Haematological Pathology
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- ItemEvaluation and validation of room temperature biospecimens transportation and storage technologies as an alternative cost effective solution to cold chain logistics and storage within biobanking and/or diagnostics(Stellenbosch : Stellenbosch University, 2017-03) Abulfathi, Fatima Adam; Swanepoel, Carmen Catherine-Ann; Grewal, Ravnit Kuar; Abayomi, Akin; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Haematological PathologyENGLISH ABSTRACT : Background: Cold chain management (CCM) is an important aspect of biobanking operation. However challenges such as constant power failure, limited access to dry ice transport and storage of human samples collected at various sites all over the world or at difficult out of reach places. and liquid nitrogen, transport logistics and courier delays especially in Africa becomes a major challenge. Ensuring samples are maintained at the proper temperature throughout all processes is imperative to maximal long term viability and usability. Thus we consider room temperature storage (RTS) technologies as an innovative, cost effective and green alternative to cold chain logistics. Methods: Various room temperature storage technologies were evaluated for the stabilization and storage of whole blood DNA and RNA, buffy coat, genomic DNA and urine DNA. The stabilizers include the Biomatrica liquid gard technology and dry matrix technology as well as DNAgenotek Hemagene buffy-coat stabilizers, Paxgene RNA and Norgen urine tubes. Samples were stored with and without a stabilizer under different temperature conditions namely room temperature, 45oC,-80oC, -20oC and liquid nitrogen (- 196oC) over different time periods to determine effect on sample integrity and quality. At the end of each time point DNA/RNA was extracted and the integrity of the samples determined by assessing the concentration, purity and integrity. Further downstream analysis such as polymerase chain reaction (PCR), quantitative real time PCR and DNA sequencing was conducted. In addition, a shipping cost analysis between satellite sites in African and our biobank was done to compare frozen and room temperature shipping. Results The study results show that sample integrity/quality for biospecimens stored at room temperature with stabilizers were comparable and more cost effective than cold chain storage systems. In addition some stabilizers showed better stabilizing properties than others. Conclusion: Room temperature storage provides an innovative and cost effective method of storage and shipping to cold chain management systems (CCM). Green technologies forms a small part of biobanking operations however its results would be beneficial as low energy options for biobanking are particular critical in low resource settings which have infrastructural challenges. In turn, it would also be a more cost-effective option for the transport and storage of human samples collected at various sites all over the world or at difficult out of reach places.
- ItemImatinib resistance : the role of pharmacogenetic variability in a South African chronic myeloid leukemia cohort(Stellenbosch : Stellenbosch University, 2023-03) De Long, Chantal; Swanepoel, Carmen; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Pathology. Haematological Pathology.ENGLISH SUMMARY: Drug-resistant cancers are often associated with poor patient outcomes and the underlying mechanism is poorly understood. Chronic Myeloid Leukemia (CML) serves as a disease model for studying cancer drug resistance, specifically to Imatinib, a tyrosine kinase inhibitor. Approximately 20-30% of patients become resistant to Imatinib. Variability in patient drug response could be due to single nucleotide variants (SNVs) in genes that encode for Imatinib-metabolizing enzymes and transporters. The overall aim of the present study is to determine whether selected SNVs located within genes CYP3A4/3A5, SLCO1A2, SLC22A4, and SLC22A1 that encode selected drug transporters (Cytochrome P450, OATP1A2, OCTN1, hOCT1), respectively, contribute to an alternative mechanism leading to Imatinib resistance in a South African cohort. A maximum of 45 samples from Imatinib-resistant CML patients were analysed along with 44 non-resistant CML patients (controls). The selected SNVs were analysed using PCR-based genotyping assays. Baseline allelic and genotypic frequencies within our CML cohort was determined and compared between Imatinib good responders and Imatinib resistant groups. In our study we observed that there were differences in allele frequencies for the following SNVs in genes SLC22A4, SLCO1A2, CYP3A4 and CYP3A5 when compared to the global/African frequencies. Furthermore, obtained results showed that the observed and expected genotype frequencies were comparable for genes SLC22A1, SLC22A4, and SLCO1A2, however, our observed genotype frequencies were different from the expected genotype frequencies for the following genes SLCO1A2, CYP3A4 and CYP3A5. Interesting findings include, the rs35191146 (ATG>AT: delG) that was linked poor Imatinib treatment outcome, however, the simultaneous presence of rs628031 (A>G: M408V) circumvented this effect. All patients within our cohort have both Met420 and Met408Val. Another interesting finding is the coincidental finding of variants SLC22A4 rs11568500 (c.616_617delinsCC), and SLC22A1 rs35191146 (c.1258_1260delATG and g.160139876_160139883delGTAAGTTG), which would be explored in future studies. Even though the selected SNVs do not affect Imatinib resistance in our cohort, our study to the best of our knowledge is the only study to determine baseline allelic and genotypic frequencies for CML patients treated with Imatinib in South Africa. Therefore, the data obtained from our study can serve as a useful tool to further investigate the pharmacogenetic variability in South Africa. In conclusion, our study adds to the body of knowledge out there related to SNV and its potential clinical relevance related to imatinib resistance especially within our diverse African cohort. This in turn highlights the need for future studies focusing on larger cohorts, with a larger selection of SNVs at more health care institutions across South Africa.
- ItemImpact of inflammation-induced oxidative stress on the integrity of immuno-haematopoietic cells and potential ameliorating interventions in an in vitro HIV model(Stellenbosch : Stellenbosch University, 2013-12) Wanjiku, Samuel Mburu; Marnewick, Jeanine L.; Abayomi, Akin; Ipp, Hayley; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Haematological Pathology.ENGLISH ABSTRACT: Chronic inflammation and immune activation are hallmarks of HIV infection, resulting in chronic oxidative stress with over-utilization of antioxidant defences, which may contribute to the loss of immune cells and faster disease progression. Low levels of antioxidants in HIV- infected individuals have been associated with frequent opportunistic infections and an increased risk of mortality. HIV infection is also associated with on-going and aberrant activation of both the innate and adaptive immune systems. An important aspect of innate immune stimulation is derived from the leakage of lipopolysaccharide (LPS) across the damaged mucosal lining of the gut in early HIV infection. The impact of this innate immune stimulation on the adaptive arm of the immune system, as represented in this study by levels of CD4+ T-cell activation and death, have not been explored previously in untreated HIV infection. Using an integrated approach of immune activation, inflammation, oxidative stress and ameliorating antioxidant intervention for the first time, this thesis reports the impact of inflammatory induced-oxidative stress on CD4+ T-cells in an in vitro HIV model. In a preliminary study, baseline levels of neutrophil respiratory burst as an in vitro indication of immune stimulation were investigated. The relationships between baseline total antioxidant status (TAS), Red blood cell (RBC) antioxidant enzyme activities (catalase, superoxide dismutase & glutathione peroxidase), lipid peroxidation and glutathione redox ratio and other markers of disease in asymptomatic, untreated HIV infection were also explored. The design and optimization of an assay that could determine the effects of LPS-induced oxidative stress on CD4+ T-cells, was a critical part of this study. The development of this assay enabled the measurement of the effects of selected antioxidant interventions N-acetyl cysteine (NAC) and vitamin C, on LPS-induced CD4+ T-cell activation and death. The results were also correlated with CD4 count, viral load and markers of inflammation (fibrinogen & D-dimers) in HIV-infected and uninfected groups. Neutrophils from HIV-infected persons at rest showed a ―primed‖ response to low stimulating agent, bacterial N-formyl peptides (fMLP), which was significantly (P = 0.04) higher than uninfected individuals. There was increased oxidative stress as evidenced by increased catalase activity, malondialdehyde (MDA) and conjugated dienes (CDs) with a corresponding decrease in antioxidant capacity in HIV-infected individuals with lower CD4 count. NAC in combination with vitamin C, significantly (P = 0.0018) reduced activation of CD4+ T-cells to a greater degree than with either antioxidant alone. NAC and vitamin C individually and in combination significantly (P = 0.05, P = 0.012 and P<0.0001) decreased the expression of the markers of apoptosis, Annexin V and 7-amino-actinomycin (7-AAD). Importantly, the antioxidant combination decreased MDA values and significantly (P = 0.01) increased the glutathione redox ratio in the HIV-infected group. Based on these results, the respiratory burst and LPS-induced activation may be important contributing factors in inflammatory-associated oxidative stress in HIV infection and contribute to the depletion of CD4+ T-cells in the asymptomatic stage of HIV infection. These results also indicate the potential inhibitory effects of NAC and vitamin C in combination as agents that may limit immune activation and inflammation-induced oxidative stress. Importantly, the study showed that at this asymptomatic stage, CD4+ T-cells of the HIV-infected group responded similarly to stimulation as the HIV negative group, indicating that antioxidant defences were still functional and that appropriate early intervention at this stage may be protective against oxidative damage to the immune cells. To the best of our knowledge, this study is the first to use an integrated approach involving not only plasma levels of antioxidant status, but also RBC antioxidant enzyme activities, oxidative damage (lipid peroxidation), inflammation, cellular levels of immune activation and potential ameliorating interventions in evaluating the problem of inflammation-induced oxidative stress in HIV infection. Based on the results of this study, it is envisaged that an insight into the immune activation, inflammatory and oxidative stress status of patients will enable long-term profiling of each patient with a view to individualized therapy. This approach may have a direct impact on patient care in resource-limited settings such as sub-Saharan Africa.
- ItemInvestigating platelet function and immune activation in HIV-infection(Stellenbosch : Stellenbosch University, 2015-04) Nkambule, Bongani Brian; Ipp, Hayley; Davison, Glenda; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Haematological Pathology.ENGLISH ABSTRACT: Introduction In the era of antiretroviral therapy (ART), people living with Human Immunodeficiency Virus (HIV) now have prolonged life spans. An emerging trend of non- acquired immune deficiency syndrome (AIDS) related complications now prevails in the aging HIV infected population. Increased levels of inflammation and chronic immune activation are associated with HIV infection. In the era of ART people living with HIV are at an increased risk of cardiovascular disease (CVD). Platelets play a pivotal role in both inflammation and immune activation and upon activation platelets degranulate and secrete various inflammatory, coagulatory and adhesion molecules. Activated platelets express surface P-selectin (CD62P) and are a key component of the coagulation pathway and serve as a link between inflammation and thrombosis. Activated platelets have been implicated in inflammatory and cardiovascular disease and have been identified as immune cells that play a crucial role in pathogen recognition and modulation of immune cells during infections. Several antiviral and antibacterial properties of platelet alpha granule contents have been established. Platelet aggregometry remains the most widely used technique to evaluate platelet function even though this technique is limited by many pre-analytical variables. Platelet flow cytometry on the other hand offers a rapid measurement of platelet function in their physiological environment with minimal artefactual activation. Few studies have however reported on standardized methods to evaluate platelet function in the context of HIV. Platelet function remains unclear and data on HIV infected treatment naïve individuals remains scarce. The aim of this project was to examine the relationship between platelet function and immune activation in patients with HIV Materials and methods This study consisted of five sub-studies, firstly platelet indices and levels of platelet activation were determined in a cohort of 330 participants (185 HIV infected ARV naïve and 145 uninfected healthy controls) using; flow cytometry and haemotology analyzers. The relationship between these indices and markers of platelet activation, disease progression and immune activation were assessed. Furthermore, levels of platelet activation and aggregation were evaluated in a cohort of 82 participants (41 HIV infected (ARV naïve) individuals and 41 uninfected healthy controls), using a novel whole blood flow cytometry based functional assay. These baseline levels were then correlated with markers of immune activation and disease progression in HIV. In a subsequent study, platelet function in a cohort consisting of 58 HIV infected (ARV naïve) and 38 uninfected controls was evaluated using flow cytometry. Platelet response was measured post stimulation with adenosine diphosphate (ADP) at concentrations known to induce reversible (0.04mM) and irreversible (0.2mM) platelet aggregation. In order to assess platelet function in HIV, platelet response was evaluated in a cohort consisting of 58 HIV infected (ARV naïve) and 38 uninfected controls. Platelets were activated using varying concentrations of ADP, arachidonic acid (AA) and collagen and platelet function was measured using flow cytometry. Levels of circulating platelet leukocyte aggregates (PLAs) were also measured using flow cytometry in a cohort consisting of 35 HIV-infected (ARV naïve) individuals and 32 uninfected healthy controls. Associations between PLAs, immune activation and disease progression in HIV infected individuals were determined. The final study evaluated platelet aggregates, platelet derived microparticles (PMPs) and microparticles (MPs) in a cohort consisting of 46 HIV infected (ARV-naïve) and 40 uninfected healthy controls. Associations between MPs, PMPs, platelet aggregates and markers of immune activation and disease progression were evaluated. Results HIV infected individuals showed decreased mean platelet volume levels (HIV mean 7.91 ± 0.85 vs. 8.52 ± 1.12, p<0.0001) that directly correlated with CD4 counts (r=-0.2898, p=0.0075) and viral load (r=0.2680, p=0.0177). Platelet distribution width (PDW) levels directly correlated (r=0.3455, p=0.0362) with active coagulation and inversely correlated (r=-0.3666, p=0.0463) with platelet aggregation. HIV infected individuals showed increased levels of platelet activation (%CD62P median 11.33[5.96-29.36] vs. control group 2.48[1.56-6.04], p=0.0001). In HIV, platelet function is retained and platelets showed increased response to submaximal concentrations of endogenous agonists. HIV infected individuals showed increased levels of circulating platelet monocyte aggregates (25.26[16.16-32.28] vs. control group 14.12[8.36- 18.83], p=0.0001) that directly correlated with markers of immune activation; %CD38/8 (r=0.54624, p=0.0155), viral load (r=0.633, p<0.009). Furthermore we report on increased levels of circulating MPs (median %MPs 1.7[0.95-2.83] vs. Control group 1.12[0.63-1.57], p=0.0160); PMPs (median %PMPs 26.64[11.33-36.62] vs. Control group 20.02[18.08-26.08], p=0.0133); activated PMPs (median CD62P MFI 3.81[3.46-4.54] vs. Control group 3.41[3.16-3.6], p=0.0037) and platelet aggregates (Median %CD62P 14.10[5.49-39.94] vs. Control group 0.17[0.10-10.99], p= 0.0097) in HIV infected asymptomatic individuals. Conclusion This study supports the potential use of the MPV and PDW as readily available markers of platelet activation and immune activation in HIV. We also showed elevated levels of activated platelets in HIV infected individuals that were hyper responsive to endogenous agonists in a concentration dependent manner. Platelet flow cytometry is a rapid and valuable technique in the evaluation of platelet function in HIV. The measurement of platelet function using flow cytometry allows the evaluation of platelet signalling pathways that may be modified in HIV infected individuals. Lastly we describe an optimized whole blood flow cytometry based assay for the evaluation of circulating microparticles (MPs), platelet derived microparticles (PMPs) and levels of activated platelets and aggregates which mimics the in vivo physiological environment of MPs. To the best of our knowledge, this study is the first to report on a novel approach in evaluating platelet function in HIV using a series of optimised whole blood flow cytometry based platelet assays. In addition, minimal work has been performed previously on platelet function in the context of HIV-infection; and particularly in a cohort of asymptomatic, untreated patients as defined for this study.
- ItemInvestigating the use of standardized EuroFlow flow cytometry panels for the characterisation and diagnosis of Chronic lymphocytic leukaemia in the Tygerberg Academic Hospital, South Africa.(Stellenbosch : Stellenbosch University, 2017-12) Musaigwa, Fungai; Swanepoel, Carmen Catherine-Ann; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Pathology. Haematological Pathology.ENGLISH ABSTRACT : Background: Flow cytometry (FC) immunophenotyping is crucial in the diagnosis and classification standardisation of haematological malignancies. FC techniques require standardisation to produce reliable and reproducible results which are important in inter-laboratory studies for laboratory methodology improvement. The aim of this study is to introduce standardised multicolour FC in the diagnosis of haematological malignancies using chronic lymphocytic leukaemia (CLL) as the proof of principle. In addition, we aim to document the incidence of CLL from the year 2011 to 2016 in the Tygerberg Academic Hospital (TAH) catchment area of Cape Town, South Africa (SA). Methods: Twenty CLL patients were recruited at TAH. Bio-specimens were prepared and analysed on the Beckman Coulter Navios flow cytometer using Euroflow™ standardised FC protocols and immunophenotypic panels with two tubes for detecting B-cell chronic lymphoproliferative disorders (B-CLPD). Tube 1 included CD20, CD4, CD45, CD8, lg-Kappa, CD56, lg-Lambda, CD5, CD19, TCRyσ, CD3 and CD38. Tube 2 included CD20, CD45, CD23, CD10, CD79b, CD19, CD200 and CD43. Combined, the two tubes identified CLL from other B-CLPD. The CLL immunophenotypic profiles were stored in a database using the compass tool of the Infinicyt™ FC software. In addition, the clinical records of patients diagnosed with CLL at TAH over a 6-year period from the year 2011 to 2016 were retrieved and analysed using descriptive statistics. Results: In comparison with the SA National Health Laboratory Service (NHLS) results at TAH, the Euroflow™ standardised multicolour FC panels and protocols are suitable for immunophenotyping CLL in this SA population. An immunophenotype database for 20 CLL diagnosed at TAH was constructed using the EuroFlow™ standardised multicolour FC panels. For the epidemiology part of the study, a total of 80 CLL patients were studied. There were slightly more females (51.2%) and the mean age at diagnosis was 67 years (37 to 95). Ninety one percent of the patients were aged 50 years and above. Males presented with the disease at a younger age (mean 63 years) than females (mean 70 years). CLL concurrent with HIV was not common (4%) and these patients were younger than 50 years. Twenty-one patients were tested for chromosomal aberrations trisomy 12 and deletion 11q, 24% and 33% were positive respectively. Deletion 13q was assessed in 25 patients and 16% were positive. Twenty patients were tested for deletion 17p and all were negative. Translocations t(8;14), t(11;14) and t (14;18) were negative in 1, 8 and 4 patients respective. Discussion: Accurate and consistent laboratory techniques and strict standardisation in FC enhances the confidence in inter-laboratory studies. Establishment of haematological malignancy immunophenotype databases would allow for faster differential diagnoses of new disease cases which is needed within our setting. Furthermore, these databases permit clear identification of atypical cases. Monitoring haematological malignancy trends is a crucial step in the management of the disease.
- ItemOptimisation of a whole blood flow cytometry assay to aid in the diagnosis of tuberculosis by detecting intracellular cytokines released by CD4+ T-cells(Stellenbosch : Stellenbosch University, 2016-03) Snyders, Candice Irene; Grewal, Ravnit Kuar; Swanepoel, Carmen Catherine-Ann; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Haematological PathologyENGLISH ABSTRACT : Background: South Africa (SA) sees 8 million new Tuberculosis (TB) cases each year and has a significant problem with Human Immunodeficiency Virus (HIV) and TB co-infection. Latent TB infection (LTBI) is described in persons infected with mycobacterium tuberculosis (M.tb) but shows no signs and symptoms of active disease. HIV+ individuals with LTBI can develop active TB infection more readily than that of HIV- individuals. Gold standard methods for diagnosing active disease have been criticized for, among other things, their lengthy turnaround times. Currently there is no gold standard for the diagnosis of LTBI. Flow cytometry allows one to measure cytokine responses in CD4+ T-cells following overnight stimulation with TB antigens ESAT-6 and CFP-10 (E/C). Studying these cytokine expression patterns will make it possible to classify patients into active disease vs. LTBI. Methods:A total of 18 TB+ patients which included 6 HIV+ patients, were recruited from Tygerberg Hospital, Western Cape. A whole blood no-centrifuge intracellular flow cytometry assay was optimised to study the cytokine expression patterns in CD4+ T-cells that have been stimulated with TB antigens and Staphylococcus Enterotoxin B (SEB), following an 18 hour overnight incubation. CD3+CD4+ T-cells were delineated into the following subsets: naïve (TN) (CD45RO-CD27+ ), central memory (TCM) (CD45RO+CD27+ ),effector memory (TEM)(CD45RO+CD27- ) and terminally differentiated effector memory cells (TDEM) (CD45RO-CD27- ). The expression patterns and effect of stimulation on cytokines IFN-γ and TNF-α as well as T-cell exhaustion marker TIM3, was assessed. Results: This study has demonstrated higher levels of IFN-γ expression in the control group compared to that of the TB positive patients (median %IFN-γ 2.960 ± 3.51 versus patient 2.370 ± 2.07; p=0.2800). TNF-α had higher expression in the patient group compared to the control subjects (median %TNF-α 2.415 ± 2.60 versus control 1.340 ± 1.86; p=0.1729). Dual expression of cytokines was almost similar in the two groups (control median % IFN-γ + TNF-α + 0.5400 ± 0.36 versus patient 0.8550 ± 0.60; p=0.3961). TIM3 expression was not significantly different between the four T-cell subsets (median TN 0.0750 ± 1.89, TCM 0.3400 ± 4.28, TEM 0.0850 ± 2.73, TDEM 0.1600 ± 1.93; p= 0.5877). When comparing the subset distribution in the patient group, TN cells were the most abundant (median 47.48 ± 20.96) followed by TEM cells (median 21.92 ± 13.25), TDEM cells (median 13.02 ± 20.13) and finally TCM cells (median 11.51 ± 8.62). These results showed a significant difference in expression between the four groups (p=<0.0001). Conclusion: Through careful titration of antibodies and relevant optimisation steps, we established a flow cytometry assay that may be used to study cytokine patterns in TB patients. The increased TNF-α only expression in the patient group is suggestive of active TB and the increased IFN-γ in the control group could indicate BCG vaccination. TIM3 would be a useful marker in a larger HIV+ cohort of patients as this will allow identification of functionally exhausted T-cells. In SA, HIV prevalence is rising and this assay proves its suitability by using minimal volumes of whole blood rather than sputum. By generating intracellular cytokine profiles one would be able to distinguish between active and LTBI which would aid in treatment management of patients.
- ItemScreening and characterisation of BCR::ABL1 kinase domain mutations in chronic myeloid leukaemia participants at Tygerberg Hospital, South Africa(Stellenbosch : Stellenbosch University, 2023-03) Shareefa, Isaacs; Swanepoel, Carmen; Chapanduka, Zivanai; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Pathology. Haematological Pathology.ENGLISH SUMMARY: To date, an increased number of drug resistant cancers have been observed. The underlying mechanisms are not well understood; therefore, we use Chronic Myeloid Leukaemia (CML) as a disease model to explore the type and frequencies of potential drug resistant causing mutations and the effect on survival at Tygerberg Hospital (TBH). The underlying genetic abnormality is a t(9::22) chromosomal translocation, and consequent fusion between the Breakpoint Cluster Region and the Non-Receptor Tyrosine Kinase Abelson genes (BCR::ABL1). Although Tyrosine Kinase Inhibitors (TKD), such as Imatinib (Gleevec), effectively decrease BCR::ABL1 mRNA transcript levels whereby the drug acts on the protein and inhibits its mode of action, point mutations within the kinase domain (KD) have shown to confer resistance to treatments. A retrospective audit was conducted on a CML cohort from 2013 to 2020, and 20 of these participants were recruited for detection of KD mutations within the ABL1 oncogene. Peripheral blood from routine diagnostic screening by the NHLS Molecular Haematology diagnostic laboratory were obtained for the mutation detection assay via bi-directional Sanger Sequencing. Identified sequence variants were then subjected to various bioinformatics tools to investigate variant, protein, and consequent effect on pathways. For the audit, a total of 165 patients with confirmed CML treated at TBH was captured. Descriptive statistics showed 46.1% of CML patients were female, and 53.9% were male. The patients were from 69 different areas in the Northern part of the Cape Metropole and surrounding Boland areas in Western Cape, South Africa. Quantitative analysis showed the youngest patient to be two years old and the oldest 88 years old, x̄ = 45-46 years. We found a weak linear correlation between age and BCR::ABL1 mRNA transcript levels. A significant difference in BCR::ABL1 mRNA transcript levels (p < 0.000) was seen in CML patients on treatment after 1 year. Of interest’s sake, 104 of the initial 165 patients who were diagnosed were still undergoing treatment. Moreover, 57 out of the 165 patients presented with possible resistance; 75% were categorized as failure and 25%at warning stage according to European Leukaemia Net guidelines. In the second part of the study, 20 participants’ samples were screened for variants. The screening sample group had a mortality rate of 20% who had a relative survival rate of less than five years. Sanger sequence analysis showed potential variants of interest in Exon 4 and of the ABL1 gene, although bioinformatics analysis alludes to the fact that these variants are not likely to play a role in resistance. In conclusion, the TBH CML cohort has a younger age at diagnosis which puts a greater strain on the public sector, and a significant number of patients are lost to follow up. More so, the age relative to CML deaths, as well as relative survival greatly differs from literature. Additionally, although synonymous mutations have recently shown to be pathological, the two variants found within our cohort has not been published to do so. CML could be a poster child for personalised medicine as the survival rate of CML patients is nearing that of the general population and treatment-free remission is attainable. Unfortunately, the paucity of CML data, drug availability and monitoring limitations are some of the obstacles that hinders South Africa from achieving these goals.