Research Articles (Haematology)

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Phenotypic characterisation of bone marrow-derived haematopoietic stem/progenitor cells from HIV-infected individuals
(Springer, 2025-04) Mistry, Priyal; Potgieter, Joachim; Pepper, Michael Sean; Durandt, Chrisna; chrisna.durandt@up.ac.za
Human immunodeficiency virus (HIV-1) infection remains a significant global public health concern, particularly in sub-Saharan Africa where the majority of HIV-1 infections are concentrated. People living with HIV (PLWH) often present with haematological abnormalities including alterations of the bone marrow (BM; dysplasia and cellularity changes) and most commonly cytopenias (anaemia, thrombocytopenia and neutropenia). From these clinical observations it is clear that HIV not only impacts the immune system but also the broader haematopoietic system. The underlying cause of HIV-mediated changes in the haematological system remains complex and multifactorial.
Late-onset hydroxyurea-induced melanonychia and tongue hyperpigmentation in a patient with polycythemia vera : a case report
(Springer, 2024-02) Letete, Nena; Vaz, Deborah
In a rare case, a 70-year-old female with polycythemia vera developed late-onset melanonychia, a seldom-documented side effect of hydroxyurea. Typically, melanonychia emerges within months of treatment, but this case is unique as it occurred four years into therapy. Notably, the patient, with darker skin, also had hyperpigmentation of her hands and tongue. Her history of hydroxyurea-associated ulcers and symptoms worsening with dose adjustment suggested drug involvement. While mucocutaneous hyperpigmentation from hydroxyurea is known, melanonychia and tongue hyperpigmentation are rarely reported, mostly in early treatment. This case highlights the importance of recognizing these side effects, especially in diverse populations and darker skin tones. The diverse skin tones seen in Sub-Saharan Africa add complexity to diagnosing such dermatological conditions, highlighting the need for awareness. Melanonychia can mimic severe conditions such as subungual melanoma, emphasizing the significance of accurate recognition and management without invasive tests. Educating clinicians and patients about these benign drug-related phenomena is essential for precise identification and management. This case contributes to understanding late-onset hydroxyurea-induced melanonychia and tongue hyperpigmentation, enhancing clinical knowledge in diverse populations.
A pilot study evaluating the ex vivo effects of varying factor VIII concentration on clot kinetics and architecture in patients with haemophilia A
(Sage, 2023) Morris, Chanel; Potgieter, Johan C.; Bester, Janette; janette.bester@up.ac.za
BACKGROUND : Haemophilia A (HA) is a bleeding disorder, due to a deficiency in factor VIII (FVIII). These patients are unable to produce a stable fibrin clot in the propagation phase of coagulation as they do not generate sufficient thrombin. The primary treatment for HA in South Africa remains replacement therapy with standard half-life FVIII clotting factor concentrate, aimed at reducing bleeding episodes. OBJECTIVES : To evaluate the effect of varying concentrations of FVIII on whole blood (WB) clot architecture and kinetics during clot formation in patients with severe HA. DESIGN : A cross-sectional study where blood from 20 patients with HA was exposed to FVIII ex vivo and compared to a control group of 20 healthy individuals. METHODS : Scanning electron microscopy (SEM) was used to study WB clot architecture and thromboelastography® (TEG®) was used to quantify WB clot kinetics. RESULTS : Scanning electron microscopy studies revealed that patients with HA have sparse, disorganized fibrin networks with limited crosslinking and red blood cells (RBCs) stacked in rouleaux formation. Haemophilia A blood spiked to a 10 to 15 IU/dL FVIII concentration showed improvements in the organisation of the fibrin network with some altered RBCs. In addition, blood spiked to a 30 to 35 IU/dL FVIII concentration showed an increase in fibrin formation with normal RBCs. Thromboelastography® showed that patients with HA had an increased clot initiation time and decreased clot strength. When spiked to a 10 to 15 IU/dL FVIII concentration the clot kinetic profile showed normalization. However, an increase in FVIII concentration higher than 30 IU/dL showed altered clot architecture and kinetics. CONCLUSION : Based on the current study, FVIII levels at 10 to 15 IU/dL improved clot kinetics but did not normalize the architecture. It may be sufficient for prevention of haemorrhages. Factor VIII levels at 30 to 35 IU/dL resulted in rapid but weaker clot formation. However, at this concentration the clot architecture was normalised which is important for haemostasis. Here it was also evident that the findings of these two modalities (TEG® and SEM) should not be separated but interpreted in conjunction with each other.
Immune and metabolic alterations in children with perinatal HIV exposure
(MDPI, 2023-02) Du Toit, Louise de Villiers; Prinsloo, Andrea; Steel, Helen C.; Feucht, Ute Dagmar; Louw, Roan; Rossouw, Theresa; louise.dutoit@up.ac.za
With the global rollout of mother-to-child prevention programs for women living with HIV, vertical transmission has been all but eliminated in many countries. However, the number of children who are exposed in utero to HIV and antiretroviral therapy (ART) is ever-increasing. These children who are HIV-exposed-but-uninfected (CHEU) are now well recognized as having persistent health disparities compared to children who are HIV-unexposed–and-uninfected (CHUU). Differences reported between these two groups include immune dysfunction and higher levels of inflammation, cognitive and metabolic abnormalities, as well as increased morbidity and mortality in CHEU. The reasons for these disparities remain largely unknown. The present review focuses on a proposed link between immunometabolic aberrations and clinical pathologies observed in the rapidly expanding CHEU population. By drawing attention, firstly, to the significance of the immune and metabolic alterations observed in these children, and secondly, the impact of their healthcare requirements, particularly in low- and middle-income countries, this review aims to sensitize healthcare workers and policymakers about the long-term risks of in utero exposure to HIV and ART.
Establishment of haemoglobin A2 reference intervals in Pretoria, South Africa : a retrospective secondary data analysis
(AOSIS, 2022-08-12) Nieuwenhuizen, Cailin; Netshidzivhani, Tshiphiri; Potgieter, Johan
BACKGROUND : Haemoglobinopathies are one of the most common inherited diseases worldwide. Quantification of haemoglobin A2 is necessary for the diagnosis of the beta thalassaemia trait. In this context, it is important to have a reliable reference interval for haemoglobin A2 and a local reference range for South Africa has not been established. OBJECTIVE : This study aimed to establish reference intervals for haemoglobin A2 using stored patient laboratory data. METHODS : This descriptive study used retrospective data to evaluate haemoglobin A2 levels determined using high-performance liquid chromatography at the National Health Laboratory Service haematology laboratory in Pretoria, South Africa. All tests performed from 01 October 2012 to 31 December 2020 were screened for inclusion; of these, 144 patients’ data met the selection criteria. The reference interval was calculated using descriptive statistics (mean and standard deviation) with a 95% confidence interval. RESULTS : Analysed data from enrolled patients showed a normal distribution. The mean age of the patients was 40 years (range: 3–84 years). The reference interval for haemoglobin A2 calculated from this data was 2.3% – 3.6%. The minimum haemoglobin A2 was 2.3% and the maximum was 3.9% with a mean of 2.95% and a standard deviation of 0.357%. CONCLUSION : A normal reference interval has been established for the population served by the laboratory that will assist with accurate diagnosis of the beta thalassaemia trait. This reference interval may also be useful to other laboratories that employ the same technology, especially smaller laboratories where obtaining a sufficiently large number of normal controls may be challenging.
Dolutegravir potentiates platelet activation by a calcium-dependent, ionophore-like mechanism
(Taylor and Francis, 2022) Madzime, Morris; Theron, Annette J.; Anderson, Ronald; Tintinger, Gregory Ronald; Steel, Helen C.; Meyer, Pieter Willem Adriaan; Nel, Jan Gert; Feldman, Charles; Rossouw, Theresa M.; theresa.rossouw@up.ac.za
Please read abstract in the article.
Ex vivo platelet morphology assessment of chronic myeloid leukemia patients before and after Imatinib treatment
(Wiley, 2022-06) Repsold, Lisa; Pool, Roger; Karodia, Mohammed; Tintinger, Gregory Ronald; Joubert, Anna Margaretha
Chronic myeloid leukemia (CML) is a myeloproliferative disease and the first line treatment is through the administration of Imatinib, a first generation tyrosine kinase inhibitor. Thrombocytosis and bleeding irregularities are common in CML, however, the morphological variations in CML patients' platelets are not well documented. In this study, ex vivo platelet morphology of control participants, as well as CML patients were assessed before and after Imatinib treatment. The topographical and structural morphology of platelets were determined via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Qualitative data of SEM and TEM revealed that CML patient's platelets were prone to aggregation and coagulation at time of diagnosis; the samples that were not aggregated at time of diagnosis showed typical discoid shaped platelets, which was comparable to control participants' platelets. TEM results of CML patients' platelets at diagnosis showed that internal granular constituents including dense bodies were decreased in comparison to control participants. In all CML patients, platelets appeared activated after 6 months of treatment with Imatinib with membrane structure abnormalities and constituent variations. Research to date has primarily focused on the effects of CML on leukocyte populations, however, the results of the current study implicate the impact of CML pathogenesis on platelets, seemingly as a result of alterations in normal hematopoiesis. In addition, the impact of Imatinib treatment on platelet morphology was also established, indicating an increase in platelet activation. Recognizing and understanding the impact of CML disease progression on platelets is of importance to aid improved patient treatment. RESEARCH HIGHLIGHTS : In the study, results from SEM and TEM indicated that CML patient's platelets were prone to aggregation at time of diagnosis, and activation after Imatnib treatment. Platelet samples that did not aggregate had decreased internal granular constituents.
Recombinant sclerostin inhibits bone formation in vitro and in a mouse model of sclerosteosis
(Elsevier, 2021-07) Dreyer, Timothy; Shah, Mittal; Doyle, Carl; Greenslade, Kevin; Penney, Mark; Creeke, Paul; Kotian, Apoorva; Ke, Hua Zhu; Naidoo, Vinny; Holdsworth, Gill; u15413706@tuks.co.za
BACKGROUND : Sclerosteosis, a severe autosomal recessive sclerosing skeletal dysplasia characterised by excessive bone formation, is caused by absence of sclerostin, a negative regulator of bone formation that binds LRP5/6 Wnt co-receptors. Current treatment is limited to surgical management of symptoms arising from bone overgrowth. This study investigated the effectiveness of sclerostin replacement therapy in a mouse model of sclerosteosis. METHODS : Recombinant wild type mouse sclerostin (mScl) and novel mScl fusion proteins containing a C-terminal human Fc (mScl hFc), or C-terminal human Fc with a poly-aspartate motif (mScl hFc PD), were produced and purified using mammalian expression and standard chromatography methods. In vitro functionality and efficacy of the recombinant proteins were evaluated using three independent biophysical techniques and an in vitro bone nodule formation assay. Pharmacokinetic properties of the proteins were investigated in vivo following a single administration to young female wild type (WT) or SOST knock out (SOST-/-) mice. In a six week proof-of-concept in vivo study, young female WT or SOST-/- mice were treated with 10 mg/kg mScl hFc or mScl hFc PD (weekly), or 4.4 mg/kg mScl (daily). The effect of recombinant sclerostin on femoral cortical and trabecular bone parameters were assessed by micro computed tomography (μCT). RESULTS : Recombinant mScl proteins bound to the extracellular domain of the Wnt co-receptor LRP6 with high affinity (nM range) and completely inhibited matrix mineralisation in vitro. Pharmacokinetic assessment following a single dose administered to WT or SOST-/- mice indicated the presence of hFc increased protein half-life from less than 5 min to at least 1.5 days. Treatment with mScl hFc PD over a six week period resulted in modest but significant reductions in trabecular volumetric bone mineral density (vBMD) and bone volume fraction (BV/TV), of 20% and 15%, respectively. CONCLUSION : Administration of recombinant mScl hFc PD partially corrected the high bone mass phenotype in SOST-/- mice, suggesting that bone-targeting of sclerostin engineered to improve half-life was able to negatively regulate bone formation in the SOST-/- mouse model of sclerosteosis. THE TRANSACTIONAL POTENTIAL OF THIS ARTICLE : These findings support the concept that exogenous sclerostin can reduce bone mass, however the modest efficacy suggests that sclerostin replacement may not be an optimal strategy to mitigate excessive bone formation in sclerosteosis, hence alternative approaches should be explored.
Apoptotic profiling of chronic myeloid leukaemia patients' platelets ex vivo before and after treatment with Imatinib
(Wiley, 2021-06) Repsold, Lisa; Pool, Roger; Karodia, Mohammed; Tintinger, Gregory Ronald; Becker, Piet J.; Joubert, Anna Margaretha
Chronic myeloid leukaemia (CML) is a malignancy of the haematopoietic stem cells. The first line of treatment for CML, especially in developing countries, remains the first-generation tyrosine kinase inhibitor, Imatinib. Patients with CML are frequently diagnosed with platelet abnormalities. However, the specific mechanism of platelet abnormalities in CML remains unclear and poorly understood. The aim of this study was therefore to determine the apoptotic profiles of CML patients ex vivo on platelets before and after treatment with Imatinib. Blood samples of healthy volunteers and CML patients at diagnosis and after 6 months treatment with Imatinib were collected. Platelet counts, viability and activation were determined. Results showed that CML patients' platelet counts were elevated upon diagnosis and these levels statistically significantly decreased after 6 months of treatment. Platelet activation was significantly increased after 6 months of treatment compared to levels at diagnosis (P-value < .05). Similarly, platelet apoptosis was also increased after 6 months of treatment. Abnormalities in platelet functioning found in this study may partly be due to clonal proliferation of haematopoietic cells in CML patients, specifically of megakaryocyte precursors as well as the inhibition of platelet tyrosine kinase's and the inhibition of platelet-derived growth factor.
Submission for special issue : The role of platelet activation in the pathophysiology of HIV, tuberculosis, and pneumococcal disease. Bedaquiline suppresses ADP-mediated activation of human platelets in vitro via interference with phosphatidylinositol 3-kinase
(MDPI, 2021-02-26) Tintinger, Gregory Ronald; Theron, Annette J.; Steel, Helen C.; Cholo, Moloko C.; Nel, Jan Gert; Feldman, Charles; Anderson, Ronald
Although bedaquiline has advanced the treatment of multidrug-resistant tuberculosis (TB), concerns remain about the cardiotoxic potential of this agent, albeit by unexplored mechanisms. Accordingly, we have investigated augmentation of the reactivity of human platelets in vitro as a potential mechanism of bedaquiline-mediated cardiotoxicity. Platelet-rich plasma (PRP) or isolated cells prepared from the blood of healthy, adult humans were treated with bedaquiline (0.625–10 μg/ml), followed by activation with adenosine 5’-diphosphate (ADP), thrombin or the thromboxane A2 receptor agonist (U46619). Expression of platelet CD62P (P-selectin), platelet aggregation, Ca2+ fluxes and phosphorylation of Akt1 were measured using flow cytometry, spectrophotometry, fluorescence spectrometry, and by ELISA procedures, respectively. Exposure to bedaquiline caused dose-related inhibition of ADP-activated, but not thrombin- or U46619-activated, expression of CD62P by platelets, achieving statistical significance at a threshold concentration of 5 μg/ml and was paralleled by inhibition of aggregation and Ca2+ mobilization. These ADP-selective inhibitory effects of bedaquiline on platelet activation were mimicked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), implicating PI3-K as being a common target of both agents, a contention that was confirmed by the observed inhibitory effects of bedaquiline on the phosphorylation of Akt1 following activation of platelets with ADP. These apparent inhibitory effects of bedaquiline on the activity of PI3-K may result from the secondary cationic amphiphilic properties of this agent. If operative in vivo, these anti-platelet effects of bedaquiline may contribute to ameliorating the risk of TB-associated cardiovascular disease, but this remains to be explored in the clinical setting.
Hypercoagulability in COVID-19
(South African Heart Association, 2020-11) Wessels, Pieter Frederik
COVID-19 is associated with a hypercoagulable state that may present as pulmonary thrombosis, pulmonary embolism, and venous and arterial thrombosis. Suggested pathogenesis include direct infection of the endothelial cell with subsequent endothelial cell dysfunction, leading to increased procoagulant activity, decreased anticoagulant activity and decreased fibrinolysis. The severe immune infl ammatory response in the lungs with cytokine release also plays a critical role (immunothrombosis). Hypoxia has a local and systemic effect on coagulation. Various markers of this state have been described, and especially the D-dimer level (and rapid changes in the D-dimer level) as a reliable prognostic marker. It is also used as indicator for initiation of anticoagulation by some experts. Due to the pleotrophic effects of heparin, it is the anticoagulant of choice for these patients (most often low molecular weight heparin, due to decreased risk of heparin induced thrombocytopenia, ease of use). No clinical trial data is available at the time of writing (28 May 2020), and suggested guidelines of experts in different countries are discussed.
ADP-mediated upregulation of expression of CD62P on human platelets is critically dependent on co-activation of P2Y1 and P2Y12 receptors
(MDPI Publishing, 2020-12) Anderson, Ronald; Theron, Annette J.; Steel, Helen C.; Nel, Jan Gert; Tintinger, Gregory Ronald; ronald.anderson@up.ac.za
This study probed the di erential utilization of P2Y1 and P2Y12 receptors in mobilizing CD62P (P-selectin) from intracellular granules following activation of human platelets with adenosine 50-diphosphate (ADP, 100 mol L1) Platelet-rich plasma (PRP) was prepared from the blood of adult humans. CD62P was measured by flow cytometry following activation of PRP with ADP in the absence and presence of the selective antagonists of P2Y1 and P2Y12 receptors, MRS2500 and PSB0739 (both 0.155–10 mol L1), respectively. E ects of the test agents on ADP-activated, CD62P-dependent formation of neutrophil:platelet (NP) aggregates were also measured by flow cytometry, while phosphatidylinositol 3-kinase (PI3K) activity was measured according to Akt1 phosphorylation in platelet lysates. Treatment with MRS2500 or PSB0739 at 10 mol L1 almost completely attenuated (94.6% and 86% inhibition, respectively) ADP-activated expression of CD62P and also inhibited NP aggregate formation. To probe the mechanisms involved in P2Y1/P2Y12 receptor-mediated expression of CD62P, PRP was pre-treated with U73122 (phospholipase C (PLC) inhibitor), 2-aminoethoxy-diphenyl borate (2-APB, inositol triphosphate receptor antagonist), calmidazolium chloride (calmodulin inhibitor), or wortmannin (PI3K inhibitor). U73122, 2-APB, and wortmannin caused almost complete inhibition of ADP-activated expression of CD62P, while calmidazolium chloride caused statistically significant, partial inhibition. PSB0739, but not MRS2500, caused potent inhibition of PI3K-mediated phosphorylation of Akt1. Optimal mobilization of CD62P by ADP-stimulated platelets is critically dependent on the co-activation of platelet P2Y1 and P2Y12 receptors. P2Y12 receptor activation is the key event in activation of PI3K, while activation of the P2Y1 receptor appears to create a high cytosolic Ca2+ environment conducive to optimum PI3K activity.
The effect of freezing and thawing of samples for anti-factor Xa testing for the determination of enoxaparin activity
(Wiley, 2021-06) Louw, Susan; Gounden, Reenelle; Vaughan, Jenifer; Ntabeni, Lumka; Mayne, Anthony; Mayne, Elizabeth S.
The quality and validity of laboratory results, especially for coagulation analyses, are influenced by pre-analytical variables including the collection technique, transportation, pre-analytical processing and storage of samples. Laboratory networks often rely on centralization of specialized testing, which necessitates on-site preparation of samples and transportation to referral centres. Low molecular weight heparin (LMWH) offers improved clinical predictability of anticoagulation and often does not require routine monitoring.
Endocrine profiling in patients with Fanconi anemia, homozygous for a FANCG founder mutation
(Wiley Open Access, 2020-08) Dillon, Bronwyn; Feben, Candice; Segal, David; Du Plessis, Johannes; Reynders, David; Wainwright, Rosalind; Poole, Janet; Krause, Amanda
BACKGROUND : Fanconi anemia (FA) is phenotypically diverse, hereditary condition associated with bone marrow failure, multiple physical abnormalities, and an increased susceptibility to the development of malignancies. Less recognized manifestations of FA include endocrine abnormalities. International discourse has highlighted that these abnormalities are widespread among children and adults with FA. To date there has been no systematic study that has evaluated the endocrine abnormalities in a cohort of patients with FA, homozygous for a founder mutation (c.637_643del (p.Tyr213Lysfs*6)) in FANCG. The objectives of the study were to evaluate endocrine gland function in patients with FA of a single FA genotype, and to determine the frequency and nature of endocrine abnormalities in this group. METHODS : Cross‐sectional, descriptive study of 24 South African patients of African ancestry with FA (homozygous for a FANCG founder mutation). Outcomes measured included growth, pubertal status, growth hormone axis screening, thyroid gland function, glucose and insulin metabolism and bone age (BA). RESULTS : Endocrine dysfunction was present in 70.8% (17 of 24), including abnormal insulin‐like growth factor 1 (IGF‐1)/insulin‐like growth factor‐binding protein 3 (IGFBP‐3) in 25.0% (6 of 24), insulin resistance in 41.7% (10 of 24), abnormal thyroid function in 16.7% (4 of 24) and short stature in 45.8% (11 of 24). No abnormalities of glucose metabolism were identified. Abnormal pubertal status was seen in three males (12.5%). Abnormal BAs were present in 34.8% (8 of 23). CONCLUSION : Endocrine abnormalities occur at a high frequency in patients with FA, homozygous for a FANCG founder mutation, similar to other FA cohorts. Our data are specific to FA patients with a single genotype, and therefore provide the first genotype‐phenotype information on endocrine abnormalities in South African patients, homozygous for a FANCG founder mutation. Recommendations regarding endocrine screening in this patient subgroup are made, including, but not limited to, baseline testing of thyroid function, fasted insulin and glucose, and IGF‐1 and IGFBP‐3.
Home therapy for inherited bleeding disorders in South Africa : results of a modified Delphi consensus process
(Health and Medical Publishing Group, 2019-09) Mahlangu, J.N.; Naidoo, Y.; Goga, Y.; Vaithlingum, M.; Joubert, J.; Sutton, C.; Potgieter, Joachim; Cruickshank, A.L.; Iorio, A.
BACKGROUND: Optimal care of patients with inherited bleeding disorders requires that bleeding episodes are treated early, or still better prevented, through extension of patient care beyond hospital-based treatment to home-based therapy. In South Africa (SA), adoption of home therapy is variable, in part owing to lack of consensus among healthcare providers on what constitutes home therapy, which patients should be candidates for it, how it should be monitored, and what the barriers to home therapy are. OBJECTIVES: To conduct a modified Delphi process in order to establish consensus on home therapy among haemophilia healthcare providers in SA. METHODS: Treaters experienced in haemophilia care were invited to participate in a consensus-seeking process conducted in three rounds. In round 1, provisional statements around home therapy were formulated as questions and collated in a structured list. In rounds 2 and 3, evolving versions of the questionnaire were administered to participants. Consensus was defined as ≥70% agreement among the participants. RESULTS: The panel composition included an equal number of physicians and non-physicians. The participation rate was 100% through all three consensus rounds. The group reached consensus for 92% of the statements. Consensus of 100% was reached on starting home therapy in paediatric patients, requiring all patients on home therapy to sign informed consent and indemnity, and providing round-the-clock support for patients on home therapy. CONCLUSIONS: The home therapy consensus statements in this report have the potential to translate to policy on home therapy and to guide the initiation, practice and evaluation of home therapy programmes in SA.
HIV and haematopoiesis
(Health and Medical Publishing Group, 2019-09) Durandt, Chrisna; Potgieter, J.C.; Mellet, Juanita; Herd, Candice Lee; Khoosal, R.; Nel, Jan Gert; Rossouw, Theresa M.; Pepper, Michael Sean
Human immunodeficiency virus (HIV) infection not only leads to a compromised immune system, but also disrupts normal haematopoiesis, resulting in the frequent manifestation of cytopenias (anaemia, thrombocytopenia and neutropenia). Although there is a definite association between the severity of cytopenia and HIV disease stage, this relationship is not always linear. For example, cytopenias such as thrombocytopenia may occur during early stages of infection. The aetiology of these haematological abnormalities is complex and multifactorial, including druginduced impaired haematopoiesis, bone marrow suppression due to infiltration of infectious agents or malignant cells, HIV-induced impaired haematopoiesis, and several other factors. In this review, we describe the frequencies of anaemia, thrombocytopenia and neutropenia reported for HIV-infected, treatment-naïve cohorts studied in eastern and southern sub-Saharan African countries. We present a rational approach for the use of diagnostic tests during the workup of HIV-infected patients presenting with cytopenia, and discuss how HIV impacts on haematopoietic stem/progenitor cells (HSPCs) resulting in impaired haematopoiesis. Finally, we describe the direct and indirect effects of HIV on HSPCs which result in defective haematopoiesis leading to cytopenias.
Clofazimine, but not isoniazid or rifampicin, augments platelet activation in vitro
(Frontiers Media, 2018-11-20) Anderson, Ronald; Theron, Annette J.; Nel, Jan Gert; Durandt, Chrisna; Cholo, Moloko C.; Feldman, Charles; Tintinger, Gregory Ronald; ronald.anderson@up.ac.za
Although the inclusion of the cationic amphiphilic, anti-mycobacterial agent, clofazimine, in the chemotherapeutic regimens of patients with multidrug-resistant tuberculosis (TB) has contributed to improved outcomes, concerns remain about the cardiotoxic potential of this agent. Accordingly, the current study was undertaken with the primary objective of investigating the effects of clofazimine, on the reactivity of human platelets in vitro, a seemingly unexplored, mechanism of cardiotoxicity. Platelet-rich plasma (PRP) prepared from the blood of healthy, adult humans was treated with clofazimine (0.625– 10 mg/L), or the primary anti-TB agents, isoniazid and rifampicin (at final concentrations of 5 and 10 mg/L), followed by addition of either adenosine 50-diphosphate (ADP) or thrombin and measurement of platelet activation according to the magnitude of expression of CD62P (P-selectin), as well as the CD62P-mediated formation of heterotypic neutrophil:platelet (NP) aggregates, using flow cytometry. Clofazimine, but neither isoniazid nor rifampicin, caused dose-related potentiation of both ADP- and thrombin-activated expression of CD62P by platelets, achieving statistical significance at threshold concentrations of 0.625 and 2.5 mg/L, respectively, as well as significant formation of N:P aggregates. These stimulatory effects of clofazimine on platelet activation were partly attenuated by pre-treatment of PRP with the membrane-stabilizing agent, a-tocopherol, possibly consistent with a membrane-disruptive mechanism. In conclusion, clofazimine, at concentrations within the therapeutic range, augments platelet activation in vitro, probably by a mechanism linked to membrane destabilization. If operative in vivo, these pro-thrombotic activities of clofazimine may predispose for development of microvascular occlusion, exacerbating an already existing high risk for development of TB-associated cardiovascular disease.
Detection of the Janus kinase 2 V617F mutation using a locked nucleic-acid, real-time polymerase chain reaction assay
(AOSIS Open Journals, 2018-01-31) Senamela, Tshiphiri; Kock, Marleen M.; Becker, Piet J.; Potgieter, Joachim
The purpose of this study was to develop a real time polymerase chain reaction (PCR) assay for the detection of the JAK2 V617F mutation that could be used in diagnostic laboratories. Sanger sequencing and a newly developed locked nucleic-acid, real-time PCR assay were used to detect the JAK2 V617F mutation. There was 100% agreement between the sequencing and PCR analysis. Both assays were able to detect the mutation in all 24 of the 60 test specimens harbouring the mutation.
Dilute Russel viper venom time analysis in a haematology laboratory : an audit
(Wiley, 2018-08) Kruger, Welma; Meyer, Pieter Willem Adriaan; Nel, Jan Gert; jan.nel@up.ac.za
INTRODUCTION : To determine whether the current set of evaluation criteria used for dilute Russel Viper Venom Time (dRVVT) investigations in the routine laboratory meet expectation and identify possible shortcomings. METHODS : All dRVVT assays requested from January 2015 to December 2015 were appraised in this cross‐sectional study. The raw data panels were compared with the new reference interval, established in 2016, to determine the sequence of assays that should have been performed. The interpretive comments were audited, and false‐negative reports identified. Interpretive comments according to three interpretation guidelines were compared. The reagent cost per assay was determined, and reagent cost wastage, due to redundant tests, was calculated. RESULTS : Only ~9% of dRVVT results authorized during 2015 had an interpretive comment included in the report. ~15% of these results were false‐negative interpretations. There is a significant statistical difference in interpretive comments between the three interpretation methods. Redundant mixing tests resulted in R 7477.91 (~11%) reagent cost wastage in 2015. CONCLUSIONS : We managed to demonstrate very evident deficiencies in our own practice and managed to establish a standardized workflow that will potentially render our service more efficient and cost effective, aiding clinicians in making improved treatment decisions and diagnoses. Furthermore, it is essential that standard operating procedures be kept up to date and executed by all staff in the laboratory.
Retrospective data analysis of all requests for flow cytometric immunophenotyping in a tertiary hospital setting
(Society of Medical Laboratory Technologists of South Africa, 2017-06) Prinsloo, Andrea; Nel, Jan Gert; Pool, Roger; andrea.prinsloo@up.ac.za
Flow cytometry is a globally accepted diagnostic tool used for the rapid identification of cells based on their surface and intracellular antigens, especially for the diagnosis of haematological malignancies. The aim of this study was to evaluate the requests received for flow cytometric immunophenotyping and to create a profile of diagnoses. In 2014 data regarding indications and diagnoses were captured from request forms received and final diagnosis reports issued by the Tshwane Academic Division (TAD) of the National Health Laboratory Service (NHLS). A total of 1234 requests were received over the one year period, of which 80.4% were performed and 16.8 % were rejected. The most common indications were leukaemia, lymphoma and cytopenia. Nineteen percent of requests received contained no correct indication or clinical history. In total, 103 and 153 diagnoses were established based on peripheral blood and bone marrow aspirate specimens respectively. Samples were mostly rejected due to sample clotting, electronic gate keeping rules and receiving the specimen more than 24 hours after collection.

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