Masters Degrees (Haematology and Cell Biology)

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Open Access
Quantification and characterisation of circulating extracellular vesicles in cervical cancer patients before, during and after treatment
(University of the Free State, 2023) Gasa, Noluthando Ncebakazi; Coetzee, M. J. C.
All cells actively release extracellular vesicles (EVs) upon activation and apoptosis. EVs mediate intercellular communication in normal physiology and pathology. Cancer cells, like normal cells, secrete cancer specific EVs into the blood stream. EVs can possibly be used to monitor disease progression and response to treatment. This research quantified and characterised circulating EVs in the plasma of ten patients with stage II cervical cancer before, during and up to six weeks after treatment. EVs were isolated from plasma by size exclusion chromatography. Flow cytometry was used to count and characterise the EVs. The total number of EVs was identified by using an anti-CD63 monoclonal antibody. Cancer derived EVs were identified using an anti-CD133, while anti-CD11b was used to identify platelet derived EVs, and anti-CD41 to identify neutrophil EVs. After the start of radiotherapy (week 1) the number of CD63+ EVs, increased as large numbers of cancer cells were killed. The number of CD63 positive events significantly decreased in week six compared to baseline. There was also a significant decrease in CD133 positive and CD41 positive events in week six compared to baseline. This study demonstrated a significant increase at the start of treatment, followed by a decrease in circulating EVs after treatment compared to baseline. These findings suggest that EVs can possibly be used to monitor a patient’s response to treatment.
Open Access
Characterisation of apparent mismatches detected during routine short tandem repeat analysis in parentage investigations
(University of the Free State, 2023) Soldati, Afika; de Kock, André; Kloppers, Jean F.
𝗕𝗮𝗰𝗸𝗴𝗿𝗼𝘂𝗻𝗱: Short Tandem Repeat (STR) analysis has proven effective for establishing parentage and biological relatedness. There are commercially available STR kits that allow for reliable PCR amplification and genotyping of STR loci. However, one or two STR loci mismatches may be identified in non-exclusion cases. In routine analysis, these discrepancies are classified as apparent STR loci mismatches. The mismatches result from various mutational mechanisms. However, the mechanisms that drive these mutations are poorly understood. Several STR loci mismatches have previously been reported to impact parentage analysis. The alleles involved in the mismatch affect the interpretation of genetic profiles and can sometimes lead to false parentage exclusions. As such, it is essential to identify and characterise the underlying cause of STR loci mismatches for further validation of the genotypic data produced within a specific DNA profiling laboratory. 𝗠𝗲𝘁𝗵𝗼𝗱𝘀: A laboratory-based descriptive-comparative study was conducted. This study consisted of 100 parentage cases with one or two STR loci mismatches from the DNA testing facility, Universitas Academic Unit, National Health Laboratory Services (NHLS) Bloemfontein from 1 January 2021 to 31 March 2022. The following 15 STR autosomal loci were included in the analysis: CSF1PO, FGA, vWA, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D10S1248, D13S317, D16S539, D18S51, D19S433, and D21S11. Both published and designed study primers were used to optimise the PCR assay conditions for the amplification of the selected STR loci using commercially available control DNA. The optimised PCR assay conditions were used to screen the samples across the 15 STR loci. Sanger sequencing and sequence analysis was conducted for each parentage case to identify and characterise the underlying cause of the observed apparent STR mismatches. Furthermore, the sequence-based alleles were evaluated for concordance with genotypes determined by Capillary Electrophoresis-based (CE-based) STR typing previously reported by the facility. 𝗥𝗲𝘀𝘂𝗹𝘁𝘀: An average concordance of 82% was observed between STR profiling and Sanger sequencing across the 15 STR loci studied. In 11 of the loci, a 100% concordance was obtained. In contrast, no concordance was observed for the D19S433 locus. The stepwise mutations observed at the various loci were 70% more frequent than other mutation models; these were attributed to DNA polymerase slippage. In comparison, 30% of the mutations were as a result of allelic dropouts, accounted for by primer-binding site sequence variants. It was observed that there were more mutations originating from paternal (n=76) rather than maternal (n=26) lineages. 𝗖𝗼𝗻𝗰𝗹𝘂𝘀𝗶𝗼𝗻: The observation of one or two STR loci mismatches in parentage analysis should not be overlooked; all the studied allelic mismatches between the parent and child were characterised successfully. The findings revealed that most of the apparent mismatches occurred due to DNA polymerase slippage. The results of this study provide evidence that sequencing of the core STR repeat and the flanking regions can provide valuable information to characterise STR loci mutational events when inconclusive parentage or kinship results are obtained. Because of the limited sample size, the findings of this study provide evidence that STR mutations are more prevalent in males than females. Furthermore, this study demonstrates the need for DNA testing facilities to have a method in place to characterise and confirm inconclusive genotypic data obtained using the available commercial STR kits.
Open Access
Validation of the Cerebrospinal Fluid (CSF) Module of the Siemens ADVIA® 2120i for Automated Cell Counts of Cerebrospinal Fluid
(University of the Free State, 2019-12) Kalambi-Matengu, Esther; Haupt, Leriska; Coovadia, Yacoob
Introduction: The majority of haematology analysers routinely utilised for whole blood specimens are now equipped with cerebrospinal fluid (CSF) and/or body fluid modules. These automated cell counters are steadily replacing manual microscopy for CSF cell counts. This switch is attributed to the automated analysers’ higher throughput, improved turnaround times, superior precision and higher accuracy compared to manual microscopy. Methods: 46 routine CSF samples were analysed using the ADVIA2120i CSF Module to acquire a red cell count (RCC), white cell count (WCC) and a four-part WCC differential. Results were compared against the mean of two CSF cell counts performed by manual microscopy using the Improved Neubauer chamber haemocytometer. Quantitative method comparison was performed using EP Evaluator® software version 8.0.0 and Microsoft Excel®. The ADVIA2120i was, furthermore, assessed for precision, linearity and carryover. Results: The ADVIA 2120i meets all acceptable claims in terms of precision, and demonstrated acceptable accuracy for RCC, mononuclear and polymorphonuclear cells, compared with the reference method. Linearity results showed that the ADVIA2120i produced results that were proportional after serial dilutions. All parameters were deemed to have acceptable carryover. Conclusions: The ADVIA2120i offers rapid, precise and accurate RCC and WCC for both normal and abnormal cell counts. Our results show that the ADVIA2120i is a suitable alternative to manual microscopy for CSF cell counts and is deemed fit for purpose.
Open Access
Evaluation of the eosin-5-maleimide flow cytometric test and other screening tests in the diagnosis of hereditary spherocytosis
(University of the Free State, 2020-06) Mothi, Hemasha; Van Marle, A.; Roodt, J. P.
Background: Hereditary spherocytosis (HS) is a genetically determined haemolytic anaemia characterised by the spherical shape of affected red blood cells. With limited confirmatory tests currently available in South Africa, the diagnosis of HS is reliant on the clinical presentation and screening tests. Objectives: The aim of this study was to compare the sensitivity and specificity of three screening tests: flow osmotic fragility test (FOFT), cryohaemolysis test (CHT) and the eosin-5-maleimide binding test (EMA-binding test) in diagnosing hereditary spherocytosis (HS). Method: All three tests were performed on 18 subjects with confirmed HS. The negative control group was comprised of 10 subjects with haemolysis and spherocytosis, and either a positive direct antiglobulin test (DAT) or normal red cell membrane studies. The tests were also performed on samples submitted for cases of suspected HS during the study period. Results: The EMA-binding test demonstrated superior sensitivity (88.9%) compared to the CHT (61.1%) and the FOFT (38.8%). The EMA-binding test specificity (90.0%) was equal to that of the FOFT and superior to the CHT (50%). Combined sensitivities and specificities for EMA-binding test and CHT, EMAbinding test and FOFT, and CHT and FOFT, were 100% and 33.3%, 94.4% and 80.0% and 88.9% and 50.0%, respectively. Conclusion: EMA-binding test is the best screening test for cases of suspected HS. If there is a high clinical index of suspicion with a negative EMA-binding test, the CHT is recommended as a second screening test.
Open Access
Comparison of ADAMTS13 and Von Willebrand factor antigen levels and activities and plasminogen levels, in the currently available plasma products for the treatment of thrombotic thrombocytopenic purpura in South Africa
(University of the Free State, 2018) Van Marle, Anneke; Joubert, Jaco
Thrombotic thrombocytopenic purpura results from a deficiency in the Von Willebrand factor cleaving protease, ADAMTS13. Treatment involves plasma exchange therapy with either fresh frozen plasma, cryosupernatant, or solvent/detergent-treated plasma (available locally as Bioplasma FDP®). The research aimed to generate in vitro data on these products, and to explore possible differences between them that may offer treatment advantages. Twenty samples per product were analysed for levels and activities of ADAMTS13 and Von Willebrand factor, and plasminogen levels (a proposed physiological backup system for ADAMTS13). Fresh frozen plasma and cryosupernatant samples were subanalysed according to ABO blood group. All samples had normal/high ADAMTS13 activity and plasminogen levels. Von Willebrand factor content was mostly normal for Bioplasma FDP®, typically deficient for cryosupernatant and mostly (unexpectedly) deficient for fresh frozen plasma. Depending on the parameter, Bioplasma FDP® was the most standardised (CV: 14.1% to 27.3%), whilst fresh frozen plasma showed great inter-individual variation (CV: 24.6% to 208.6%). Statistically significant differences were found across products (p values ≤ 0.0095) and ABO blood groups (p value: 0.0001). In conclusion, all three products can be used for treatment of thrombotic thrombocytopenic purpura. Product choice depends on the need for additional viral safety, costs, product availability and the perceived impact of within-product variations.
Open Access
Presence of glyphosate in food products in South Africa of which maize or soybean is the primary constituent
(University of the Free State, 2017-02) Koortzen, B. J.; Viljoen, C. D.; Sreenivasan, S.
English: South Africa is considered a major GM crop producing country. The predominant GM trait in maize and soybean in South Africa is herbicide tolerance. Herbicide tolerant (HT) crops allow for the application of herbicide during the growing season to selectively kill weeds without damaging the crop. Glyphosate is the most widely used herbicide on HT crops in South Africa and the world. Glyphosate is absorbed by HT crops after application. Studies have detected levels of up to 2.2 mg/kg in HT maize and 26 mg/kg in HT soybean. Glyphosate is not removed from grain by washing, cooking or processing. As a result of this, glyphosate can also be detected in processed food products. It has also been found that glyphosate can be detected in animal tissue and urine after exposure to the herbicide through feed. Similarly, glyphosate has also been detected in the urine of humans, either as a result of occupational exposure, through diet and/or water. Pure glyphosate is considered safe by regulatory authorities and international bodies. However, recent studies have found that glyphosate in formulation at low concentrations results in endocrine disruption as well as DNA and chromosomal damage. As a result of this, the IARC re-classified glyphosate as a “probable human carcinogen”. Glyphosate is used on HT maize, a major staple food, and HT soybean, an important source of protein, in South Africa. Thus, the aim of this study was to determine whether glyphosate is present in commercially available South African food products containing maize and/or soybean as a primary constituent. The majority of food products tested in this study contained glyphosate. The level of glyphosate ranged from 0.027 mg/kg to 2.257 mg/kg that is below the MRL and ADI established in South Africa. However, recent studies have shown that glyphosate in formulation is genotoxic at the levels found in maize and soybean containing foods in South Africa. The results from this study found that the level of glyphosate in food products in South Africa is comparable to the limited number of studies from the UK and USA. This study is unique to other published studies since it focused on food products likely to be consumed daily. This study has confirmed that South Africans are exposed daily to low levels of glyphosate through food products. The GM HT events in the food products were quantified in order to explain the variation in the levels of glyphosate. It was determined that 57 products contained one or more GM HT event in a range of 0.25% to 100%. However, there was no correlation between the level of glyphosate and percentage GM HT event in the products, even when GM HT negative samples were excluded from the analysis. This suggests that either glyphosate is not applied to some GM HT crops, when weed control is not required, or that the herbicide is applied to non-GM crops as a desiccant prior to harvesting. Most of the food products used in this study were labelled in terms of GM content. The percentage GM HT event(s) in the food products was used to determine compliance to mandatory GM labelling in South Africa since the data was available. Results indicated that the majority of companies in South Africa are compliant with the Consumer Protection Act (2008) concerning GM labelling. However, most of the products labelled “GMO free”, did not comply with the expectation of discerning consumers. To conclude, this is the first study to investigate the extent of glyphosate in South African food products. This study has confirmed that South African consumers are exposed to low levels of glyphosate as a result of consuming maize and soybean food products. The level of glyphosate detected in the food products although considered low, is at a concentration reported to cause genotoxic effects at cellular level by in vitro studies. It is currently not known what the safety implications are of chronic exposure to glyphosate, through the consumption of a staple like maize meal and soybean in South Africa. The question of whether glyphosate in food is safe in the long term needs to be addressed through future research.
Open Access
Molecular screening of Coloured South African breast cancer patients for the presence of BRCA mutations using high resolution melting analysis
(University of the Free State, 2016-08) Oosthuizen, Jaco; Van der Merwe, N. C.; Foulkes, W. D.
English: The populations of South Africa (SA) exhibit a rainbow of genetic diversity due to the high contribution of ancestral genetic admixture. The economic structure of this third world country has limited the exploration of this genetic diversity with regards to familial breast cancer (BC) testing. As the SA Coloured woman has a lifetime risk of 1 in 22 to develop BC, the main aim of the study involved targeting the highly penetrant genes BRCA1 and BRCA2 for comprehensive mutation analysis. This was done in order to determine the range of variants and mutations present within BC patients representing this group. In order to perform such a comprehensive screen, High Resolution Melting Analysis (HRMA) was optimised and validated for use in conjunction with the protein truncation test (PTT), genotyping assays using real-time based PCR, single stranded conformational analysis (SSCP) and DNA Sanger sequencing to determine the presence of potential disease-causing mutations. A total of 229 Coloured BC patients were included based on a specific selection criteria. This criteria included being affected with BC and either having a positive family history of the disease, or an early age at onset (<45 years) or bilateral disease. All male BC patients were included. Twelve different pathogenic or class 5 mutations were detected for a total of 33 patients. These mutations were identified using genotyping analysis, PTT and HRMA. These mutations were confirmed using Sanger sequencing. These mutations included all three the Afrikaner founder mutations, together with the Xhosa/Coloured mutation detected for the Xhosa and Coloured population residing in the Western Cape. A total of 50 variants were identified using HRMA, ranging from single base changes to a 12bp deletion occurring within the coding region of BRCA2. The clinical significance of these variants were classified using computer-based analysis. Variants of unknown significance (VUS) were investigated using a multiple evidence-based approach in order to confirm their clinical status. These included using the BIC, ClinVar, the ENIGMA guidelines, and the 1000 Genomes project database. This was done in order to investigate whether the variant was novel or allocated to a specific population cluster. The majority of the variants was class 1 polymorphisms, exhibiting normal variation. The portfolio of variants reflected 300 years of admixture between the Bantu-speaking Black African populations of the North Western Cape province, the European settlers and the slaves from the East as global, Eastern and African polymorphisms were observed. Numerous new pathogenic mutations were identified, ranging from likely pathogenic (class 4) to class 5. Many of these mutations proved to be restricted to the southern tip of SA. Based on these results, recommendations can be made regarding the composition of targeted mutation panels for the diagnostic testing of SAC BC patients and their families.
Open Access
Investigating the genetic profile of the E-cadherin gene in squamous carcinoma of the esophagus
(University of the Free State, 2003-06) Mabina, Boitumelo Desiree; Pretorius, G. H. J.; Van der Merwe, N. C.
Open Access
Co-expression and functional assay of human Rb and E2F1 proteins in yeast
(University of the Free State, 1999-09) Wollenschlaeger, Alex; Pretorius, G. H. J
The mammalian cell cycle is composed of a myriad interactions occurring in a defined sequence dictated by the flow of entropy. The decision to study the cell cycle requires entry into a world where events take place, not because they want to, but because they have to. Cells do not 'decide' to perform certain actions, but are driven by the laws of nature, the same laws that are responsible for the existence of the universe. Study of this maelstrom of reactions is truly analogous to opening Pandora's proverbial box. A peek inside and all the inner workings of the cell start to spill out. Unfortunately, this is where things start to get complicated. Serendipitously, simpler alternatives are available. The yeast cell cycle is remarkably similar to that of higher animals. The Rb and E2F 1 proteins are integral components of the mammalian cell cycle, and as such, they are found in aberrant forms in numerous malignancies. This necessitates an adequate means for the determination of the cellular status of these proteins, as prognosis and diagnosis of several neoplastic disorders are dependentthereon. This study aimed to develop a yeast-based strategy for functional analysis of the human tumour suppressor protein Rb. This goal was impeded by one factor; the yeast cell cycle is too similar to that of humans. Several yeast strains, derived from W303-lA, were constructed that each contain a reporter gene -the lacZ gene of E. coli- regulated by E2F recognition elements introduced within the upstream CyC1 promoter. The theory was that in the absence of Rb, ectopically expressed human E2F 1 protein would be able to bind the RE and activate transcription of the reporter gene, ultimately resulting in a readily observable product. In the presence of functional Rb, E2Fl would be bound by the tumour suppressor protein, and thus be incapable of activating the reporter gene. This would provide an assay of Rb status, based not on tedious sequencing analysis of the genetic material, but on the actual functional activity of the protein. When dealing with Mother Nature, though, we are oftentimes reminded that she has thought of everything. S. cerevisiae contains an E2F-like activity, capable of binding the exact RE introduced into the reporter gene promoter. This was confirmed by experiments in this study, where the reporter gene was activated in the absence of ectopically expressed E2F1 protein. The endogenous yeast E2F-like protein is thus able to activate transcription of the reporter gene, negating the effect that would be observed by ectopic expression of human E2F 1, and thus, all Rb-expression was performed in the absence of eo-expressed human E2F 1. Since it is impossible to distinguish between the yeast and human E2F activities, it is impossible to create a functional assay for Rb activity in the W303-1Aderived chimeras constructed. As mentioned previously, it could be possible to overcome this problem by knocking out the yeast-borne E2F activity, but this approach is restricted by two barriers. Firstly, the yeast equivalent of the E2F-family is yet to be cloned. This problem can be approached with a transposon-based strategy. The endogenous E2F activity is capable of activating transcription of the lacZ reporter gene, and in so doing, provides a convenient assay for YE2F integrity. Through the use of a plasmid containing an inducible transposase it should be possible to disrupt the YE2F -encoding gene through integration of a transposon. This would be accompanied by an inability of the yeast to activate the reporter gene. The transposon, containing flanking genomic DNA, could then be retrieved and provide the basis for cloning the gene coding for YE2F. Since the reporter gene is specific for E2F-like binding, retrieval of non-specific factors should be negated. A second problem is that, since the E2F proteins play an important role in the progression of the cell cycle in higher animals, it is possible that the yeast would not survive knocking out its homologue. Relegation of YE2F to the role of bystander could possibly wreak havoc with the delicate mechanism that is the cell cycle. Still, it would be interesting to further pursue this idea. Ectopic expression of human Rb in the various strains used in this study provided some interesting results. Transformation of pAWl, followed by galactose-based induction of Rb expression resulted in observable differences in growth characteristics of certain strains. Those most affected were W-lf and W-1r, which each contain a single repeat of the E2F RE within the upstream promoter of the reporter gene, albeit in forward and reverse orientations, respectively. The effect was particularly evident in W-1r, where expression of Rb resulted in complete cessation of growth, probably due to binding of yeast E2F-like activity. These results could not, however, be reconciled with those obtained from cell cycle analysis with the aid of flow cytometry. These experiments did not show any significant effect of Rb expression on the cell cycle of the examined strains. This is possibly due to an insufficient period of observation, but this could, unfortunately, neither be confirmed nor dismissed. From the results obtained in this study it appears that construction of an apposite reporter system for the functional assay of Rb is perhaps more tricky than would be expected. The interference of endogenous proteins is a cause for concern in a development strategy such as this, and serves as a caveat for future studies in this field.
Open Access
A yeast-based assay for detection of mutations in the human p16 gene
(University of the Free State, 1999-05) Botha, Chantal; Pretorius, G. H. J.
Cyclin-dependent kinases (CDKs) are crucial regulators of the cell cyele. CDKs themselves are subject to control by both cyelins and CDK inhibitors. Among the inhibitors, p 16 is very prominent, since it has been found to be mutated or lost in a variety óf tumours, We are interested in mutations involved in the progression of leukemia from the chronic to the acute phase. The p 16 gene has been implicated in this progression, therefore we needed an assay for p 16 status that could be applied to screen patients in chronic phase regularly. Traditional mutation screening makes use of physical methods such as Single Stranded Conformational Polymorphism (SSCP) analysis. These methods are generally labour intensive and are not always informative. If tests for the actual function for the gene products could be devised, it could be used to screen tumour samples for the status of these genes. We have decided to develop a yeast-based test that would directly assay for activity rather than just nucleotide changes. The assay is based on the yeast two-hybrid system, where protein-protein contact is reflected in colony colour. We have designed a primer set to amplify the p16 reading frame by RT-PCR from small amounts of leukocyte mRNA. This cDNA is then cotransformed with a gapped plasmid containing terminal p 16 overlaps, allowing homologous recombination to splice the reading frame into the plasmid. The host strain also contains a CDK4- expressing plasmid and if the amplified p16 can still bind to CDK4, the colonies would turn blue. We have successfully constructed and tested the system and found it to be very sensitive, being able to assay p 16 from as little as 300 microliters of whole blood.
Open Access
Selection and characterization of a novel factor XI inhibiting peptide by using phage display technology
(University of the Free State, 2002-11) Motloi, Nthabiseng Cecilia; Meiring, S. M.
English: The role of factor XI in hemostasis can be seen as a combination of a procoagulant action (the formation of fibrin) and an antifibrinolytic action (the protection of fibrin). High levels of factor XI lead to a prolonged down regulation of fibrinolysis and therefore a risk of thrombosis (Meijers et ai, 2000). Under disease conditions associated with Disseminated Intravascular Coagulation (DIC), the continuous exposure to excess TF typically exhaust the available tissue factor pathway inhibitor (TFPI), leading to rampant thrombin generation by factor XI feedback and therefore also a risk of thrombosis (0sterud and Bjerlid, 2001). I selected possible .inhibitors of factor XI using phage display technology. I started the phage display selection by biopanning in immuno-tubes and eluted the factor XI binding phages non-specifically from the immuno-tube. I did four selection rounds, to enrich the factor XI binding phages. I found only two strong factor XI binding phage clones from a linear 12-mer phage library. Both phage clones bound dose dependently and with a high affinity to factor XI. Both clones also lengthened the partial thromboplastin time (aPTT) dose dependently. The amino acid sequences of the peptides displayed on these two clones indicate that both peptides contain three amino acid sequences of HMWK and thrombin. One clone also contains a three amino acid sequence of factor XII. None of them contains a three amino acid sequence of factor IX. I synthesize a linear peptide with the corresponding sequence as the peptide displayed on the clone that was prevented from binding to factor XI by both factor IX and thrombin. I characterized the peptide by studying its effect on the aPTT. This peptide lengthens the aPTT dose dependently. The lengthening in aPTT of our peptide however indicates that I have selected an inhibitor of the contact system factors of coagulation. In summary, this study shows that the phage display can be used to select novel factor XI inhibitors from random peptide libraries. With further studies, this peptide may be developed as an antithrombotic.
Open Access
DNA characterization of the FGA locus in the human genome
(University of the Free State, 2002-11) Asfaw, Estifanos Kebede; De Kock, André; Pretorious, G. H. J.
English: The human alpha fibrinogen (FGA) short tandem repeat locus is found in the long arm of chromosome 4. It is located in the third intron of the alpha fibrinogen gene. This complex highly polymorphic tetranucleotide repeat locus together with other STR DNA markers is extensively used in personal identification in medical and forensic sciences. STRs are also used to study genetic variation in distinct ethnic groups and in disease diagnosis. More than 80 alleles have been reported for this locus from various population frequency studies. A few sequence studies have also reported 11 sequence variants to date. The FGA locus was found to have high heterozygosity and power of discrimination. The aim of this study was to characterise the sequence of microvariant and off-ladder complete and microvariant alleles of the FGA locus that were observed during routine paternity analyses. The characterization of the sequence of as many as possible of alleles observed in our study population would also be attempted. A total of 62 DNA specimens were selected and sequence characterized either for one or both alleles. The DNA specimens were 52 from Negroid, 5 mixed ancestry, 4 Caucasian and 1 SAN origin. The PCR reaction was used to amplify the selected alleles. The band of interest was cut from the gel and purified in consecutive PCR and purification steps till separate single bands were obtained. The purified single bands were sequenced using a BigDye terminator ready reaction kit in both forward and reverse reactions separately. These products were precipitated with ethanol acetate and subjected to capillary electrophoresis on an ABIPrism 310 Genetic Analyser using POP6 polymer. The results were analysed using the "Sequence Analysis Software version 2.1". The data obtained were checked, printed and compared with STR analysis results and FGA sequence reports. From the selected 62 specimens a total of 76 complete and microvariant alleles, the size of which ranged between 16.1 (224bp) to 44.2 (337bp) were found. These represent 27 different alleles (13 complete and 14 interalleles). In this study 2 novel (previously undescribed) alleles (40.2 and 41.2) were found. Three sequence variants (26, 28 and 43.2) with two variants each were observed. Two alleles 43.2 and 44.2 that had reported sequence variants were found to have different sequence structures from the published sequences. Forty-nine of the 76 sequenced alleles were within ladder and the remaining 29 were off-ladder. Only 8.41% (4/49) of the within ladder alleles had been wrongly assigned allelic numbers with routine STR analysis. The difference between the routine assignment and the sequencing of these alleles was only 1 or 2 bp. In contrast, all of the 29 off-ladder alleles were wrongly assigned. In this instance the difference was 2 or more base pairs. Although this study was conducted on conveniently selected DNA samples, it had significant results. Three sequence variants, 2 newalleles and, 1 allele, which had been reported, but sequence had not been described was found. Additionally, two other alleles with reported sequences were found, but their sequence structure differed from the published sequences. The samples in this investigation were not representative of the population groups that are found in the Free State province and we suggest further population-based studies of STR loci that are commonly used in paternity and forensic investigation. The information obtained from such studies will disclose the frequency of sequence variant alleles.
Open Access
The evaluation of tirofiban hydrochloride in a high shear rate arterial thrombosis model in baboons
(University of the Free State, 2009-11) Janse van Rensburg, Walter James; Meiring, S. M.; Roodt, J. P.
English: Background: Acute coronary syndrome (ACS) is a major cause of mortality and morbidity world-wide, and is responsible for roughly 2.5 million hospital admissions world-wide annually. ACS is commonly associated with platelet thrombus formation on disrupted atherosclerotic plaques, therefore effective and safe anti-platelet drugs are needed to help treat and prevent ACS. The current most popular anti-platelet drugs are associated with increased bleeding risk and reduced efficacy, thus drugs with a wider therapeutic window (more efficacy with less bleeding) need to be developed. Tirofiban hydrochloride is a small, short half-life molecule that inhibits platelet aggregation by antagonising the glycoprotein IIb/IIIa receptor on platelets preventing fibrinogen and von Willebrand factor to cross-link platelets, thereby inhibiting the final pathway of platelet aggregation. Tirofiban hydrochloride was believed to be a very promising drug due to its short half-life, as an antidote strategy is not needed to reverse adverse bleeding events, but it soon fell out of favour when it was found not to be as effective as for example abciximab in preventing ischaemic events. This was possibly due to the recommended dose being suboptimal. Methods and Results: We studied the efficacy of tirofiban hydrochloride to inhibit platelet thrombus formation on an injured and partially occluded artery by evaluating the effect of escalating doses on cyclic flow reduction (CFR) formation in a high shear arterial thrombosis model in baboons, and also evaluated its safety in two different bleeding models. We then compared our results to results found in the same model using clopidogrel. A significant effect on the number of CFRs was only observed after injection of three times (30 μg/kg bolus plus 0.45 μg/kg/min infusion) the therapeutic dose tirofiban, but it was a weak inhibitor at this dose. Only after injection of nine times (90 μg/kg bolus plus 1.35 μg/kg/min infusion) the recommended therapeutic dose, a strong complete inhibition was observed. A further dose of 27 times (270 μg/kg bolus plus 4.05 μg/kg/min infusion) the recommended therapeutic dose was given to evaluate the effect of an overdose on the bleeding tendency. A significant prolongation in bleeding time (3.05 minutes to 11.90 minutes) was observed after injection of nine times the therapeutic dose, an average 2.7 ± 2.44 fold increase in blood loss was also observed at this dose. A maximum increase in blood loss of an average of 3.4 ± 1.77 fold was seen after injection of 27 times the therapeutic dose. The efficacy of tirofiban hydrochloride was comparable to that of clopidogrel found in earlier studies, but the blood loss was much less when compared to the average 4.3 ± 2.6 fold increase with clopidogrel at 2.5 mg/kg and 8.0 ± 5.0 fold increase at 5 mg/kg. Conclusion: Tirofiban hydrochloride is an effective anti-platelet drug, but only offers adequate protection against arterial thrombosis at a dose between three and nine times the recommended therapeutic dose. However, it still remains safer in terms of bleeding than the most common anti-platelet drugs used today. We recommend that further in vivo studies be done to determine the optimal dose for tirofiban hydrochloride treatment, and that new clinical trials be done with higher dose tirofiban hydrochloride.
Open Access
Molecular screening for the presence of large deletions or duplications in BRCA using Multiplex ligation-dependent probe amplification in South Africa
(University of the Free State, 2016-07) Moeti, Pakiso James; Van der Merwe, N. C.; Dajee, B. K.
English: Germline BRCA gene mutations are associated with hereditary breast and ovarian cancers (OVC). Identification of these mutations greatly improves the preventive strategies and management of patients affected with the disease. The large majority of alterations identified within BRCA1 and BRCA2 are point mutations and small insertions/deletions. However, an increasing number of large genomic rearrangements (LGRs) are internationally being reported. Their contribution to familial breast cancer risk varies for different populations, for in some countries it represents a founder mutation (such as the Netherlands), whereas in others this type of mutation is totally absent. The main objective was to optimize and validate this new technique for use within the diagnostic laboratory and to screen various South African (SA) population groups for the presence of these larger genomic rearrangements present within BRCA1 and BRCA2. A total of 129 patients, who tested negative for the presence of smaller pathogenic BRCA1 or BRCA2 mutations were included in the study. The patients represented the Black, Indian and Coloured populations of South Africa. The selection criteria included being affected with breast cancer, have a minimum of one other family member affected with the disease or an early age at onset (diagnosed before the age of 45). Genomic DNA was extracted from peripheral blood samples. Multiplex Ligation-dependent probe amplification (MLPA) was performed using the SALSA® MLPA® probemixes P002-C1, SALSA® MLPA® P002-D1 and SALSA® MLPA® P087-C1 for BRCA1 and SALSA® MLPA® probemixes P045-B3 and SALSA® MLPA® P077-A3 for BRCA2. The data obtained were analyzed by using the GeneMarker® software v 2.6.4. Screening for the presence of LGRs within BRCA1 and BRCA2, did not reveal any genomic rearrangements present within these genes. Although no patients were identified that carried this type of deletions or duplications, the use of the five and two for BRCA2, of which one represented the confirmation set) were successfully validated for use on the diagnostic platform. The data furthermore highlighted the dramatic effect that small deletions or duplications within these genes might have when situated within the critical ligation site of the specific probe set. The presence of these smaller mutations could result in false positive results. The results of this study serve as a warning to pathology laboratories within SA, as the most common Afrikaner founder mutation situated within BRCA2 exon 17 affects the ligation of the probe set for exon 17. The presence of this mutation resulted in a reduced signal for exon 17, therefore a false positive result. This places emphasis on the confirmation of all potential positive results by using an alternative method or different probemix in order to prevent reporting of a false positive result. The data gathered corresponded to that of previous SA studies and supported the tentative hypothesis that LGRs do not seem to play a significant role within the various SA populations. It does not contribute significantly to the familial BC risk within SA.
Open Access
Molecular screening of the South African Indian population for BRCA1 and BRCA2 using high resolution melting analysis
(University of the Free State, 2016-01) Combrink, H. M. V. E.; Van der Merwe, N. C.; Visser, B.
English: The lifetime risk for developing breast cancer within the Indian population of South Africa is one in 17. Disease causing mutations in BRCA1/2 increase the risk of developing this disease by up to 80%. The main objective of this study was to screen this unique population for mutations in BRCA1/2. This was achieved by optimising High Resolution Melting Analysis (HRMA) as the screening technique for the smaller exons while the Protein Truncation Test (PTT) was used to screen exon 11 for BRCA1/2 respectively. In order to optimise HRMA, a full BRCA1/2 screen was performed on 24 patients from four different South African ethnic groups using Single-Stranded Conformation Polymorphism/ Heteroduplex Analysis (SSCP/HA). These results were compared to a HRMA screen performed on the same patients. No differences were observed between the sensitivity of the three techniques and the turnaround time (TAT) was considerably less for HRMA. The entire cohort used in this study came from 50 unrelated South African Indian patients. A full BRCA1/2 screen was performed on these patients. A total of nine different pathogenic mutations were detected. Four of the disease causing mutations (BRCA1 c.1360_1361delAG, p.Ser454Terfs; c.3593T>A, p.Leu1198Ter and BRCA2 c.5279C>G, p.Ser1760Ter; 5563C>G, p.Ser1855Ter) were detected using PTT, whereas the other five mutations (BRCA1 185delAG, p.Leu22_Glu23LeuValfs; c.191G>A, p.Cys64Tyr; c.5365_5366delGCinsA, p.Ala1789_Ile1790LeuTrpfs and BRCA2 c.9435_9436delGT, Val3145_Phe3146=fs; c.8754+1G>A, IVS21+1G>A) were detected using HRMA. Three unrelated patients were carriers of the splice site mutation found within BRCA2 exon 21. The research that was conducted, contributed to the knowledge pool for predictive testing in the clinical setting of South Africa and gave insight into possible diagnostic tests that could be designed for this population.
Open Access
Screening black South African females with Type 2 Diabetes Mellitus for mutations in the peroxisome proliferator-activated receptor gamma gene
(University of the Free State, 2016-01) Nienaber, Elzette; Marx, G. M.; Goedhals, D.
English: Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic disorder which is caused by a combination of an inadequate response to insulin secretion and a resistance to insulin action. The peroxisome proliferator-activated receptor gamma (PPARG) gene has been established as one of the major genes to have an impact on the risk of T2DM. The Pro12Ala polymorphism is one of the most common mutations found within PPARG and has been described in many different populations. However, it has not yet been established whether the Pro12Ala variant has a significant association with T2DM in the black, female South African population. The aim of this study was to screen for novel T2DM genetic risk factors in the PPARG gene and to determine the presence of previously identified T2DM genetic risk factors in black South-African women with T2DM. Quantitative PCR was performed on 184 black female South African participants that consisted of 93 patients diagnosed with T2DM and 91 control participants. Quantitative PCR was used to screen for the presence of the Pro12Ala polymorphism in the PPARG gene. Next Generation Sequencing (NGS) was performed on eight patients and eight control samples which were individually matched according to age and body mass index (BMI). NGS was used to identify novel polymorphisms which might be associated with T2DM and to detect the prevalence of previously described variants within the PPARG gene. The qPCR genotyping results showed that of the 184 participants, 183 had the Pro/Pro genotype and only one had the heterozygous Pro/Ala genotype. The Ala/Ala genotype was not detected in this study population. Although the study sample is only a small representation of the total population, it can be derived from the results that it is likely that the Ala/Ala genotype is rare in the population. Additionally, NGS results identified two variants within three individuals of the selected sample. The one variant (rs41516544) did not show any clinical relevance and is probably just a rare population variant. The other variant (rs3856806) is a well-described polymorphism and has been associated with having a protective effect against T2DM and was present in a control participant. This variant might be significant in its association to T2DM in the black South African population but will have to be further investigated in future studies.
Open Access
Screening for genetic variants implicated in monogenic forms of hypertension in a hypertensive urban black Free State cohort
(University of the Free State, 2016-02) Smith, Tanja; Viljoen, C. D.
English: Non-communicable diseases (NCDs), also known as chronic diseases of lifestyle, are the leading cause of death worldwide. Amongst the risk factors for NCDs, hypertension (blood pressure (BP) ≥140/90 mmHg) is one of the leading causes of death in South Africa. The prevalence of hypertension in an urban black population in Mangaung in the Free State is reported to be much higher than the average for South Africa. Monogenic forms of hypertension are a group of physiological disorders where the elevated BP is thought to be primarily due to a genetic component. Several genes that play a role in the sodium reabsorption pathway have been implicated in the syndromes associated with monogenic hypertension, including the chimeric CYP11B1/CYP11B2, NR3C1, HSD11B2, SCNN1B, SCNN1G, and WNK4. In a previous study involving the Mangaung population, it was found that BP correlated positively with adiposity, as well as with sodium intake. In addition, genetic analysis indicated that a genetic variant (A6986G in CYP3A5) implicated in primary hypertension, could be an independent risk factor for hypertension in 2% of the Mangaung population. It is not known, however, if genetic variants implicated in monogenic forms of hypertension could play a role in the Mangaung population. The aim of this study was to screen for genetic variants implicated in monogenic forms of hypertension in a black hypertensive cohort from Mangaung. In this study, a generic CTAB method was successfully used to extract DNA from blood spotted onto FTA® paper, which resulted in successful PCR amplification. Thereafter, a long range PCR assay was successfully optimized in order to amplify the chimeric CYP11B1/CYP11B2. Conventional PCR assays to amplify selected target regions in NR3C1 (exons 6, 7, 9, 10 and 11), HSD11B2 (exons 3, 4, and 5), SCNN1B (exon 13), SCNN1G (exon 13) and WNK4 (exons 7 and 17), were also successfully optimized. High resolution melting (HRM) analysis was optimized in an attempt to identify samples with potential sequence variants, thereby reducing the cost of sequencing. However, HRM analysis was only successful in identifying samples with sequence variants for NR3C1 exon 10, NR3C1 exon 11, and SCNN1B exon 13. As a result of the difficulty experienced identifying sequence variants using HRM analysis, it was decided to use DNA sequencing instead to screen for sequence variants in this study cohort. Long range PCR was used to screen for the presence of the chimeric CYP11B1/CYP11B2 in this hypertensive cohort. The long range PCR assay allowed the conclusive identification of the chimeric CYP11B1/CYP11B2 in at least one hypertensive individual and could potentially explain the elevated BP in this individual. However, multiple fragments were produced using the long range PCR assay. It is suspected that the high degree of similarity reported between CYP11B1 and CYP11B2 could have resulted in non-specific amplification. Using DNA sequencing, 53 sequence variants in genes implicated in monogenic forms of hypertension were identified in this hypertensive cohort. Of these, one variant (Asn767Asn) has previously been associated with glucocorticoid resistance and four variants (Arg563Gln, Thr594Met, Leu649Leu, and Ala547Ala) have previously been associated with elevated BP. Out of the 53 sequence variants identified in this study, 26 were novel. The number of sequence variants identified in genes implicated in monogenic forms of hypertension is surprisingly high. Several authors have suggested that monogenic forms of hypertension could be more common in the general population. To conclude, several sequence variants in genes implicated in monogenic forms of hypertension were identified in a hypertensive cohort from Mangaung. It was found that 84 out of 90 hypertensive individuals had one or more sequence variants in genes implicated in monogenic forms of hypertension. The data from this study suggests that monogenic forms of hypertension may play an important role in the development of hypertension in the Mangaung population.
Open Access
The application of high-resolution melting curve analysis for the detection of mutations in the MCR-ABL kinase domain of patients with chronic myeloid leukaemia
(University of the Free State, 2011-12) Wienand, Kirsty; Viljoen, C. D.; Louw, V. J.
CML is a haematological malignancy that is characterized by the BCR-ABL fusion oncogene that encodes a constitutively active tyrosine kinase. The treatment of choice for CML is a tyrosine kinase inhibitor and molecular monitoring of patients forms an integral part of disease management. When the expected response to tyrosine kinase inhibitor is not achieved within internationally accepted time frames, acquired resistance to tyrosine kinase inhibitors is suspected. Acquired resistance to tyrosine kinase inhibitors is primarily due to mutations in the BCR-ABL kinase domain. Types of mutations include single base mutations, insertions, deletions as well as duplications. Characterization of these mutations is important for treatment, since the type and position of the mutation may have an effect on how the patient responds to treatment. Although several methods have been described for detecting mutations, DNA sequencing is mostly used. Sequencing is currently the only technique that can simultaneously detect single base mutations, insertions and deletions in the BCR-ABL kinase domain. However, sequencing is costly as some patient samples do not have mutations and the lack of response to treatment is due to non-compliance. Thus, a screening method to exclude samples without mutations would make mutational analysis more cost-effective. High resolution melting (HRM) is a relatively new technique that is being used to screen for mutations, prior to sequencing. HRM has recently been used to screen the region of BCR-ABL encoding for the kinase domain for single base mutations. However, it was unknown whether HRM could be used to identify insertions, deletions or duplications in the kinase domain. This study has shown that HRM can be used to screen for mutations including insertions, deletions and duplications the region of BCR-ABL encoding for the kinase domain, prior to sequencing. HRM was performed on 40 patient samples, 10 of which had confirmed mutations in BCR-ABL in the region of the kinase domain. Of the 10 samples with mutations, three had single base mutations, one with a previously described insertion, seven had novel deletion variants. Furthermore, HRM detected a tandem duplication of the kinase domain in two patient samples that was not previously been possible with sequencing. There was 100% congruency between the detection of mutations using HRM and sequencing results, indicating similar sensitivity. HRM proved successful to indicate the presence of deletion variants. However, the deletion variants were detected in the HRM region preceding the area affected by the deletion. It was confirmed that the detection of the deletion variants was due to the PCR extension of HRM 1 amplicon into the HRM area of the deletion. It has been suggested that the insertion, deletions and duplications detected in this study may result in acquired resistance to tyrosine kinase inhibitor. In conclusion, this was the first study to use high-resolution melting to detect insertions, deletions and duplications in the region of BCR-ABL encoding for the kinase domain, indicating the suitability of the assay for screening for mutations prior to sequencing.
Open Access
Development and application of a real-time PCR method to detect selected single nucleotide polymorphisms associated with hypertension in a black South African population
(University of the Free State, 2014-08) Du Toit, Egardt; Viljoen, C. D.
English: Hypertension is one of the leading causes of death and disability in the world. In 95% of individuals with hypertension, the condition arises from the interaction of multiple environmental factors with physiological systems. Environmental factors that have been found to increase blood pressure include obesity, aging, high salt and alcohol consumption, low potassium and calcium intake, stress and insulin resistance. Physiological systems that regulate blood pressure include the autonomic nervous system, the renal system, hormonal system and the cardiovascular system. Various genes in these systems including the β1-adrenergic receptor (ADRB1), α-adducin (ADD1), angiotensinogen (AGT), aldosterone synthase (CYP11B2), CYP3A5 and G protein-coupled receptor kinase 4 (GRK4) have been implicated in hypertensive blood pressure due to the presence of single nucleotide polymorphisms (SNPs). The occurrence of such SNPs in blood pressure regulatory systems is thought to result in altered gene expression or protein function. In South Africa, the prevalence of hypertension has been determined to be approximately 39.9% in males and 34.9% in females. The Assuring Health for all in the Free State (AHA-FS) study determined that the prevalence of hypertension was approximately 48.3% in the Mangaung population. The AHA-FS study also found that 37.6% and 51.2% of individuals in the study cohort were overweight or obese, respectively, and that high body mass could be an important risk factor for hypertension. The aim of this study was to determine whether genes in the sympathetic nervous system, renal system and hormonal systems could contribute to the high prevalence of hypertension in the Mangaung population. Previously identified SNPs associated with hypertension in ADRB1 (A145G and G1165C), ADD1 (G217T), AGT (G-217A, C521T and T704C), CYP11B2 (C-344T), CYP3A5 (A6986G) and GRK4 (G448T, C679T and C1711T) were genotyped in a cohort of the AHA-FS study, which comprised black individuals from Mangaung, Free State. Six of the 11 candidate SNPs did not appear to be associated with hypertension in the black population of Mangaung. These included G1165C (ADRB1), G-217A and T704C (AGT), G448T, C679T and C1711T (GRK4). None of the latter SNPs were associated with statistically significant elevations in either systolic or diastolic blood pressure. The association remained negative even after the cohort was stratified into underweight to normal weight and overweight to obese groups. The lack of association between these SNPs and hypertensive blood pressure in the black population group in Mangaung compared to other population groups could be attributed to population differences in environmental factors, ethnicity, cohort size and/or epistasis. Five SNPs were associated with hypertension in black individuals from Mangaung. The latter included SNPs in CYP3A5 (A6986G), ADRB1 (A145G), AGT (C521T), CYP11B2 (C-344T) and ADD1 (G217T). The hypertensive A allele of the A6986G SNP of CYP3A5 has been associated with systolic hypertension in homozygous individuals of the study. Similarly, an association between the A allele and hypertension in African-Americans and Swedish Caucasians has also been found. Thus, the A allele of the A6986G SNP seems to cause hypertension susceptibility in different populations, including the Mangaung population group. As for the A145G SNP of ADRB1, hypertensive systolic blood pressure was associated with individuals that were homozygous for the hypertensive A allele of the A145G SNP. A meta-analysis determined that individuals expressing the A allele of A145G had a 24% higher risk for developing hypertension. In the Mangaung population, hypertension risk associated with the A allele of A145G was especially increased in overweight to obese individuals. The presence of the hypertensive T allele of the C521T SNP of AGT was associated with hypertensive diastolic blood pressure in the Mangaung population. Similar results were found in Hutterite, Russian and Tartar population groups. Furthermore, in the Mangaung population the hypertension risk conferred by the T allele was significantly increased in overweight and obese individuals. This suggests that the T allele of C521T may be involved in particularly overweight and obesity related hypertension. The hypertensive T allele of the C-344T SNP of CYP11B2 was only associated with hypertensive systolic and diastolic blood pressure in the overweight to obese individuals of the Mangaung population. In another study conducted on black South African individuals the T allele was also associated with hypertension. The cohort of the latter study furthermore had an overweight to obese body mass index average. It therefore appears that the T allele of C-344T could primarily be a risk factor for overweight and obesity related hypertension. Interestingly the normotensive G allele of ADD1 was implicated in obesity related hypertension, instead of the hypertensive T allele. In contrast, another study on black South Africans found that the T allele was associated with hypertension. However, results from a previous study on African-Americans suggested that the T allele may be protective against hypertension. It has been proposed that other unidentified polymorphisms, which also could affect hypertension susceptibility, could be in linkage disequilibrium with the G217T SNP and that allelic variation of the other polymorphic loci could contribute to the inconsistent findings of association studies. Several individuals in the cohort from the AHA-FS study could not be genotyped for the candidate SNPs. Attempts to obtain conventional PCR product for individuals where genotyping failed did not prove entirely successful. In the cases where no PCR amplicon could be amplified even after attempts at assay optimization, it was concluded that primer mismatch, especially at the 3‟ end of the primer may be the likely cause. Where PCR amplicon was successfully amplified, sequencing proved difficult due to short amplicon size. However, partial sequence data revealed additional SNPs in some individuals in the probe binding region that would account for failed genotyping. A limitation of this study was that only selective SNPs in genes associated with hypertension were genotyped in the cohort. Furthermore, since the study did not investigate the role of potentially novel SNPs in candidate genes, it is possible that additional unidentified SNPs in these genes may also contribute to hypertension. Despite these limitations, this study is currently the most comprehensive of its kind on the Mangaung population. Future research could focus on additional genes as well as screening these genes for novel SNPs, especially in the genes where the SNPs investigated were not associated with hypertension in the Mangaung cohort. In conclusion, the A6986G SNP in CYP3A5 appears to be an independent risk factor for hypertension, whereas A145G in ADRB1, C521T in AGT, C-344T in CYP11B2 and G217T in ADD1 may be associated with hypertension related to overweight and obese body weights. To our knowledge, this is the most comprehensive study investigating a combination of several gene SNPs associated with hypertension in a black Mangaung population.
Open Access
Characterization of a human inhibitory antibody fragment against tissue factor
(University of the Free State, 2011-11) Vermeulen, Jan-G.; Meiring, S. M.; Van Heerden, E.
English: Tissue factor is a transmembrane glycoprotein that functions as the primary initiator of coagulation in response to mechanical or chemical damage. Due to its key position within the coagulation cascade it also plays an important role in the pathology of thrombosis and thrombotic complications associated with cardiovascular disease as well as in non-thrombotic disorders and diseases such as obesity, diabetes, cancer and HIV-AIDS. Recognising the potential in tissue factor inhibition as a novel approach to antithrombotic therapy, our laboratory utilized phage display technology in a previous study, in order to identify a 26 kDa single chain antibody fragment which functionally inhibits human tissue factor. In the current study, the tissue factor inhibitory single chain variable fragment (TFIscFv) was expressed by means of the pIT2 plasmid vector by Escherichia coli HB2151. This expression system was utilised in an up-scale setting in an attempt to improve the TFI-scFv yield. Although functional TFI-scFv was successfully purified from the culture media by means of Protein A affinity chromatography, the process was hampered by large sample volumes, low levels of expression as well as the high cost involved in Protein A purification. Due to an initial focus on improving TFI-scFv yield through the processing of larger sample volumes rather than the improvement of the expression system, immobilised nickel affinity chromatography was investigated as a more cost effective alternative to Protein A affinity chromatography. It was found that the original expression system was incompatible with immobilised nickel affinity chromatography as the protein was expressed into the culture media. The culture media contained nickel chelating elements that stripped the nickel from the column and consequently prevented TFIscFv purification. Subsequently the TFI-scFv gene was isolated, cloned into an over-expression system and modified to redirect the expression to the bacterial cytoplasm. Although TFI-scFv was successfully redirected to the bacterial cytoplasm and purified by means of nickel affinity chromatography, it was found that expression was hampered due to the presence of rare codons. The detrimental effect of rare codons on TFI scFv yield was addressed through the modification of the expression host by the coexpression of the pRARE plasmid as well as by the rare codon optimization of the TFI-scFv gene sequence for expression in E. coli. Although the co-expression of the pRARE plasmid only slightly improved TFI-scFv yield, a sufficient amount of TFIscFv was generated for functional testing. The modified TFI-scFv displayed a similar inhibition effect with reference to the original construct. The rare codon optimisation resulted in a substantial increase in TFI-scFv yield but consequently resulted in the loss of solubility and the production of inclusion bodies. Although the loss of TFIscFv solubility is unwanted, the high level of expression achieved provides an ideal platform for the further development and characterization TFI-scFv in animal thrombosis models.