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PrPSc detection in formalin-fixed paraffin-embedded tissue by ELISA
Eric M Nicholson, Justin J Greenlee, Amir N Hamir
BMC Research Notes , 2011, DOI: 10.1186/1756-0500-4-432
Abstract: Formalin-fixed paraffin-embedded tissues from TSE transmission studies of scrapie in sheep, chronic wasting disease in white-tailed deer or transmissible mink encephalopathy in cattle were cut at 5 μm thickness. Samples containing the tissue equivalent of as little as one 5 μm section can be used to readily discriminate positive from negative samples.This approach cannot replace IHC but may be used along with IHC as both a more rapid and readily high throughput screen where fresh or frozen tissues are not available or impractical.Due to the lack of a defined immune response or nucleic acid component of the infectious agent, approaches for transmissible spongiform encephalopathy (TSE) diagnosis rely upon methods of immunodetection including immunohistochemistry (IHC), Western blotting and enzyme-linked immunosorbent assay (ELISA)-based approaches for detection of the infectious agent. [1-3] Generally speaking, IHC relies upon formalin fixed paraffin embedded tissues, while Western blotting and ELISA utilize fresh or frozen tissues. Recently, methods have been reported that allow detection of PrPSc in formalin fixed tissues by Western blot [4-6]. Here we report an extension of this approach to allow ELISA-based detection of PrPSc in formalin-fixed paraffin-embedded tissues.This study utilized archived paraffin-embedded tissue samples from studies of scrapie in sheep, chronic wasting disease (CWD) in white-tailed deer (WTD) and transmissible mink encephalopathy (TME) in cattle as part of TSE research conducted at the National Animal Disease Center-USDA-ARS (Ames, IA). Animals were cared for and euthanized under National Animal Disease Center approved institutional animal care and use protocols. Samples were collected in 10% neutral buffered formalin prior to standard processing into paraffin blocks, with time in formalin ranging from 7 days to ~450 days. Previous studies of formalin fixed tissues report a marked sensitivity decrease for Western blots on tissues left in
Development and Validation of an Immunohistochemical Method for Diagnosis of Bovine Tuberculosis in Formalin-Fixed, Paraffin-Embedded Tissues
J. Martinez-Burnes,O. Castillo-Martinez,D. Solano-Terreros,N.I. de la Cruz-Hernandez,J. Campuzano-Granados,H. Barrios-Garcia,A. Snydelaar Hardwicke,C. Almazan-Garcia,J.A Guizarnotegui-Blanco,J. Ramos-Vara
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2012.2856.2860
Abstract: The objective of the study was to develop an indirect Immunoperoxidase Method for bovine tuberculosis diagnosis in formalin fixed, paraffin embedded tissues and its validation compared to mycobacterial isolation and Ziehl Neelsen staining. About of 33 bovine lymph nodes with isolation of Mycobacterium bovis and 11 negative lymph nodes from tuberculosis free ranches were used. Sections of all lymph nodes examined were stained with Ziehl Neelsen and Immunohistochemistry (IHC). Rabbit anti-M. bovis polyclonal antibodies and horse anti-rabbit were used as primary and secondary antibodies. The immunologic reaction was detected with an immunoperoxidase DAB Method and counterstained with hematoxylin. Results show complete agreement between Immunohistochemistry and mycobacterial culture. From the 33 positive isolation cases, all of them (100%) were positive by IHC. From the 11 negative cases, all of them were negative to Mycobacterium by IHC. Regarding Ziehl Neelsen of the 33 positive isolation cases, 30 (90.9%) had acid-fast bacilli and from the 11 negative isolation cases none had acid fast bacilli. Results show that IHC represents a fast, sensitive and specific diagnostic tool for bovine tuberculosis in formalin fixed, paraffin embedded tissues allowing simultaneous observation of tissue lesions and antigens.
Quantitative Proteomic Analysis of Formalin Fixed Paraffin Embedded Oral HPV Lesions from HIV Patients
Mohit Raja Jain, Tong Liu, Jun Hu, Marlene Darfler, Valerie Fitzhugh, Joseph RinaggioHong Li
The Open Proteomics Journal , 2008, DOI: 10.2174/1875039700801010040]
Abstract: Human immunodeficiency virus (HIV) infection is associated with dysplastic changes in oral human papilloma virus (HPV) lesions, suggesting changes in keratinocytes. In the present study, we seek to identify proteomic changes in oral HPV lesions between HIV(+) and HIV(-) patients. While fresh tissues represent the most desirable samples for proteomic investigations, they are often difficult to obtain in large numbers under clinical settings. We therefore have developed a new method to identify protein changes in formalin fixed and paraffin-embedded (FFPE) oral HPV lesions utilizing iTRAQ technology in conjunction with Liquid Tissue sample preparation method. Using this method, we identified nine proteins that were differentially expressed in oral HPV lesions as a result of HIV infection. The quantitative proteomic method presented here will be valuable for others who plan to analyze FFPE tissues.
Recurrent Respiratory Papillomatosis: HPV Genotypes and Risk of High-Grade Laryngeal Neoplasia  [PDF]
Turid Omland, Kathrine A. Lie, Harriet Akre, Lars Erik Sandlie, Peter Jebsen, Leiv Sandvik, Dag Andre Nymoen, Davit Bzhalava, Joakim Dillner, Kjell Br?ndbo
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0099114
Abstract: Patients with recurrent respiratory papillomatosis (RRP) in Norway treated between 1987 and 2009 were recruited to this cohort study. They were followed from disease onset and data recorded until January 2012. Here, we describe the distribution of human papillomavirus (HPV) genotypes, the prevalence of multiple HPV infections, and the risk of high-grade laryngeal neoplasia and respiratory tract invasive carcinoma in a large cohort of patients with RRP. We also examined whether HPV genotype, gender, age or clinical course are risk factors for this development. Clinical records and histological specimens were reviewed. Using formalin-fixed paraffin-embedded biopsies, HPV genotyping were performed by quantitative polymerase chain reaction assays identifying 15 HPV types. HPV-negative specimens were analyzed by metagenomic sequencing. Paraffin blocks were available in 224/238 patients. The DNA quality was approved in 221/224 cases. HPV DNA was detected in 207/221 patients and all were HPV 6 or HPV 11 positive, comprising HPV 6 in 133/207, HPV 11 in 40/207 cases and HPV 6/11 in 15/207 cases. Co-infection with one or two high-risk HPV types together with HPV 6 or HPV 11 was present in 19/207 patients. Metagenomic sequencing of 14 HPV-negative specimens revealed HPV 8 in one case. In total, 39/221 patients developed high-grade laryngeal neoplasia. 8/221 patients developed carcinoma of the respiratory tract (six patients with laryngeal carcinoma and two patients with lung carcinoma). High-grade laryngeal neoplasias were found more frequently in HPV-negative versus HPV-positive patients, (RR = 2.35, 95% CI 1.1, 4.99), as well as respiratory tract carcinomas (RR = 48, 95% CI 10.72, 214.91). In summary, the majority of RRP were associated with HPV 6 and/or 11. HPV-negative RRP biopsies occurred more frequently in adult-onset patients, and were associated with an increased risk of laryngeal neoplasia and carcinoma in the respiratory tract.
Deep Clonal Profiling of Formalin Fixed Paraffin Embedded Clinical Samples  [PDF]
Tara Holley, Elizabeth Lenkiewicz, Lisa Evers, Waibhav Tembe, Christian Ruiz, Joel R. Gsponer, Cyrill A. Rentsch, Lukas Bubendorf, Mark Stapleton, Doug Amorese, Christophe Legendre, Heather E. Cunliffe, Ann E. McCullough, Barbara Pockaj, David Craig, John Carpten, Daniel Von Hoff, Christine Iacobuzio-Donahue, Michael T. Barrett
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0050586
Abstract: Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.
Proteomic Analysis of Matched Formalin-Fixed, Paraffin-Embedded Specimens in Patients with Advanced Serous Ovarian Carcinoma  [PDF]
Ashlee L. Smith,Mai Sun,Rohit Bhargava,Nicolas A. Stewart,Melanie S. Flint,William L. Bigbee,Thomas C. Krivak,Mary A. Strange,Kristine L. Cooper,Kristin K. Zorn
Proteomes , 2013, DOI: 10.3390/proteomes1030240
Abstract: Objective: The biology of high grade serous ovarian carcinoma (HGSOC) is poorly understood. Little has been reported on intratumoral homogeneity or heterogeneity of primary HGSOC tumors and their metastases. We evaluated the global protein expression profiles of paired primary and metastatic HGSOC from formalin-fixed, paraffin-embedded (FFPE) tissue samples. Methods: After IRB approval, six patients with advanced HGSOC were identified with tumor in both ovaries at initial surgery. Laser capture microdissection (LCM) was used to extract tumor for protein digestion. Peptides were extracted and analyzed by reversed-phase liquid chromatography coupled to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database. Differences in protein abundance between samples were assessed and analyzed by Ingenuity Pathway Analysis software. Immunohistochemistry (IHC) for select proteins from the original and an additional validation set of five patients was performed. Results: Unsupervised clustering of the abundance profiles placed the paired specimens adjacent to each other. IHC H-score analysis of the validation set revealed a strong correlation between paired samples for all proteins. For the similarly expressed proteins, the estimated correlation coefficients in two of three experimental samples and all validation samples were statistically significant ( p < 0.05). The estimated correlation coefficients in the experimental sample proteins classified as differentially expressed were not statistically significant. Conclusion: A global proteomic screen of primary HGSOC tumors and their metastatic lesions identifies tumoral homogeneity and heterogeneity and provides preliminary insight into these protein profiles and the cellular pathways they constitute.
Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples  [PDF]
Craig April,Brandy Klotzle,Thomas Royce,Eliza Wickham-Garcia,Tanya Boyaniwsky,John Izzo,Donald Cox,Wendell Jones,Renee Rubio,Kristina Holton,Ursula Matulonis,John Quackenbush,Jian-Bing Fan
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0008162
Abstract: We have developed a gene expression assay (Whole-Genome DASL?), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens.
Elevated Pressure Improves the Extraction and Identification of Proteins Recovered from Formalin-Fixed, Paraffin-Embedded Tissue Surrogates  [PDF]
Carol B. Fowler,Ingrid E. Chesnick,Cedric D. Moore,Timothy J. O'Leary,Jeffrey T. Mason
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0014253
Abstract: Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.
Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples
Barcelos, Denise;Franco, Marcello F.;Le?o, Sylvia Cardoso;
Revista do Instituto de Medicina Tropical de S?o Paulo , 2008, DOI: 10.1590/S0036-46652008000600002
Abstract: development and standardization of reliable methods for detection of mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. in this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on pcr performance in paraffin embedded specimens. tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. specimens were submitted to pcr for amplification of the human beta-actin gene and separately for amplification of the insertion sequence is6110, specific from the m. tuberculosis complex. amplification of the beta-actin gene was positive in all samples. no amplicons were generated by pcr-is6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. in conclusion, combined inhibitory factors interfere in the detection of m. tuberculosis in stored material. it is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.
An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays  [cached]
Guo Huifang,Liu Wenbin,Ju Zhenlin,Tamboli Pheroze
Proteome Science , 2012, DOI: 10.1186/1477-5956-10-56
Abstract: Introduction Protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA) is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA. Results Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples. The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p < 0.05) in at least one cell or tissue type, with only 23 markers common in all three sample sets. In addition, FFPE preparations yielded biologically meaningful observations related to pathway signaling status in cell lines, and classification of renal tissues. Conclusions With optimized protein extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type.
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