Masters Degrees (Haematological Pathology)

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Browsing Masters Degrees (Haematological Pathology) by Subject "Flow cytometry -- Diagnostic use"

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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.