonas aeruginosa, and Paenibacillus larvae. Very good inhibitory effects of freeze-dried sunflower bee pollen were found against Paenibacillus larvae, Brochotrix thermosphacta, and Enterococcus raffinosus. The best antifungal activity of the sunflower bee pollen was found in the frozen bee pollen extracts against Aspergillus ochraceus and freeze-dried bee pollen extracts against Aspergillus niger.

Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing-thawing.

PubMed

Fraser, L; Strzezek, J

2007-06-01

Whole ejaculate or sperm-rich fraction, collected from four sexually mature boars, was frozen in an extender containing lactose-hen egg yolk with glycerol (lactose-HEY-G) or extender containing lactose, lyophilized lipoprotein fractions isolated from ostrich egg yolk and glycerol (lactose-LPFo-G), and Orvus Es Paste, respectively. The sperm samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Sperm DNA integrity was assessed using a modified neutral comet assay. Sperm characteristics such as motility, plasma membrane integrity (SYBR-14/PI), mitochondrial function (rhodamine 123) and acrosome integrity were monitored. Freezing-thawing caused a significant increase (P<0.05) in sperm DNA fragmentation, irrespective of the procedures of ejaculate collection and extender type. Sperm DNA fragmentation was significantly lower (P<0.05) in the whole ejaculate compared with the sperm-rich fraction, indicating that spermatozoa maintained in the whole seminal plasma prior to its removal for freezing-thawing procedure were less vulnerable to cryo-induced DNA fragmentation. Furthermore, spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender exhibited lower (P<0.05) DNA fragmentation than those frozen in the absence of cryoprotective substances. The levels of sperm DNA damage, as expressed by comet tail length and tail moment values, were significantly higher (P<0.05) in sperm samples frozen in the absence of cryoprotective substances. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. It can be suggested that evaluation of DNA integrity, coupled with different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, may aid in determining the quality of frozen-thawed boar semen.

Inactivation kinetics of Vibrio vulnificus in phosphate-buffered saline at different freezing and storage temperatures and times.

PubMed

Seminario, Diana M; Balaban, Murat O; Rodrick, Gary

2011-03-01

Vibrio vulnificus (Vv) is a pathogen that can be found in raw oysters. Freezing can reduce Vv and increase the shelf life of oysters. The objective of this study was to develop predictive inactivation kinetic models for pure cultures of Vv at different frozen storage temperatures and times. Vv was diluted in phosphate-buffered saline (PBS) to obtain about 10(7) CFU/mL. Samples were frozen at -10, -35, and -80 °C (different freezing rates), and stored at different temperatures. Survival of Vv was followed after freezing and storage at -10 °C (0, 3, 6, and 9 d) and at -35 and -80 °C (every week for 6 wk). For every treatment, time-temperature data was obtained using thermocouples in blank vials. Predictive models were developed using first-order, Weibull and Peleg inactivation kinetics. Different freezing temperatures did not significantly (α = 0.05) affect survival of Vv immediately after freezing. The combined effect of freezing and 1 wk frozen storage resulted in 1.5, 2.6, and 4.9 log10 reductions for samples stored at -80, -35, and -10 °C, respectively. Storage temperature was the critical parameter in survival of Vv. A modified Weibull model successfully predicted Vv survival during frozen storage: log10 Nt = log 10No - 1.22 - ([t/10{-1.163-0.0466T}][0.00025T(2) + 0.049325]). N(o) and N(t) are initial and time t (d) survival counts, T is frozen storage temperature, Celsius degree. Vibrio vulnificus can be inactivated by freezing. Models to predict survival of V. vulnificus at different freezing temperatures and times were developed. This is the first step towards the prediction of V. vulnificus related safety of frozen oysters.

Effect of Holder pasteurization and frozen storage on macronutrients and energy content of breast milk.

PubMed

García-Lara, Nadia Raquel; Vieco, Diana Escuder; De la Cruz-Bértolo, Javier; Lora-Pablos, David; Velasco, Noelia Ureta; Pallás-Alonso, Carmen Rosa

2013-09-01

The aim of this study was to explore the effect of Holder pasteurization and frozen storage at -20°C after pasteurization on fat, total nitrogen, lactose, and energy content of breast milk. Both procedures are routinely practiced in human milk banks. A total of 34 samples of frozen breast milk, donated by 28 women, were collected. Once thawed, an aliquot of each sample was analyzed before pasteurization; the remaining milk was pasteurized (Holder method) and split into 8 aliquots. One aliquot was analyzed after pasteurization and the remainder frozen at -20°C and analyzed 30, 60, 90, 120, and 180 days later. For every aliquot, fat, total nitrogen, lactose, and energy content were determined using the device human Milk Analyzer. We observed a significant reduction in fat (3.5%; -0.17 (-0.29; -0.04) g/dL) and energy content (2.8%; -2.03 (-3.60; -0.46) g/dL) after pasteurization. A significant decrease over time was observed for fat, lactose and energy content. No significant changes were observed for nitrogen content. Mean differences between day 0 postpasteurization and day 180 were -0.13 (-0.21; -0.06) g/dL for fat, -0.08 (-0.13; -0.03) g/dL for lactose, and -1.55 (-2.38; -0.71) kcal/dL for energy content. The relative decreases were 2.8%, 1.7%, and 2.2%, respectively. Overall (postpasteurization + frozen storage), a 6.2% and 5% decrease were observed for fat and energy, respectively. Holder pasteurization decreased fat and energy content of human milk. Frozen storage at -20°C of pasteurized milk significantly reduced fat, lactose, and energy content of human milk.

Influence of Freezing and Storage Procedure on Human Urine Samples in NMR-Based Metabolomics

PubMed Central

Rist, Manuela J.; Muhle-Goll, Claudia; Görling, Benjamin; Bub, Achim; Heissler, Stefan; Watzl, Bernhard; Luy, Burkhard

2013-01-01

It is consensus in the metabolomics community that standardized protocols should be followed for sample handling, storage and analysis, as it is of utmost importance to maintain constant measurement conditions to identify subtle biological differences. The aim of this work, therefore, was to systematically investigate the influence of freezing procedures and storage temperatures and their effect on NMR spectra as a potentially disturbing aspect for NMR-based metabolomics studies. Urine samples were collected from two healthy volunteers, centrifuged and divided into aliquots. Urine aliquots were frozen either at −20 °C, on dry ice, at −80 °C or in liquid nitrogen and then stored at −20 °C, −80 °C or in liquid nitrogen vapor phase for 1–5 weeks before NMR analysis. Results show spectral changes depending on the freezing procedure, with samples frozen on dry ice showing the largest deviations. The effect was found to be based on pH differences, which were caused by variations in CO2 concentrations introduced by the freezing procedure. Thus, we recommend that urine samples should be frozen at −20 °C and transferred to lower storage temperatures within one week and that freezing procedures should be part of the publication protocol. PMID:24957990

Influence of Freezing and Storage Procedure on Human Urine Samples in NMR-Based Metabolomics.

PubMed

Rist, Manuela J; Muhle-Goll, Claudia; Görling, Benjamin; Bub, Achim; Heissler, Stefan; Watzl, Bernhard; Luy, Burkhard

2013-04-09

It is consensus in the metabolomics community that standardized protocols should be followed for sample handling, storage and analysis, as it is of utmost importance to maintain constant measurement conditions to identify subtle biological differences. The aim of this work, therefore, was to systematically investigate the influence of freezing procedures and storage temperatures and their effect on NMR spectra as a potentially disturbing aspect for NMR-based metabolomics studies. Urine samples were collected from two healthy volunteers, centrifuged and divided into aliquots. Urine aliquots were frozen either at -20 °C, on dry ice, at -80 °C or in liquid nitrogen and then stored at -20 °C, -80 °C or in liquid nitrogen vapor phase for 1-5 weeks before NMR analysis. Results show spectral changes depending on the freezing procedure, with samples frozen on dry ice showing the largest deviations. The effect was found to be based on pH differences, which were caused by variations in CO2 concentrations introduced by the freezing procedure. Thus, we recommend that urine samples should be frozen at -20 °C and transferred to lower storage temperatures within one week and that freezing procedures should be part of the publication protocol.

Evaluating ex vivo fluorescence confocal microscopy images of basal cell carcinomas in Mohs excised tissue.

PubMed

Longo, C; Rajadhyaksha, M; Ragazzi, M; Nehal, K; Gardini, S; Moscarella, E; Lallas, A; Zalaudek, I; Piana, S; Argenziano, G; Pellacani, G

2014-09-01

Fluorescence confocal microscopy (FCM) is an emerging technology for rapid imaging of excised tissue, without the need for frozen- or fixed-section processing. Basal cell carcinomas (BCCs) can be detected in Mohs excisions although few studies have described the major BCC findings as seen on FCM. To describe the major BCC findings of excised tissue during Mohs surgery and to correlate them with histopathology. Freshly excised tumours and frozen-thawed discarded tissue of BCC during Mohs surgery were analysed by means of FCM. A side-by-side correlation between FCM images and histological sections was performed. The FCM features of overlying skin and adnexal structures were also described. Sixty-four BCC cases were analysed. Distinct BCC types appeared unique in terms of shape and size of tumour islands [bigger in nodular (18/25), smaller and rounded in micronodular (7/7) and tiny cords for infiltrative ones (24/30)] and for the presence of clefting, palisading and increased nucleus/cytoplasm ratio. An excellent correlation was found between FCM and histological findings (Cohen's κ statistics = 0·9). In six cases, the presence of sebaceous glands and intense stroma reaction represented possible confounders. Fluorescence confocal microscopy is a fast and new imaging technique that allows an excellent visualization of skin structures and BCC findings during Mohs surgery. © 2014 British Association of Dermatologists.

Safety of lumbar spine radiofrequency procedures in the presence of posterior pedicle screws: technical report of a cadaver study.

PubMed

Gazelka, Halena M; Welch, Tasha L; Nassr, Ahmad; Lamer, Tim J

2015-05-01

To determine whether the thermal energy associated with lumbar spine radiofrequency neurotomy (RFN) performed near titanium and stainless steel pedicle screws is conducted to the pedicle screws or adjacent tissues, or both, thus introducing potential for thermal damage to those tissues. Cadaver study. Cadaver laboratory equipped with fluoroscopy, surgical spine implements, and radiofrequency generator. No live human subject; a fresh frozen (and thawed) cadaver torso was used for the study. Titanium and stainless steel pedicle screws were placed in the lumbar spine of a fresh frozen cadaver torso with real-time fluoroscopic guidance. Conventional RFN cannula placement was performed at the level of pedicle screws and a control (nonsurgically altered) lumbar level. Neurotomy was performed with conventional radiofrequency lesioning parameters. Temperatures were recorded at multiple sites through thermistor probes. Direct contact of the radiofrequency cannula with the pedicle screws during conventional RFN produced a substantial increase in temperature in the surrounding soft tissues. A small increase in temperature occurred at the same sites at the control level. Titanium and stainless steel pedicle screws are capable of sustaining large increases in temperature when the radiofrequency probe comes in contact with the screw. These results are suggestive that pedicle screws could serve as a possible source of tissue heating and thermal injury during RFN. Wiley Periodicals, Inc.

Fusobacterium nucleatum in gastroenterological cancer: Evaluation of measurement methods using quantitative polymerase chain reaction and a literature review.

PubMed

Yamamura, Kensuke; Baba, Yoshifumi; Miyake, Keisuke; Nakamura, Kenichi; Shigaki, Hironobu; Mima, Kosuke; Kurashige, Junji; Ishimoto, Takatsugu; Iwatsuki, Masaaki; Sakamoto, Yasuo; Yamashita, Yoichi; Yoshida, Naoya; Watanabe, Masayuki; Baba, Hideo

2017-12-01

The human microbiome Fusobacterium nucleatum , which primarily inhabits the oral cavity, causes periodontal disease and has also been implicated in the development of colorectal cancer. However, whether F. nucleatum is present in other gastroenterological cancer tissues remains to be elucidated. The present study evaluated whether quantitative polymerase chain reaction (qPCR) assays were able to detect F. nucleatum DNA and measure the quantity of F. nucleatum DNA in esophageal, gastric, pancreatic and liver cancer tissues. The accuracy of the qPCR assay was determined from a calibration curve using DNA extracted from cells from the oral cavity. Formalin-fixed paraffin-embedded (FFPE) tumor tissues from 20 patients with gastroenterological [esophageal (squamous cell carcinoma), gastric, colorectal, pancreatic and liver] cancer and 20 matched normal tissues were evaluated for F. nucleatum DNA content. The cycle threshold values in the qPCR assay for F. nucleatum and solute carrier organic anion transporter family member 2A1 (reference sample) decreased linearly with the quantity of input DNA ( r 2 >0.99). The F. nucleatum detection rate in esophageal, gastric and colorectal cancer tissues were 20% (4/20), 10% (2/20) and 45% (9/20), respectively. F. nucleatum was not detected in liver and pancreatic cancer tissues. The qPCR results from the frozen and FFPE tissues were consistent. Notably, F. nucleatum was detected at a higher level in superficial areas compared with the invasive areas. F. nucleatum in esophageal, gastric and colorectal cancer tissues was evaluated by qPCR using FFPE tissues. F. nucleatum may be involved in the development of esophageal, gastric and colorectal cancer.

Immunofluorescence Staining — EDRN Public Portal

Cancer.gov

Direct immunofluorescence method is used to detect the deposit of immunoglobulins, complement components, fibrinogen, etc. in tissues. This technique is usually performed on frozen sections. The primary antibody is conjugated to fluorescein binds directly with the antigen and can be detected by the fluorescent tag using a fluorescent microscope.

Trends of brominated diphenyl ethers in fresh and archived Great Lakes fish (1979-2005)

USGS Publications Warehouse

Batterman, Stuart; Chernyak, Sergei; Gwynn, Erica; Cantonwine, David; Jia, Chunrong; Begnoche, Linda J.; Hickey, James P.

2007-01-01

While few environmental measurements of brominated diphenyl ethers (BDEs) were completed prior to the mid-1990s, analysis of appropriately archived samples might enable the determination of contaminant trends back to the introduction of these chemicals. In this paper, we first investigate the stability of BDEs in archived frozen and extracted fish samples, and then characterize trends of these chemicals in rainbow smelt (Osmerus mordax) and lake trout (Salvelinus namaycush) in each of the Great Lakes between 1979 and 2005. We focus on the four most common congeners (BDE-47, 100, 99 and 153) and use a change-point analysis to detect shifts in trends. Analyses of archived fish samples yielded precise BDE concentration measurements with only small losses (0.8% per year in frozen fish tissues, 2.2% per year in refrigerated extracts). Trends in fish from all Great Lakes showed large increases in BDE concentrations that started in the early to mid-1980s with fairly consistent doubling times (generally 2–4 years except in Lake Erie smelt where levels increased very slowly), though concentrations and trends show differences by congener, fish species and lake. The most recent data show that accumulation rates are slowing, and concentrations of penta- and hexa-congeners in trout from Lakes Ontario and Michigan and smelt from Lake Ontario started to decrease in the mid-1990s. Trends in smelt and trout are evolving somewhat differently, and trout concentrations in the five lakes are now ranked as Michigan > Superior = Ontario > Huron = Erie, and smelt concentrations as Michigan > Ontario > Huron > Superior > Erie. The analysis of properly archived samples permits the reconstruction of historical trends, congener distributions, biomagnification and other information that can aid the understanding and management of these contaminants.

Calnexin, an ER stress-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer.

PubMed

Ryan, Deborah; Carberry, Steven; Murphy, Áine C; Lindner, Andreas U; Fay, Joanna; Hector, Suzanne; McCawley, Niamh; Bacon, Orna; Concannon, Caoimhin G; Kay, Elaine W; McNamara, Deborah A; Prehn, Jochen H M

2016-07-01

Colorectal cancer (CRC) is a leading cause of cancer mortality in the Western world and commonly treated with genotoxic chemotherapy. Stress in the endoplasmic reticulum (ER) was implicated to contribute to chemotherapeutic resistance. Hence, ER stress related protein may be of prognostic or therapeutic significance. The expression levels of ER stress proteins calnexin, calreticulin, GRP78 and GRP94 were determined in n = 23 Stage II and III colon cancer fresh frozen tumour and matched normal tissue samples. Data were validated in a cohort of n = 11 rectal cancer patients treated with radiochemotherapy in the neoadjuvant setting. The calnexin gene was silenced using siRNA in HCT116 cells. There were no increased levels of ER stress proteins in tumour compared to matched normal tissue samples in Stage II or III CRC. However, increased calnexin protein levels were predictive of poor clinical outcome in the patient cohort. Data were validated in the rectal cancer cohort treated in the neoadjuvant setting. Calnexin gene-silencing significantly reduced cell survival and increased cancer cell susceptibility to 5FU chemotherapy. Increased tumour protein levels of calnexin may be of prognostic significance in CRC, and calnexin may represent a potential target for future therapies.

Decellularized Versus Fresh-Frozen Allografts in Anterior Cruciate Ligament Reconstruction: An In Vitro Study in a Rabbit Model.

PubMed

Dong, Shikui; Huangfu, Xiaoqiao; Xie, Guoming; Zhang, Yang; Shen, Peng; Li, Xiaoxi; Qi, Jin; Zhao, Jinzhong

2015-08-01

The common fresh-frozen allografts that are used for anterior cruciate ligament (ACL) reconstructions behave slower during the remodeling process and produce weaker tendon-bone integrations than do autografts. Decellularization of allogenic tendons results in a clean and porous collagen scaffold with low antigenicity and high compatibility, which may be more suitable for ACL reconstructions. Allograft decellularization will result in a tissue structure with suitable mechanical characteristics for ACL reconstruction, thereby promoting graft remodeling and enhancing tendon-bone healing. Controlled laboratory study. Decellularized allograft tissues were prepared with a pH-modified decellularization process and evaluated for their biocompatibility and biomechanical character in vitro. Eighty New Zealand White rabbits were divided into 2 groups, with 40 in each group, to receive ACL reconstruction with either fresh-frozen (common) allografts or decellularized allografts on both knees. At 2, 4, 8, and 12 weeks postoperatively, the rabbits were euthanized for biomechanical testing, micro-computed tomography analysis, and histologic analysis. The pH-modified decellularized allograft tissues kept excellent biocompatibility and biomechanical character during the in vitro study. Biomechanical testing indicated that the decellularized allograft had significantly higher ultimate load (P = .02) and stiffness (P = .01) levels than the common allograft at 12 weeks, and there was no significant difference between the 2 groups at any other time point. The micro-CT evaluation determined significantly higher bone mineral density (P < .01) in the decellularized allograft group than that in the common allograft group at 12 weeks, but no difference between the 2 groups was observed at any other time point. Regarding bone volume/total volume, there was no difference between the 2 groups at any time point. Fibroblast ingrowths, vascular formation, and connective tissue formation in the tendon-bone interface were better in the decellularized group within 8 weeks. New bone formation was more common in the decellularized allograft group. The collagen birefringence was restored more quickly in the decellularized allograft group than in the common allograft group at all time points. The use of pH-modified decellularized allografts compared with the common allografts resulted in better cellularity, vascularity, collagen matrix remolding, new bone formation around the graft, enhanced tendon-bone healing, and higher ultimate failure load and stiffness of the graft after ACL reconstruction in the rabbit model. The pH-modified decellularized allograft may be a better graft option than the common fresh-frozen allograft for knee ligament reconstructions. © 2015 The Author(s).

Silver nanoparticle based surface enhanced Raman scattering spectroscopy of diabetic and normal rat pancreatic tissue under near-infrared laser excitation

NASA Astrophysics Data System (ADS)

Huang, H.; Shi, H.; Feng, S.; Lin, J.; Chen, W.; Huang, Z.; Li, Y.; Yu, Y.; Lin, D.; Xu, Q.; Chen, R.

2013-04-01

This paper presents the use of high spatial resolution silver nanoparticle based near-infrared surface enhanced Raman scattering (SERS) from rat pancreatic tissue to obtain biochrmical information about the tissue. A high quality SERS signal from a mixture of pancreatic tissues and silver nanoparticles can be obtained within 10 s using a Renishaw micro-Raman system. Prominent SERS bands of pancreatic tissue were assigned to known molecular vibrations, such as the vibrations of DNA bases, RNA bases, proteins and lipids. Different tissue structures of diabetic and normal rat pancreatic tissues have characteristic features in SERS spectra. This exploratory study demonstrated great potential for using SERS imaging to distinguish diabetic and normal pancreatic tissues on frozen sections without using dye labeling of functionalized binding sites.

A pragmatic regional interdependence approach to primary frozen shoulder: a retrospective case series.

PubMed

Wong, Christopher Kevin; Strang, Bryanna L; Schram, Galen A; Mercer, Elizabeth A; Kesting, Rebecca S; Deo, Kabi S

2018-05-01

Although the shoulder is known to move together with the scapula and other upper quarter joints, the current frozen shoulder clinical practice guidelines describe only physical therapy study treatments directed to the shoulder. None received a strong recommendation, highlighting the need for alternate interventions. This retrospective case series describes a pragmatic regional interdependence approach to frozen shoulder with impairment and functional outcomes, noting whether final ROM approached normal. Five consecutive patients referred with frozen shoulder diagnoses attended 11-21 sessions over 5-10 weeks with one physical therapist. Treatment addressed inter-related regions (shoulder, shoulder girdle, scapulothoracic/humerothoracic, and spine) following a pragmatic approach using impairment-based interventions (joint/soft tissue mobilization, muscle stretching/strengthening) as well as patient education, modalities and warm up that addressed individual presentations. All patients improved on all outcomes. Mean shoulder ROM at discharge, the impairment outcome, demonstrated large effect size increases: flexion (117 ± 10-179 ± 12, d  = 5.9), abduction (74 ± 8-175 ± 9, d  = 9.3), external rotation (23 ± 7-89 ± 2, d  = 12.0). The Disability of Arm Shoulder Hand functional outcome score upon follow up demonstrated a large effect size improvement ( d  = 1.5) from 40.0 ± 19.4-6.2 ± 3.7. Final ROM approached normal. This case series utilized a regional interdependence approach to frozen shoulder that included manual therapy interventions directed to consistent upper quarter body segments. Shoulder ROM was returned to near normal with functional improvements evident months after discharge. A pragmatic regional interdependence approach addressing multiple joints related to shoulder function may benefit other people with frozen shoulder. 4.