Dissertations / Theses on the topic 'Fixed and Paraffin Embedded Tissues'
To see the other types of publications on this topic, follow the link: Fixed and Paraffin Embedded Tissues.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 36 dissertations / theses for your research on the topic 'Fixed and Paraffin Embedded Tissues.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Guo, Tong. "Proteome analysis of formalin-fixed and paraffin-embedded tissue." College Park, Md. : University of Maryland, 2008. http://hdl.handle.net/1903/8062.
Full textAbstract:
Thesis (Ph. D.)--University of Maryland, College Park, 2008.Thesis research directed by: Dept. of Chemistry and Biochemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
2
Clift, Sarah J. "Standardization and validation of an immunoperoxidase test for African horsesickness virus using formalin-fixed, paraffin-embedded tissues." Diss., University of Pretoria, 2009. http://hdl.handle.net/2263/24626.
Full textAbstract:
The aim of this study was to standardize and validate an immunohistochemical test for the routine diagnosis of African horsesickness in horses. Hamblin developed the primary anti- African horsesickness virus serum that I used and the avidin-biotin complex detection system was employed. During the standardization process I demonstrate that lung, heart and spleen samples are the most reliable. I also show that it is not necessary to take multiple samples per organ, because the AHSV-positive signal is generally widespread throughout the lung and heart, in particular. In order to validate the technique, samples from 118 negative and 128 positive horse cases, including all nine known serotypes, were immunostained. All of the positive cases were confirmed by means of virus isolation. Negative horse samples were obtained from countries where African horsesickness does not occur. None of the negative cases stained positive and all the positive cases were correctly identified. Therefore, there was 100 % concordance between immunohisto chemistry (when applied to formalin-fixed, paraffin-embedded heart and/or lung and/or spleen tissues from positive horse cases that had been archived for less than 10 years) and virus isolation results. Heart and lung had consistently more positive signal than spleen. The Hamblin antiserum did not cross-react with closely-related orbiviruses (specifically equine encephalosis virus and bluetongue virus) in selected horse and sheep tissues, respectively. Characteristic positive staining was observed in lung, heart and spleen samples from two dogs that died of African horsesickness. Positive signal was not affected by long-term storage in formaldehyde (up to 365 days). Also, specific positive staining could be detected in heart and/or lung and/or spleen samples in more than 95 % of positive horses where tissue blocks had been stored for between 10 and 83 years. The principal target cells in the horse and dog cases were microvascular endothelial cells, intravascular monocyte-macrophages and, to a lesser extent, interstitial macrophages in lung, spleen and liver, in particular. Positive staining is intracytoplasmic with a bead/dot and/or granular character. Beads, dots or granules may occur singly or in clusters. Occasionally, linear deposits of positive signal delineate segments of capillary vessels. The veterinary pathologist must look for characteristic positive signal in target cells, because, occasionally, certain bacteria (Rhodococcus equi and Helicobacter sp.) cross-react with the Hamblin antiserum. Clearly, the test is highly sensitive, specific and robust, sufficiently so for the routine diagnosis of African horsesickness virus.Dissertation (MSc)--University of Pretoria, 2008.
Paraclinical Sciences
unrestricted
3
Clift, Sarah Jane. "Standardization and validation of an immunoperoxidase test for African horsesickness virus using formalin-fixed, paraffin-embedded tissues." Pretoria : [s.n.], 2008. http://upetd.up.ac.za/thesis/available/etd-05132009-173308/.
Full text
4
Viljoen, Rabia. "Optimisation of sample preparation for DNA extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases." Master's thesis, Faculty of Health Sciences, 2021. http://hdl.handle.net/11427/33072.
Full textAbstract:
A retrospective case review revealed an increase in sudden unexpected death (SUD) admittance at Salt River Mortuary (SRM) between 2014 and 2018, and that 40 % of SUD occurred in young individuals between the ages of 1 and 40 years old (SUDY). Despite extensive investigations, the cause of death remained undetermined in 26 % of SUDY cases. These dormant cases may benefit from retrospective post-mortem molecular autopsies for investigation into genetic causes of death. Often, formalin fixed paraffin embedded tissues (FFPETs) are the only archival sources of DNA available for retrospective analyses. This study aimed to optimise DNA recovery from FFPETs for potential use in molecular autopsies of unresolved SUDY cases. To this end, DNA was extracted from FFPET sections using the QIAamp® DNA FFPE tissue kit; the thickness and number of sections were varied. DNA was assessed using spectrophotometry, real-time PCR and digital capillary electrophoresis. Results showed that finer sectioning (1-µm thick as compared to 3-µm and 5-µm thick), improved DNA concentrations, purities and DNA fragment lengths. Increasing the number of 1-µm thick sections from 30 to 100, significantly improved DNA yield. DNA was not significantly more degraded for FFPETs stored for up to three years, which holds promise in the effectiveness of the technique for aged samples. The DNA extraction method developed in this study yielded a median of 320 ng (287 ng - 698 ng) of DNA with 55 % of DNA fragments being at least 400 bp in size. These results are especially informative for downstream molecular analyses, indicating that genotyping or sequencing assays need to be designed to target amplicons less than 400 bp in size. The degraded nature of the FFPET samples also suggests that massively parallel sequencing might be suited for downstream molecular analysis for determining cause of death in unresolved SUDY cases.
5
Hutamo, Kutlwano Aggrineth. "Typing of Mycobacterium bovis in formalin-fixed, paraffin-embedded tissues from selected wildlife species in the Kruger National Park, South Africa." Diss., University of Pretoria, 2012. http://hdl.handle.net/2263/29674.
Full textAbstract:
Mycobaterium bovis is the causative agent of bovine tuberculosis (BTB) and it is a member of the Mycobacterium tuberculosis complex (MTBC). This bacterium has a wide host range of which, cattle is considered as the maintenance host. Humans, goats, wildlife, cats, dogs and lions are also susceptible to the bacterium and are considered putative spillover hosts as infection is not confined in these hosts. Mycobacterium bovis is prevalent in developing countries especially in farmed animals. This presents a problem since BTB is a zoonosis. People living in close contact with infected cattle or those who drink unpasteurized milk are at risk of infection. About 10% of cases of human tuberculosis are thought to be caused by M. bovis. In some instances, wildlife provides a reservoir for the pathogen and transmits it to cattle in farms and poses further risk to humans at the wildlife/livestock/human interface. Certain countries like the United Kingdom where BTB was previously eradicated are experiencing substantial increase in BTB infection. This is thought to be a result of wildlife reservoirs that infect farmed animals, especially cattle. Such reservoirs make eradication of the disease extremely difficult and require programmes to be put in place to control spread of the disease. This makes M. bovis a pathogen of economic importance since the programmes may be costly. In addition, wildlife that is infected cannot be exported and this further affects the economy negatively. In order to control the spread of the pathogen, it is essential to determine the source of infection. However, it is difficult to determine the source or to track the spread of BTB especially in wildlife where animals have unrestricted movement. The inability to conduct epidemiological studies of BTB may be a result of the lack of molecular typing methods that allow bacteria to be identified to strain level rapidly and fairly simpler than culture, thus providing much needed information about the pathogen. In recent years, typing of M. bovis isolates to strain level has been made possible by the development of PCR-based technologies such as IS6110 typing and spoligotyping. These technologies were however, found to be unsuitable for differentiating certain species in the MTBC. Newer technologies based on the variable number of tandem repeats (VNTRs) in organisms have been developed and allow for the differentiation of members in the MTBC, which have a high level of genome homology. These technologies include multiple-locus variable number tandem repeat analysis (MLVA) and mycobacterial interspersed repetitive unit (MIRU)-VNTR analysis. It was also discovered that mycobacteria have genomic regions of difference (RD) that could be used to identify the different species of bacteria in the MTBC. Retrospective studies may play a key role in tracing the source of diseases and following the pattern of transmission. However, in most instances, no fresh samples are available for such studies. For this reason, formalin-fixed paraffin-embedded (FFPE) tissue from wildlife in the Kruger National Park (KNP) was used for conducting a retrospective study aimed at determining the epidemiology of M. bovis in the KNP. However, amplification of DNA derived from FFPE tissue for PCR based techniques has been found to be a difficult exercise and not many standard protocols have been developed and validated for the use of such DNA. In this study, different methods of extraction were used to obtain DNA from FFPE tissue since it is difficult to obtain high quality DNA from such tissue, which is degraded. Formaldehyde, the main component of formalin which is used to fix tissue samples, causes degradation and cross-linking of DNA. In addition, previous studies are inconsistent with regards to the best method to use when extracting DNA from FFPE tissue. Three PCR-based techniques were used to type or identify the isolates in order to standardize a protocol for use in typing isolates from FFPE tissue. These techniques included analysis of the RDs, VNTR based methods i.e. MLVA and MIRU-VNTR and spoligotyping. Since there are many factors that influence the quality of FFPE tissue, samples confirmed BTB positive by VNTR analysis, spoligotyping and IS6110 analysis were used in order to optimize a PCR for FFPE tissue. Furthermore, in order to serve as control samples for spoligotyping and analysis of the RDs, DNA obtained from fresh tissue was also used in the study. Despite the various methods used to extract and to type DNA, the DNA from FFPE tissue provided unspecific results that did not allow for an informative retrospective study of M. bovis. This may be due to the fact that the DNA used had a high degree of degradation from prolonged fixation in formalin. Although M. bovis could not be typed in FFPE tissues, it could be identified by analysis of the regions of difference, more specifically the RD9 region. Amplification of RD9 is thus recommended for use in retrospective studies for diagnostic purposes, especially in cases where highly degraded DNA is used. This region (RD9) should however, only be used as a presumptive diagnosis since RD9 also identifies M. africanum, M. microti, M. pinnipedii, M. caprrae and M. bovis BCG. However, RD9 specifically excludes M. tuberculosis. In the SA context, particularly in the KNP, this allows for some sound inferences since the animals are likely to be infected with M. bovis as opposed to M. tuberculosis. This study highlighted statements in previous studies where it was stated that fixation of tissue in formalin should be done in such a way to reduce degradation of DNA in FFPE tissue in order to allow for its use in retrospective molecular studies which may be very insightful in determining the epidemiology of diseases that are difficult to track and/or control. CopyrightDissertation (MSc)--University of Pretoria, 2012.
Veterinary Tropical Diseases
unrestricted
6
Leslie, Jane Alison. "A pilot study for the use of pancreatic ductal adenocarcinoma formalin-fixed paraffin-embedded tissue in BAC CGH microarrays." Thesis, University of Liverpool, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479043.
Full text
7
Damjanovic, Vesterlund Justina. "Establishment of immunohistochemical double staining on formalin fixed paraffin embedded tissue samples with Pax 5, PD1, PDL1 and PDL2." Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-297184.
Full text
8
Matilda, Rentoft. "The use of formalin fixed paraffin embedded tissue and global gene expression profiling for increased understanding of squamous cell carcinoma of the tongue." Doctoral thesis, Umeå universitet, Patologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-54005.
Full textAbstract:
Head and neck cancer is the 6th most common malignancy worldwide, with tumours of the tongue being one of the most prevalent sites. Despite advances in surgery and radiotherapy, the five-year survival has not changed during the last decades and remains at approximately 50%. Identification of novel biomarkers for more personalized treatment is important for increasing survival in these patients. One of the most commonly used methods in the search for new biomarkers is microarray analysis. A substantial limitation with this technique is the requirement for fresh frozen samples from which high quality RNA can be extracted. This becomes particularly problematic when attempting to discover differences associated with individual sub-types or rare cancers. Recent developments, including the DASL microarray platform, have provided the possibility of analysing RNA of poorer quality from formalin fixed paraffin embedded (FFPE) samples. FFPE is the standard way of preserving tissue from patients and millions of samples are stored around the world. In this thesis we have evaluated the use of FFPE samples and global gene expression profiling for increasing basic knowledge in a subgroup of oral cancer patients with tumours of the tongue. As confirmation of microarray results using qPCR is of outmost importance for conclusive data evaluation, we first aimed at finding a housekeeping gene stably expressed across malignant and non-malignant FFPE oral tissue. TUBA6, which belongs to the tubulin family was detected as being the most stable out of eight possible genes and was thus used for qPCR normalization throughout the following studies. We have performed three separate microarray experiments. Initially only a focused DASL array covering 502 cancer related genes was available and we used it to analyze a smaller cohort of patients and controls (n=36). A similar cohort (n=29) was also analyzed for expression of 836 micoRNAs. In 2009 a whole genome DASL array was launched, covering over 20,000 genes, and all tongue tumour samples available between 1997 and 2010 (n=87) were analysed using this array. Similar to other research groups we observed very high replicate reproducibility using both DASL arrays. When using the microRNA array and the whole genome DASL array an effect of sample quality on the detected expression level of individual genes was noticed. While the expression of some genes severely decreased with a decrease in sample quality others were not changed. This will impair normalization, leading to a residual non-biological variation within the data. Based on our findings we have presented some recommendations for minimizing the effect of sample quality and maximizing the level of biologically relevant information obtained from these experiments, e.g. ensuring that samples in groups to be compared are of the same quality range. For the microRNA data we also introduced an additional normalization step to the standard normalizations. We could show that lists of differentially expressed genes generated when taking these precautions were enriched for genes involved in cancer related processes and contained for tongue carcinoma previously identified changes. A number of differentially expressed genes, novel for tongue carcinoma, were also confirmed in high quality fresh frozen samples, including BCL2A1 (apoptosis), CXCL10 (immune response), SLC2A6 (energy transport) and miR-424 (angiogenesis). In conclusion microarrays can be used to analyze FFPE samples but should be performed with care. Standard normalization methods will not remove the variation introduced by samples being of different quality, leading to spurious results. Taking a few precautions, however, led to the identification of differentially expressed genes relevant in tumour development and maintenance. The recommendations we make can facilitate design of future studies using FFPE samples. The genes we identified as being differentially expressed in tumour tissue now need to be further evaluated for their potential as biomarkers in tongue carcinoma.
9
Lüder, Ripoli Florenza [Verfasser]. "Comparison of fresh frozen vs. formalin-fixed, paraffin-embedded specimens and the expression profiling of 16 target genes in neoplastic and non-neoplastic canine mammary tissues using a multiplex branched-DNA assay / Florenza Lüder Ripoli." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2016. http://d-nb.info/1125394560/34.
Full text
10
Rossouw, Sophia Catherine. "Optimisation of proteomics techniques for archival tumour blocks of a South African cohort of colorectal cancer." University of Western Cape, 2020. http://hdl.handle.net/11394/8036.
Full textAbstract:
Philosophiae Doctor - PhDTumour-specific protein markers are usually present at elevated concentrations in patient biopsy tissue; therefore tumour tissue is an ideal biological material for studying cancer proteomics and biomarker discovery studies. To understand and elucidate cancer pathogenesis and its mechanisms at the molecular level, the collection and characterisation of a large number of individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardised methods of formalin fixation and paraffin embedment, these archived, FFPE tissues are important collections of pathology material, often accompanied by important metadata, such as patient medical history and treatments. FFPE tissue blocks are conveniently stored under ambient conditions for decades, while retaining cellular morphology due to the modifications induced by formalin.
2022
11
Djidja, M.-C., S. Francese, Paul M. Loadman, Chris W. Sutton, P. Scriven, E. Claude, M. F. Snel, J. Franck, M. Salzet, and M. R. Clench. "Detergent addition to trypsin digest and Ion Mobility Separation prior to MS/MS improves peptide yield and Protein Identification for in situ Proteomic Investigation of Frozen and FFPE Adenocarcinoma tissue sections." Wiley, 2009. http://hdl.handle.net/10454/4565.
Full textAbstract:
noThe identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI-mass spectrometry imaging (MALDI-MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin-fixed paraffin-embedded breast cancer tissue sections were used. An improved protocol for on-tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin-fixed paraffin-embedded tumour tissue sections. A novel approach combining MALDI-MSI and ion mobility separation MALDI-tandem mass spectrometry imaging for improving the detection of low-abundance proteins that are difficult to detect by direct MALDI-MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI-MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified
12
Di, Cesare Sebastian. "Retrospective transcriptional and genomic analysis of formalin-fixed paraffin-embedded human uveal melanoma." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96723.
Full textAbstract:
Many of the underlying genetic causes of Uveal Melanoma (UM) metastatic disease still remain to be elucidated. This particular cancer is the most common primary malignant intraocular tumor in adults, comprising approximately 5% of all diagnosed melanoma cases. Despite its rarity, approximately 50% of patients diagnosed with UM will develop metastatic disease. Due to the poor understanding of the genetic mechanisms that cause UM metastatic disease, further investigations into elucidating these mechanisms are necessary. Fresh biopsied ocular tumor tissues are difficult to obtain for the purpose of performing microarray experiments on extracted nucleic acids. Present technology now allows for extraction of total RNA from formalin-fixed paraffin embedded (FFPE) tissue to run genomic cancer arrays by the cDNA mediated Annealing Sectioning and Ligation (DASL) method. We aimed to correlate gene transcript differences between two UM clinical-histopathological parameters (Metastasis, Cell Type). ANOVA testing identified 106 genes with a significant change in transcript abundance (p < 0.05, Welch T-test) between tumors from patients that died of metastasis to those that did not. Similarly, 64 genes significantly differed in transcript abundance when the spindle and epithelioid cell type parameters were compared. As a predictor of metastasis-dependant death, 12 genes were shown to correctly predict this parameter in 27/37 tumors (73%). As a predictor of UM cell type we successfully predicted the classification of 27/31 (87.1%) non-mixed tumors with a significant difference in transcript abundance between 10 genes.We sought to validate these transcriptional findings at the DNA and protein level using comparative genomic hybridization arrays (aCGH) and immunohistochemistry. LIG4 (predictor of metastasis) and ErbB3 (predictor of UM cell type) correlated well at both levels in independent patient sample populations.In addition, DASL analysis revealed an up-regulation of PAI-1 serum bio-marker expression in patients that developed metastatic disease. ELISA analysis of UM patient plasma revealed a positive correlation of increased PAI-1 plasma protein expression with increasing tumor height (n = 11, r = 0.6525, p = 0.0295). To the best of our knowledge, this is the first study to have performed and validated a retrospective transcriptional analysis using RNA extracted from FFPE primary UM.À ce jour, l'étude des facteurs génétiques reliés au mélanome uvéal (MU) est en pleine croissance. Ce type de cancer représente la principale forme de tumeur intraoculaire maligne au sein de la population adulte, comprenant approximativement 5% de tous les cas de mélanome diagnostiqués. Plus de 50 % des individus chez qui on a diagnostiqué la présence d'un mélanome uvéal développeront des métastases dans divers organes, principalement au foie. On connaît mal le mécanisme biologique de cette maladie et c'est pour cette raison que l'étiologie du mélanome uvéal est nécessaire.Il est difficile d'obtenir des échantillons de biopsie de tissus cancéreu oculaires pour extraire les acides nucléiques. A ce jour, la technologie nous permet d'extraire l'ARN de tissus fixés dans la formaldehyde et enrobés de paraffine (FFPE « Formaldehyde Fixed and Paraffin embedded »), en totalité pour ensuite analyser les génomes par la méthode cDNA mediated Annealing Sectioning and Ligation (DASL). Notre but est d'établir la corréler les differences des gènes transcrit entre deux paramètres clinico-hispopathologiques MU (Métastase, Type de cellule). A partir des tests ANOVA, nous avons identifié 106 gènes possédant un changement de transcription significatif (p < 0.05, Welch T-test) entre les tumeurs des patients qui sont décédés de métastases et ceux qui ont survecu. De plus, 64 génes possédant un changement de transcription significatif lorsque les paramètres des cellules fusiformes et des cellules épithélioïdes ont été comparés. 12 gènes ont correctement prédit la mort dépendant des métastases dans 27 des 37 tumeurs testés. Grace à cette méthode, notre groupe a réussi à prédire la classification de 27/31 tumeurs non-mixtes entre 10 gènes pour des cellules de type MU.Nous avons validé nos découvertes au niveau de l'ADN et des protéines en utilisant des tests d'hybridization comparative génomique (aCGH) et de l'immunohistochimie. Une bonne corrélation de LIG4 (métastase) et ErbB3 (cellules de type MU) est obtenue dans nos échantillons de population de patients indépendants.De plus, les analyses DASL ont révélé une expression de haute-régulation de sérum PAI-1 bio marqueur dans les échantillons des patients qui ont développé de métastases. D'après les analyses ELISA du plasma des patients avec du MU, une corrélation positive entre l'augmentation d'expression de protéines de plasma PAI-1 avec une augmentation de l'hauteur des tumeurs (n = 11, r = 0.6525, p = 0.0295).D'après nos connaissances, ce travail est la première étude qui ce validé l'analyse rétrospective transcription avec de l'ARN extrait d'échantillons FFPE MU primaire.
13
Nil, Zelha. "Genome Wide Variation Analysis Of Formalin Fixed Paraffin Embedded Pulmonary Metastatic Tumor Samples Of Osteosarcoma Patients." Master's thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614014/index.pdf.
Full textAbstract:
Osteosarcoma (OS) is a type of cancer that starts in the bone. It generally occurs in the cells called osteoblasts which form matrix of the bone. It is the most common malignant tumor of bone with an incidence rate of 19% among all cancer types. The vast majority of OS patients have pulmonary metastases at the time they are diagnosed, and about half develop lung disease later. Moreover, pulmonary metastatic tumors lead to poor prognosis and increased death rate. Although mutations in the genes coding for p53, Rb, fos and myc were detected in pulmonary metastatic tumors of OS, there is no unique genetic pathway identified for progression of pulmonary metastasis.
In this research, a genome wide association study (GWAS) using FFPE samples from lung tissue of 9 patients with pulmonary metastatic OS was performed. Among 358 associated SNPs, rs6499861, rs10884554 and rs12154602 were found to be associated with metastatic OS most significantly. Moreover, second wave analysis of GWAS results provided the significant genes and pathways associated with metastatic OS.
A methodology for copy number aberration and LOH analysis of SNP array data of a FFPE sample was generated using R-aroma package. Results were obtained by three different methods, namely, CalMaTe, TumorBoost and Virtual Normal algorithm. Among these, CalMaTe was found to produce less noisy data than VN Algorithm during total copy number segmentation. LOH analysis could only be performed for one sample with the second method due to poor data quality of the other samples.
According to the results of copy number aberration and LOH analysis of one tumor sample T8, copy number gains in 1p31.1, 6p21.32, 7p14.3, 11q22.1, 12p12.1, and 18q12.1 chromosomal regions and copy number losses in 2p16.2, 8q24.13, 17q23.3 and 17q21.31 chromosomal regions have been found. Moreover, LOH events were observed in 2q14.3, 11q13.4, 18p11.21, 19q12, 20p13 and 23q21.1 chromosomal regions.
Identification associated SNPs and significant copy number changes may be helpful in investigation of potential diagnostic and prognostic markers in metastatic osteosarcoma.
14
Claesson, Cim. "Is Multiplex Ligation-dependent Probe Amplification a good method for screening formalin fixed paraffin embedded neuroblastoma tumors?" Thesis, Högskolan i Skövde, Institutionen för vård och natur, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-5442.
Full textAbstract:
Neuroblastoma is one of the most enigmatic solid tumors for scientists and pediatric oncologists. Neuroblastoma is primary a childhood form of cancer, consisting of neuroectodermal cells that originate from the neural crest and is destined for the adrenal medulla and sympathetic nervous system. The Neuroblastoma group at The University of Gothenburg received formalin-fixed paraffin-embedded tumor samples from Vietnam and this project was to examine if the quality of the DNA from, is good enough to run comparative genome hybridization array experiment on by using a cheaper technique Multiplex Ligation-dependent Probe Amplification technique. Multiplex Ligation-dependent Probe Amplification (MRC Holland) is a multiplex PCR method that can detect abnormalities such as deletions and amplifications. By using probes consisting of one short synthetic arm with a PCR primer sequence Y at the 3´end, and one long probe with a stuffer sequence, and a PCR primer sequence X at the 5´end that can hybridize and ligate. If these probes ligate it is possible to amplify them by PCR just using specific primers for the X and Y sequences. The resulting amplification products can then be analyzed bycapillary electrophoresis. These patient that the DNA was derived from had all stage 4 neuroblastoma, and that is why they all present many aberrations. Among the fascinating data from this experiment, there are many patients with both 11q deletions and has an extreme amplification of MYCN. In Sweden only a few cases has been discovered. In this material even though all patients are stage 4 patients, 16 have this combination.
15
Gordon, Ann Elizabeth-Chamberlain. "Criminal paternity DNA testing of microscopically-identified chorionic villi in formalin-fixed paraffin-embedded products of conception." Diss., Connect to online resource - MSU authorized users, 2006.
Find full text
16
Pearson, Joyce. "Immunophenotypic classification of canine malignant lymphoma in formalin-fixed, paraffin wax-embedded specimens using CD3 and CD79a cell markers." Diss., University of Pretoria, 1999. http://hdl.handle.net/2263/29493.
Full textAbstract:
Inconsistent use of nomenclature in different canine malignant lymphoma (CML)classification systems, which lead to incorrect diagnosis and prognosis, necessitated a retrospective study of 103 cases of CML. Histological classification was done according to the Working Formulation, on H&E sections, after standard processing. Immunophenotyping, using CD3 (T cell) and CD79a (8cell) markers, was carried out on the same sections. Intermediate grade lymphomas were the largest category (49.5%), with 16.5%high grade lymphomas. More than half (53.3%) of the lymphomas were of 8 cell immuno phenotype, and 36.5% were T cell lymphomas. Only 9.7% of the total number of lymphomas exhibited double negative staining. Only two categories, the immunoblastic and medium-sized macro nucleolated (MMC) category (Fournel-Fleury et al.). exhibited constant (8 cell) immunophenotype. All the other categories exhibited mixed immunophenotype. The Working Formulation, with omission of the follicular tyoes (due to the rarity thereof in CML) and addition of the MMC category and immunophenotyping, is best for classifying CML.Dissertation (MMed Vet (Pathology))--University of Pretoria, 1999.
Anatomy and Physiology
Unrestricted
17
Pearson, Joyce. "Immunophenotypic classification of canine malignant lymphoma in formalin-fixed, paraffin wax-embedded specimens using CD3 and CD79a cell markers." Pretoria : [s.n.], 2009. http://upetd.up.ac.za/thesis/available/etd-11162006-121839/.
Full text
18
Ward, Kathleen Anne. "The application of the polymerase chain reaction to the study of the human papillomavirus in fresh and paraffin embedded tissues." Thesis, Queen's University Belfast, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295438.
Full text
19
Naven, Marc. "Development of a pipeline and protocols for next generation sequencing of blood and formalin-fixed, paraffin-embedded tumour DNA samples." Thesis, Cardiff University, 2015. http://orca.cf.ac.uk/91435/.
Full textAbstract:
Using existing software and six novel scripts, we developed a pipeline for variant calling using exome re-sequencing data of germline blood deoxyribonucleic acid (DNA) samples. We observed >99% (6,723/6,739) concordance between calls from our pipeline and previous genotyping. We identified >93% of single nucleotide variants (SNVs) and >94% of insertion/deletions called by a commercial partner using the same sequencing reads. Using a subset of genes, we showed that around half of predicted pathogenic variants could be validated in vitro. Together, these data showed that our pipeline was reliable for variant calling. Next generation sequencing (NGS) of DNA from formalin-fixed, paraffin-embedded (FFPE) tumours is technically challenging. We sought to determine the sensitivity of NGS to detect known somatic hotspot mutations (n=25) in 19 FFPE advanced colorectal cancers and to optimise it for the identification of novel somatic variants. Analysis using Illumina’s MiSeq software identified 100% of somatic mutations with 93% specificity, whereas the Genome Analysis Tool Kit’s (GATK) HaplotypeCaller identified 80-92% of somatic mutations with 100% specificity. We investigated the background mutator profile and found that normal FFPE DNA had 3-fold more SNVs than matched blood DNA, with an excess of FFPE-associated C:G>T:A substitutions (27.1 vs. 5.3%). Uracil DNA glycosylase treatment reduced this excess. Only ~5% of variants were consistently called in replicate runs and were likely to be genuine somatic variants. We detected potential candidates for genetic biomarkers of cetuximab-resistance in colorectal cancers that were previously shown to be wild type for KRAS, NRAS, BRAF and PIK3CA. We found that 7/21 (33%) of the CRCs analysed harboured mutations at either codon 12 or 61, whileother CRCs carried truncating KRAS mutations. NRAS also carried a codon 12 mutation in 1 CRC, and PIK3CA possessed mutations at codons 542 and 545. PTEN was found to harbour 5 truncating mutations at codons 71, 140, 155, 178 and 189, potentially leading to loss of function.
20
McCosker, Helen Clare. "Prognostic significance of IGF and ECM induced signalling proteins in breast cancer patients." Thesis, Queensland University of Technology, 2012. https://eprints.qut.edu.au/53580/1/Helen_McCosker_Thesis.pdf.
Full textAbstract:
Breast cancer is a leading contributor to the burden of disease in Australia. Fortunately, the recent introduction of diverse therapeutic strategies have improved the survival outcome for many women. Despite this, the clinical management of breast cancer remains problematic as not all approaches are sufficiently sophisticated to take into account the heterogeneity of this disease and are unable to predict disease progression, in particular, metastasis. As such, women with good prognostic outcomes are exposed to the side effects of therapies without added benefit. Furthermore, women with aggressive disease for whom these advanced treatments would deliver benefit cannot be distinguished and opportunities for more intensive or novel treatment are lost. This study is designed to identify novel factors associated with disease progression, and the potential to inform disease prognosis. Frequently overlooked, yet common mediators of disease are the interactions that take place between the insulin-like growth factor (IGF) system and the extracellular matrix (ECM).
Our laboratory has previously demonstrated that multiprotein insulin-like growth factor-I (IGF-I): insulin-like growth factor binding protein (IGFBP): vitronectin (VN) complexes stimulate migration of breast cancer cells in vitro, via the cooperative involvement of the insulin-like growth factor type I receptor (IGF-IR) and VN-binding integrins. However, the effects of IGF and ECM protein interactions on the dissemination and progression of breast cancer in vivo are unknown. It was hypothesised that interactions between proteins required for IGF induced signalling events and those within the ECM contribute to breast cancer metastasis and are prognostic and predictive indicators of patient outcome.
To address this hypothesis, semiquantitative immunohistochemistry (IHC) analyses were performed to compare the extracellular and subcellular distribution of IGF and ECM induced signalling proteins between matched normal, primary cancer, and metastatic cancer among archival formalin-fixed paraffin-embedded (FFPE) breast tissue samples collected from women attending the Princess Alexandra Hospital, Brisbane. Multivariate Cox proportional hazards (PH) regression survival models in conjunction with a modified „purposeful selection of covariates. method were applied to determine the prognostic potential of these proteins.
This study provides the first in-depth, compartmentalised analysis of the distribution of IGF and ECM induced signalling proteins. As protein function and protein localisation are closely correlated, these findings provide novel insights into IGF signalling and ECM protein function during breast cancer development and progression. Distinct IGF signalling and ECM protein immunoreactivity was observed in the stroma and/or in subcellular locations in normal breast, primary cancer and metastatic cancer tissues. Analysis of the presence and location of stratifin (SFN) suggested a causal relationship in ECM remodelling events during breast cancer development and progression. The results of this study have also suggested that fibronectin (FN) and ¥â1 integrin are important for the formation of invadopodia and epithelial-to-mesenchymal transition (EMT) events. Our data also highlighted the importance of the temporal and spatial distribution of IGF induced signalling proteins in breast cancer metastasis; in particular, SFN, enhancer-of-split and hairy-related protein 2 (SHARP-2), total-akt/protein kinase B 1 (Total-AKT1), phosphorylated-akt/protein kinase B (P-AKT), extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (ERK1/2) and phosphorylated-extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (P-ERK1/2).
Multivariate survival models were created from the immunohistochemical data. These models were found to fit well with these data with very high statistical confidence. Numerous prognostic confounding effects and effect modifications were identified among elements of the ECM and IGF signalling cascade and corroborate the survival models. This finding provides further evidence for the prognostic potential of IGF and ECM induced signalling proteins. In addition, the adjusted measures of associations obtained in this study have strengthened the validity and utility of the resulting models.
The findings from this study provide insights into the biological interactions that occur during the development of breast tissue and contribute to disease progression. Importantly, these multivariate survival models could provide important prognostic and predictive indicators that assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy. The outcomes of this study further inform the development of new therapeutics to aid patient recovery. The findings from this study have widespread clinical application in the diagnosis of disease and prognosis of disease progression, and inform the most appropriate clinical management of individuals with breast cancer.
21
Price, Brendon. "Human Papillomavirus DNA extraction and genotype analysis by multiplex real time polymerase chain reaction from formalin fixed paraffin wax-embedded cervical carcinoma specimens." Master's thesis, Faculty of Health Sciences, 2019. http://hdl.handle.net/11427/31189.
Full textAbstract:
Introduction: Cervical squamous cell carcinoma is most commonly caused by persistent infection by high risk human papillomavirus (hrHPV) genotypes. The exact type of hrHPV varies geographically and is the basis for HPV–based vaccination for cervical squamous cell carcinoma prevention. Little is known regarding local hrHPV genotypes within the Western Cape population of South Africa. Aims and objectives: This was a pilot study aiming to extract of high quality genomic DNA from archival FFPE cervical squamous cell carcinoma cases and identify hrHPV genotypes by multiplex real time PCR (RT-PCR). Materials and methods: A retrospective search identified a total of 57 cases of cervical squamous cell carcinoma for the period 2004-2014. This was reduced to a final number of 23 that exhibited sufficient tumour burden for DNA extraction. The most common age group was 40-49 years. HIV status was as follows: two HIV-positive, 14 HIV-negative and 7 HIV unknown. DNA was extracted from archival FFPE cervical squamous cell carcinoma samples using QIAGEN QIAamp® DNA FFPE Tissue kit. Housekeeping genes were detected by endpoint PCR using standard primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequences to determine the quality and integrity of extracted DNA for downstream PCR amplification experiments. HrHPV DNA amplification was optimised using a touchdown PCR technique with L1 consensus gene GP5+/GP6+ primers. HrHPV genotypes were detected using a four colour multiplex hrHPV genotyping kit. Samples showing positive results in overlapping probe filter detection spectra were subjected to DNA Sanger sequencing for final confirmation of specific hrHPV genotype. Results: Standard xylene DNA extraction methods using QIAamp® system yielded adequate amounts of DNA with average final concentration of 463.2 ng/l and A260/A280 ratio of 1.86. Housekeeping genes were successfully detected in all samples, confirming that no significant DNA degradation of target sequences occurred within the archival time range of 2004-2014. HPV L1 detection via GP5+/GP6+ primers with endpoint PCR was not achieved via standard cycling conditions and required the use of a touchdown technique with gradually decreasing annealing temperatures. This method successfully identified HPV L1 sequences in 22 out of 23 cases. Multiplex RT-PCR with four colour hydrolysis probes identified hrHPV genotypes in 22 of 23 cases with relative frequencies of HPV genotypes: 16>>18=39=45>33. Most cases showed infection with a single hrHPV genotype (HPV 16 and one case with HPV 33) with four cases demonstrating two genotypes (two with HPV 16&18, one with 16&33 and one with 39&45) and one case with three genotypes (HPV 16, 39, 45). Interestingly, none of the HIV-positive cases showed multiple hrHPV genotype infection. Four hrHPV cases with overlapping spectra for HPV 18/31 and 45/59 were subjected to Sanger sequencing for confirmation of genotype. Three of four cases showed 100% match for genotypes 18 and 45 with the final case demonstrating only co-infective HPV 16.Conclusion: Commercial DNA extraction kits yield adequate amounts of intact, amplifiable DNA in archival FFPE cervical carcinoma specimens. Touchdown PCR is necessary for HPV detection in extracted FFPE DNA cases using GP5+/GP6+ L1 primers. RT-PCR using multicolour hydrolysis probes is a rapid, sensitive technique for hrHPV genotype screening of cervical squamous cell carcinoma specimens. A three colour detection system rather than four colour kit is recommended for future studies in order to avoid extra cost in DNA sequencing cases with overlapping spectra. This pilot study demonstrates hrHPV genotype prevalence similar to that in other populations and suggests that vaccination with currently available formulations would provide a sufficiently wide coverage of HPV genotypes. Future studies will include application of the FFPE DNA extraction, endpoint PCR and RT-PCR techniques to the remainder of the cases in the original cohort.
22
Ragonesi, Leanne M. "Structural characterisation of the term placenta: Maternal obesity and gestational diabetes mellitus." Thesis, Queensland University of Technology, 2017. https://eprints.qut.edu.au/113719/1/Leanne_Ragonesi_Thesis.pdf.
Full textAbstract:
This thesis investigated placental histopathologies and perinatal outcomes from women with normal glycaemia, women with maternal obesity and women with gestational diabetes mellitus. The incidence of placental maturational defects were higher in the placentae from women with maternal obesity and women with gestational diabetes mellitus, suggesting that obesity and gestational diabetes mellitus may be associated with structural changes to the placenta that may affect function. In addition, this study optimised a method for extracting and amplifying microbial DNA from formalin-fixed tissue, in order to perform microbial examination in parallel with histopathology analysis using the same tissue specimen.
23
Ranbaduge, Nilini Sugeesha. "Mass Spectrometry-Based Clinical Proteomics for Non-Small Cell Lung Cancer." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469103007.
Full text
24
Farrugia-Jacamon, Audrey. "Investigations moléculaires dans la mort subite du sujet de moins de 35 ans." Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00804339.
Full textAbstract:
Les canalopathies cardiaques congénitales constituent la principale hypothèse diagnostique dans les cas de mort subite inexpliquée chez les sujets de moins de 35 ans. Notre travail a eu pour objectif demettre au point une stratégie de détection post-mortem des mutations sur les gènes connus pour être impliqués dans les canalopathies cardiaques, applicable en routine, à partir de la principale source d'ADN post-mortem disponible en France à savoir les prélèvements fixés au formol et inclus en paraffine (FFIP). A partir d'une cohorte de 12 cas, deux techniques de détection de variants génétiques ont été évaluées, une technique de criblage par l'analyse des courbes de fusion haute résolution et une technique de génotypage par spectrométrie de masse MALDI-TOF, respectivement sur le gène KCNQ1 et le gène RyR2. Quelle que soit la technique utilisée, il n'est pas possible de s'affranchir du séquençage de type Sanger afin d'explorer les séquences d'intérêts qui n'ont pu être optimisées avec l'une ou l'autre des méthodes à la fois sur les prélèvements congelés et FFIP. L'arrivée des séquenceurs de nouvelles générations ouvrent ainsi de nouvelles perspectives dans ce domaine.
25
Jhuang, Jie-Yang, and 莊傑仰. "Identify the common DNA viral pathogens from formalin-fixed paraffin-embedded myocardium tissues of myocarditis." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/21645895247955016339.
Full textAbstract:
碩士國立臺灣大學
法醫學研究所
95
Fatal acute myocarditis is a common cause of alleged medicolegal investigation. Forensic pathologists frequently encounter these death investigations, so it is an important disease we have to understand. The etiology of myocarditis is usually inferred from clinical information and preliminary laboratory studies. This study was undertaken to evaluate the molecular analysis of endomyocardial archive tissues in identifying the possibility of common DNA viral pathogens. We collected all available clinical record and endomyocardial archive tissues for patients who had myocarditis recorded as the clinical diagnosis at the National Taiwan University Hospital from 2001 to 2005. Findings for all available patients（6 men and 6 women；median age, 26 years）with myocarditis that fulfilled the Dallas criteria were included in this study. Twelve subjects who had died naturally except heart diseases served as control group from forensic autopsy. Nested polymerase chain reaction （PCR）was used for detection of DNA viral genomes （human herpes virus 1, human herpes virus 2, Epstein-Barr virus, and human herpes virus 5）from endomyocardial biopsied tissues. DNA viral nucleic acid were found in the hearts of 2 patients （16.7%）, including human herpes virus 5（2 patient）. In the control group, no viral genome was detected. In patients with unexplained myocarditis, viral infection really contributes to be an important etiology. We can’t realize which kinds of DNA viral infection based on only microscopic examination of endomyocardial biopsies. Serological tests can help these but it is time-consuming and not very specific. Nested polymerase chain reaction may be an sensitive and specific tool to identify viruses. Then, this may be given as a guide in treating patients in the future, such as viral vaccine prevention. It may be useful in forensic cases to identify the underlying virus infection.
26
Hong, Chih-Kang, and 洪志岡. "The prevalence of common RNA enteroviral pathogens from formalin-fixed paraffin-embedded myocardium tissues of myocarditis." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/89510507403561154089.
Full textAbstract:
碩士國立臺灣大學
法醫學研究所
99
The etiological factor of sudden cardiac death due to acute myocarditis has long been an important issue in forensic medicine. Due to the progression of molecular technique, there are increasing researches about the etiology of myocarditis. Although the cause of myocarditis in any given pations, systemic diseases, drugs, and toxins have been associated with the development of this disease. Viruses are an important cause of myocarditis in North America and Europe. The prevalence of viral myocarditis is still unclear in Taiwan. The purpose of this study was to investigate the prevalence of myocarditis infected by RNA enterovirus in Taiwan. The formalin fixed paraffin embedded myocardial tissue blocks of myocarditis were obtained from endomyocardial biopsy (9 samples), heart transplantation (1 sample) and forensic autopsy specimen die of myocarditis (1 sample). Tissue blocks were studied by using immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) for enterovirus. This study showed that histological section from 7 of 11 myocarditis cases were positive for the viral capsid protein VP1 by immunohistochemical staining. In 4 of 7 VP1 immunohistochemical positive staining specimens, viral genome of enterovirus was detected by RT-PCR using 5’ NTR genomic fragment. These results showed most RNA fragment recovered by RT-PCR which fall between 152-470 nt. The product of RT-PCR probe should less than 200 bp which is the best candidate in these recovery. Also the study provided data of rough enteroviral prevalence in myocarditis (more than 36.4%). Further evidence of prevalence about enterovirus involvement in myocarditis needs more large scale of such cases using the method provided in the study to get in Taiwan.
27
Hsieh, Chao-Han, and 謝昭漢. "mRNA expressions of matrix metalloproteinases and tissue inhibitor metalloproteinases on formalin fixed paraffin embedded human breast tumor tissues using RT-PCR." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/t3uet5.
Full textAbstract:
碩士國立中興大學
動物科學系所
99
Formalin fixed paraffin embedded (FFPE) is the most commonly used method worldwide for tissue storage; this resource represents a vast repository of tissue material with a long-term clinical follow-up. Although, FFPE preserves the tissue integrity it may cause extensive damage to nucleic acids stored within the tissue. Hence, the primary goal of this experiment is to set up the best condition for detecting mRNA expression in FFPE tissues by RT-PCR. To optimize the RT-PCR condition, we compare the GAPDH mRNA expression in fresh frozen tissues and tissues with formalin fixation for 1, 2 and 3 days under different proteinase K reaction time, primer concentration, commercial RT kits, amplicon size of primer treatment. We concluded that a shorten fixation time to less than one day, and extended proteinase K reaction time to 24 hours produced better RNA quality and recovery. The appropriate primer amplicon size is smaller than 100 bp and the concentration of primer is better at 5 μM in 1.5 μl. Biomi Biotech RT kit is more sensitive than Invitrogen Superscript RT kit in detecting small amplicon size primer. Matrix metalloproteinases (MMPs) can degrade extracellular matrix, which is regulated by tissue inhibitors of metalloproteinases (TIMPs). Hence, those two factors are considered to play an important role in cancer metastasis and invasion. We applied the above optimal condition for RT-PCR to measure the RNA expressions of matrix metalloproteinases (MMP) -2, -9, -14 and tissue inhibitor of metalloproteinases (TIMP) -1, -2 on FFPE human breast cancer tissues. Thirty cases of FFPE human breast tumor from 2009 and two cases of FFPE human breast tumor from 2007 were adopted in this experiment. FFPE of eleven cases of intraductal papilloma (IP), eleven cases of ductal carcinoma in situ (DCIS), and ten cases of invasive ductal carcinoma (IDC) were included in current study. The RT-PCR results were quantified. The statistics of the results show that RNA expressions of MMP-9 and TIMP-2 were significantly lower in DCIS. TIMP-1 RNA expression was not detected in all samples studied. Correlation analysis show that the expression between MMP-2 and MMP-9, MMP-2 and TIMP-2, MMP-9 and TIMP-2 are highly related. The correlation analysis of MMP-2, MMP-9, MMP-14 and TIMP-2 are highly related in DCIS. Our results suggest that MMP-2, MMP-14 and TIMP-2 are important in extracellular matrix degradation in DCIS and MMP-9 is more relevant with invasive ductal carcinoma.
28
Hamoud, Mohamed Mamdouh. "Studies on infectious bursal disease virus (IBDV) dsRNA extracted from formalin fixed paraffin embedded tissue." 2006. http://purl.galileo.usg.edu/uga%5Fetd/hamoud%5Fmohamed%5Fm%5F200608%5Fphd.
Full text
29
Upreti, Deepa. "Diagnostics for Rift Valley fever virus." Thesis, 2018. http://hdl.handle.net/2097/39109.
Full textAbstract:
Master of ScienceDepartment of Diagnostic Medicine/Pathobiology
A. Sally Davis
Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic Phlebovirus that is a significant threat to ruminants and humans. RVFV is categorized as an overlap Select Agent by the Department of Health and Human Services and US Department of Agriculture. Therefore, the study of RVFV’s pathogenesis and the development of novel diagnostic tools for the prevention and control of outbreaks and virus spread is crucial. RVF is endemic to sub-Saharan Africa but has spread beyond the continent to the Arabian Peninsula indicating the competence of the virus to emerge in new areas. Thus, the high likelihood of RVF’s spread to other non- endemic countries also spurs the need for development and implementation of rapid diagnostic tests and surveillance programs. In the US, RVFV is a Select Agent, requiring BSL-3 enhanced containment practices for research work. First, we developed a method for the detection of RVFV RNA by reverse transcriptase real-time PCR (RT-qPCR) using non-infectious, formalin- fixed, paraffin-embedded tissues (FFPET). The results from FFPET RT-qPCR were compared to prior results for fresh-frozen tissues (FFT) RT-qPCR, as well as immunohistochemistry and histopathology completed on the same FFPET blocks. We developed a novel technique using a rapid and low cost magnetic bead extraction method for recovery of amplifiable RVFV RNA from FFPET. FFPET RT-qPCR can serve as an alternative tissue-based diagnostic test, which does not require a BSL-3 research facility. Second, we assessed the diagnostic accuracy and precision of a recombinant RVFV nucleoprotein based competitive ELISA (cELISA) assay to detect RVFV antibodies. The cELISA results were compared to the virus neutralization test, the gold standard serological assay for RVFV. This prototype cELISA is easy to implement, sensitive, specific, and safe test for the detection of antibodies to RVFV in diagnostic and surveillance applications. RVF is an important transboundary disease that should be monitored on a regular basis. The diagnostic tests developed and validated in this thesis could be used in endemic or non-endemic countries for the early detection of RVF and assist with the implementation of countermeasures against RVFV.
30
Chen, Yu-chen, and 陳育辰. "Establishment of quantification of hepatitis B virus covalently closed circular DNA in formalin-fixed, paraffin embedded liver tissue." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/66077715732613647316.
Full textAbstract:
碩士國立成功大學
分子醫學研究所
95
Hepatitis B virus (HBV) is a small noncytopathic hepatotropic virus that causes acute and chronic hepatitis, liver cirrhosis and development of hepatocellular carcinoma. Treatment of HBV infected patients with nucleotide or nucleoside analogues including lamivudine, adefovir dipivoxil, and entecavir effectively inhibit serum viral load, induce normalization of serum transaminase level, and improve liver necroinflammation and fibrosis. However, evidence from in vitro studies revealed that covalently closed circular DNA (cccDNA) of HBV was considerably resistant to lamivudine treatment. In addition to lamivudine, adefovir dipivoxil could only partially but not completely inhibit HBV cccDNA formation. Unlike adefovir and lamivudine, entecavir was shown to be capable of reducing cccDNA levels in liver in several in vitro studies. Therefore, aims of this study are (1) to establish the method to quantify cccDNA in formalin-fixed, paraffin embedded (FFPE) liver tissues, which are more accessible than fresh frozen tissues; (2) to compare results of FFPE liver tissues with fresh frozen liver tissues. We collected paired liver tissue with FFPE liver tissues and fresh frozen liver tissue from chronic hepatitis B patients who had enrolled in clinical trials with antiviral treatment, such as lamivudine, adefovir dipivoxil, and entecavir. For each FFPE liver tissues, we collected samples consisting of 20 sections with 10μm in thickness. Following DNA extraction, we successfully quantified total HBV DNA levels by specific real-time PCR assays. Moreover, we quantified cccDNA in FFPE liver tissues by using quantitative nested PCR with newly designed outer primers. This new method can only specifically amplify cccDNA verified by enzyme treatment. In this step, we discovered that DNase may have enzymatic activity on cccDNA quantification process. Futhermore, we examined the necessity of the DNase treatment. The results demonstrated that DNase can’t influence the amount of cccDNA in fresh frozen tissue but can reduce great amount of cccDNA in paraffin embedded tissue. Therefore, this method without DNase treatment was able to specifically to amplify cccDNA in paraffin embedded tissue. In the future, this method will apply to investigate the dynamics of cccDNA level before and after different antiviral treatment.
31
Kuo, Shanny Hsuan, and 郭. 軒. "Molecular Detection of Feline Coronaviruses in Formalin-Fixed and Paraffin-Embedded Tissue (FFPE) by nested RT-PCRs: a Diagnosis-Aiding Approach." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/dc943h.
Full textAbstract:
碩士國立臺灣大學
分子暨比較病理生物學研究所
105
Feline infectious peritonitis (FIP), caused by feline coronavirus (FCoV), is a lethal disease in cats. The clinical signs are non-specific and antemortem diagnosis remains challenging and frustrating. Appling histopathology combined with immunohistochemical (IHC) staining is considered as the gold standard for FIP diagnosis. However, the sensitivity of the IHC method depends much on the numbers of intralesional antigen-bearing cells. Due to the limitations of small sampling sizes as well as the equivocal IHC staining pattern in some specimens, formalin-fixed and paraffin-embedded tissue (FFPE) biopsies frequently submitted for histopathological examination for FIP are the most challenging specimens for pathologists. It has been demonstrated that the consensus PCR targeting 3’UTR alone is non-specific for diagnosis of FIP in fresh tissues. Moreover, two recently described mutations, the substitution of methionine (M) to leucine (L) amino acid mutation at position 1058 (M1058L) and the substitution of serine (S) to alanine (A) amino acid mutation at position 1060 (S1060A) in spike (S) gene, which together can distinguish feline infectious peritonitis virus (FIPV) from feline enteric coronavirus (FECV) in >95% of serotype I FCoV-infected cases in freshly-collected specimens, have suggested a potential diagnostic value. The aim of this study was to compare the uses of a consensus nested RT-PCR (nRT-PCR) targeting 3’UTR and a nRT-PCR targeting the two mutations in S gene in aiding the diagnosis of FIP in FFPE tissues. After evaluation of the RNA quality in FFPE tissues by a RT-PCR targeting the housekeeping gene of feline GAPDH, a total of 38 histopathologically and immunohistochemically confirmed FIP cases and 22 non-FIP cases were used as the source of RNA and examined nRT-PCRs. We have successfully extracted RNA and amplified FCoV genes in 31/38 (82%) FIP cases using consensus nRT-PCR, whereas 17/38 (42%) FIP cases were detected using the S-specific nRT-PCR. Following subsequent sequencing, 16 out of 17 serotype 1 cases had one of the two mutations (M1058L and S1060A) in the S gene. None of the FFPF tissues from these non-FIP cats were positive by both methods. We have demonstrated that in combined with histopathology and IHC staining, both consensus nRT-PCR and S-specific nRT-PCR were capable of detecting viral RNA from FFPE samples where IHC signals were equivocal and possibly misinterpreted as negativity. Both methods serve as a useful tool in supporting FIP diagnosis and for the retrospective study of FIP in archival FFPE tissues.
32
Govender, Kerushini. "Preliminary validation of Mycobacterium tuberculosis complex-specific PCR tests for the detection of M. bovis and M. tuberculosis in formalin-fixed, paraffin-embedded tissues of captive and free-ranging wildlife." Diss., 2013. http://hdl.handle.net/2263/37366.
Full textAbstract:
Bovine tuberculosis is a global cause for concern in livestock, free-ranging wildlife,
zoological collections and the human population. Large amount of time, effort and
resources are spent on its diagnosis and control methods. This study was aimed at
determining the sensitivity and specificity of the IS6110 specific PCR test on formalin
fixed, paraffin embedded (FFPE) tissue blocks, compared to that of the gold
standard method culture and to differentiate M. bovis from other members of the M.
tuberculosis complex using the RD4 region of difference specific PCR test. A total of
141 FFPE tissue blocks of wild animals from game reserves, the National Zoological
Gardens and routine tuberculosis (TB) surveys in Kruger National Park were tested.
Among the 50 known TB positive samples (35 M. bovis culture positive, twelve M.
tuberculosis culture positive and three diagnosed tuberculosis positive on
histopathology examination) the IS6110 PCR had an overall sensitivity of 22%. The
positive predictive value of the IS6110 test (91.67%) was quite high implying that
although sensitivity was low, one can be highly confident that a positive test result is
a true reflection of the positive disease status. The overall sensitivity of the RD4 PCR
was 20%. The positive predictive value of the RD4 test (41.67%) was low, implying
that a positive test result may be unreliable. The sensitivities of the M. tuberculosis
and M. bovis culture positive samples were compared and a significant difference
was noted. Sensitivities of the IS6110 and RD4 assays in M. tuberculosis culture
positive samples were 66.67% and 33.33%, respectively; sensitivities of the IS6110
and RD4 assays in M. bovis culture positive samples were 8.57% and 17.14%,
respectively. Difference in bacterial load in tissues infected with the two
mycobacterial species may account for this finding (i.e. M. bovis infections have a
lower bacteria load). Of the 91 known TB negative samples, the specificity of the
IS6110 (98.90%) and RD4 (84.62%) PCR tests were high, but the negative
predictive values of 69.67% and 65.81%, respectively, suggest that the probability of
negative test results being incorrect still exists. The resultant sensitivity was
increased when parallel interpretation was applied to histopathology examination
and the IS6110 or RD4 PCR tests and when applied to the IS6110 and RD4 PCR
tests. Both histopathology examination and PCR tests produce rapid results and
their combination can be used in routine diagnostics. The RD4 PCR assay was
unable to distinguish M. bovis from other members of the MTB complex and based
on the findings of this study the RD4 PCR cannot add value to the diagnosis of
suspect tuberculosis samples at this stage, but successful troubleshooting relating to
1) extraction method, 2) DNA inhibitors, 3) contamination and 4) multisampling protocol, may enable its use in future.Dissertation (MSc)--University of Pretoria, 2013.
gm2014
Veterinary Tropical Diseases
Unrestricted
33
Votavová, Hana. "Odlišení primárně mediastinálního a difuzního velkobuněčného B-lymfomu s využitím metody real-time kvantitativní polymerázové řetězové reakce." Doctoral thesis, 2011. http://www.nusl.cz/ntk/nusl-299441.
Full textAbstract:
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. It is a molecular and prognostic heterogeneous disease. Three main genetic subtypes are called germinal center-like DLBCL (GC-like DLBCL), non-germinal center-like DLBCL (nonGC-like DLBCL) and primary mediastinal B-cell lymphoma (PMBL). These subtypes can be reliably distinguished only with usage of gene expression profiling (GEP). The GEP method can be applied only when fresh frozen tissue is available. The method is technically difficult and expensive. Thus, it is not used routinely. Since the DLBCL subtypes differ in prognosis, it is extremely important to be able to distinguish them. The presented thesis is focused on distinguishing of PMBL diagnosis in the group of DLBCL. Easily stored formalin-fixed, paraffin-embedded tissue (FFPE) and gene expression analysis using real-time quantitative polymerase chain reaction (RTqPCR) are used. In the first step, PMBL and DLBCL cases were distinguished with an internationally accepted clinical-pathological method. The agreement between clinical-pathological diagnosis and GEP is only 76%. In the presented text a genetic algorithm for PMBL/DLBCL distinguishing is suggested. It uses three carefully chosen genes and their expression is measured with RTqPCR. Both, the...
34
Bee-Piao and 黃碧標. "Proteins Extracted from Formalin-Fixed and Paraffin-Embedded Samples for Biomarkers Mining." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/86200918270217423126.
Full textAbstract:
碩士中山醫學大學
生化暨生物科技研究所
97
Formalin-fixed and paraffin-embedded (FFPE) samples are widely used in pathological examination for carcinoma and various diseases, but the FFPE sections are rare and difficult to be further investigated. The present study aimed to establish an extraction approach to obtain proteins from FFPE samples, which could be useful in mining potential protein markers. A satisfactory protocol of the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival FFPE tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE pig liver tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human。Carcinoma FFPE samples, confirmed by two pathological doctors and then treated with different methods to extract proteins. To evaluate the effects of each extraction approach on FFPE sample, the treated FFPE slices were further dehydrated with ethanol, reacted with xylene, embedded with paraffin and sectioned. The efficiency of each extraction approach was both evaluated by CBB-stained protein profile on the SDS-acrylamide gel and the H-E stained profile of re-fixed and embedded samples. The CBB-stained protein profiles revealed that DNase treatment following 2% SDS/Tris heating extraction was the most effective approach to extract proteins from FFPE samples in this study. These findings were consistent with the HE-stained profiles of treated FFPE samples. Moreover, the CBB-stained protein profiles presented seven protein bands with different levels among CHC and normal biliary duct. The proteins with different level might be potential markers to distinguish CHC from the normal biliary duct. The present study established a protein extraction approach combining DNase treatment and SDS/Tris heating extraction for FFPE samples. By using the approach, proteins extraction for FFPE samples should be more effectively, the findings which may be useful and helpful for mining potential carcinoma markers from FFPE sections.
35
Zhang, XIAO. "EVALUATION OF RNA QUALITY FROM FORMALIN FIXED AND PARAFFIN EMBEDDED SAMPLES:APPLICATIONS AND LIMITATIONS." Thesis, 2008. http://hdl.handle.net/1974/1740.
Full textAbstract:
RNA molecules isolated from FFPE samples are highly fragmented and modified, and generally deemed unsuitable for downstream gene expression profiling. With the development of molecular biology, there has been growing interest in profiling archival FFPE samples. Successful profiling of transcripts from FFPE samples would greatly expand tissue sources for large scale gene expression studies; also it would pave the way for future applications on the type of tissue readily available in the clinical setting. So far, there is a lack of systemic studies evaluating the quality of RNA isolated from routinely processed FFPE samples, and it has remained difficult to assess how well FFPE-derived RNA mirrors the status of RNA isolated before fixation. In this project, the similarity of miRNA and mRNA profiles between matched frozen and FFPE lymphoid hyperplasia tissues (N=7 for miRNA comparison, N=4 for mRNA comparison) were evaluated. We found consistently good correlation (mean of Pearson coefficient=0.939, mean of Spearman coefficient=0.905, mean of Kendall tau=0.744) between matched frozen and FFPE-derived miRNA profiles, suggesting FFPE samples may retain miRNA expression information quite well. This has major positive implications for research using FFPE samples, as miRNA profiling becomes more prominent in bioprofiling studies. On the contrary, mRNA isolated from FFPE samples showed less correlation (Spearman coefficient less than 0.75) with its frozen counterpart on the Agilent microarray platform. With a post extraction heat treatment aimed at reversing base modifications and cross linking structures, obvious global mRNA quality improvement was observed in cases where samples appeared to be heavily cross linked, but was less effective and even detrimental in cases where cross linking was less prominent. This research suggests that the extent of cross linking may be critical in terms of determining whether a particular FFPE tissue will become a useful source of mRNA for global profiling studiesThesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-26 10:49:50.044
36
Nourizadeh, Alireza. "APC, BRAF and KRAS mutations, and MLH1, MGMT and CDKN2A expression analysis in Nepalese colorectal cancer patients. : -." Thesis, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-15719.
Full textAbstract:
Colorectal cancer (CRC) is a common malignancy which develops due to old age and lifestyle factors, low percent of patients afflicted by a genetic disorders. Half of all colorectal cancer patients are diagnosed after metastasis. The high rate of the late detection, emphasizes on the requirement of convenient and inexpensive diagnostic methods for comprehensive screening programs. The aim of this study was to discover proto-oncogenes mutation and assessment of tumor suppressor genes expression. Formalin fixed paraffin embedded (FFPE) histologically verified colorectal cancer samples were used. APC, KRAS and BRAF mutations were investigated using polymerase chain reaction (PCR) fragments and direct sequencing. Gene expression assessment of MLH1, MGMT and CDKN2A were achieved via quantitative polymerase chain reaction (qPCR). In the present study we could detect a novel transversion heterozygous mutation in APC gene codon 1365 in three patients. BRAF codon 600 mutation were detected in one patient. KRAS codon 12 mutation was discovered in one sample and also a novel transition mutation in codon 15 was detected in 6 patients. In 80% of cases, MLH1 and MGMT expression were undetectable, in remaining 20%, MLH1 expression were reduced, but MGMT showed both reduced and increased expression compared to control. In 100% of patients CDKN2A expression was undetectable. The rate of mutations in predetermined hotspot codons and amount of uncommon mutations into APC, BRAF and KRAS in Nepalese patients indicates the requirement of further investigation in CRC patients from that part of the world. Also, the expression rate of MLH1, MGMT, CDKN2A and deficiency of an information source emphasizes the necessity of whole genome CRC expression profiling data to comparison and conclusion.-
-