Haematological Pathology

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Browsing Haematological Pathology by browse.metadata.advisor "Swanepoel, Carmen Catherine-Ann"

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    Evaluation 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 Pathology
    ENGLISH 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.
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    Investigating 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.
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    Optimisation 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 Pathology
    ENGLISH 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.