Dissertations / Theses on the topic 'Formalin-fixed, paraffin-embedded tissues- FFPE'

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.

Philosophiae Doctor - PhD
Tumour-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

no
The 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

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

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.

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. Copyright
Dissertation (MSc)--University of Pretoria, 2012.
Veterinary Tropical Diseases
unrestricted

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.

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 studies
Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-26 10:49:50.044

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...

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.

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.

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.

Master of Science
Department 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.

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

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.

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