Verification of a Benchtop Hematology Analyzer with a 5-Part Differential Count: There is Nothing Wrong with Being Small.

BACKGROUND: The benchtop ADVIA 560 AL hematology analyzer (Siemens Healthineers Tarrytown, NY, USA) offers a small footprint and ease of operation making it suitable for satellite laboratories and intensive care units. A verification study of this analyzer was performed. METHODS: Between- and intra-run precision, carry-over, linearity, and throughput were evaluated on the ADVIA 560 AL. Accuracy was assessed on 94 patient samples by comparing the results obtained on the ADVIA 560 AL to the results on the reference Sysmex XN1000 analyzer (Sysmex Corporation, Kobe, Japan). RESULTS: The ADVIA 560 AL showed acceptable imprecision on control material and minimal bias in comparison to the XN 1000 on patient samples with a throughput of 60 samples per hour. The percentage carryover was not significant and the linearity was within acceptable limits. CONCLUSIONS: The ADVIA 560 AL bench-top analyzer is suitable for acute care centers and satellite laboratories owing to its small footprint, ease of use, and reproducible and accurate results.

Assessing Biomarkers in Viral Infection.

Current biomarkers to assess the risk of complications of both acute and chronic viral infection are suboptimal. Prevalent viral infections like human immunodeficiency virus (HIV), hepatitis B and C virus, herpes viruses, and, more recently, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may be associated with significant sequelae including the risk of cardiovascular disease, other end-organ diseases, and malignancies. This review considers some biomarkers which have been investigated in diagnosis and prognosis of key viral infections including inflammatory cytokines, markers of endothelial dysfunction and activation and coagulation, and the role that more conventional diagnostic markers, such as C-reactive protein and procalcitonin, can play in predicting these secondary complications, as markers of severity and to distinguish viral and bacterial infection. Although many of these are still only available in the research setting, these markers show promise for incorporation in diagnostic algorithms which may assist to predict adverse outcomes and to guide therapy.

Selected inflammatory and coagulation biomarkers pre-viral suppression in people with human immunodeficiency virus (HIV) infection without overt cardiovascular disease: Is there a need to redefine reference indices?

BACKGROUND: Human immunodeficiency virus (HIV) infection is prevalent in Africa and causes morbidity and mortality despite antiretroviral therapy (ART). Non-communicable complications of HIV infection include cardiovascular disease (CVD) with thromboses throughout the vascular tree. Ongoing inflammation and endothelial dysfunction in people living with HIV (PLWH) probably contribute significantly to HIV-related CVD. OBJECTIVES: A systematic review was conducted to inform interpretation of 5 biomarkers commonly measured in PLWH namely interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-α), D-dimers, and soluble intracellular and vascular adhesion molecules-1 (sICAM-1 and sVCAM-1) to attempt to define a range for these values in ART naïve PLWH without overt CVD or additional comorbid diseases. METHODS: A systematic search was conducted for all studies documenting the levels of the above biomarkers in ART naïve PLWH published on the PubMed database from 1994 to 2020. RESULTS: The number of publications that reported medians above the assay values was: 4/15 for D-dimer; 0/5 for TNF-α, 8/16 for IL-6, 3/6 for sVCAM-1, and 4/5 for sICAM-1. CONCLUSION: The clinical utility of biomarkers is reduced by the lack of standardisation of the measurement of these parameters, absence of normal reference indices and the lack of uniformity of study protocols in different research centres. This review supports the ongoing use of D-dimers to predict thrombotic and bleeding events in PLWH since the weighted averages across study assays suggest that the median levels do not exceed the reference range. The role of inflammatory cytokine monitoring and measurement of endothelial adhesion markers is less clear.

Distinguishing and overlapping laboratory results of thrombotic microangiopathies in HIV infection: Can scoring systems assist?

BACKGROUND: Patients with Human Immunodeficiency Virus (HIV) infection are at risk of thrombotic microangiopathies (TMAs) notably thrombotic thrombocytopenic purpura (TTP) and disseminated intravascular coagulation (DIC). Overlap between laboratory results exists resulting in diagnostic ambiguity. METHODS: Routine laboratory results of 71 patients with HIV-associated TTP (HIV-TTP) and 81 with DIC with concomitant HIV infection (HIV-DIC) admitted between 2015 and 2021 to academic hospitals in Johannesburg, South Africa were retrospectively reviewed. Both the PLASMIC and the International Society of Thrombosis and Haemostasis (ISTH) DIC scores were calculated. RESULTS: Patients with HIV-TTP had significantly (P < .001) increased schistocytes and features of hemolysis including elevated lactate dehydrogenase (LDH)/upper-limit-of-normal ratio (median of 9 (interquartile range [IQR] 5-12) vs 3 (IQR 2-5)) but unexpectedly lower fibrinogen (median 2.8 (IQR 2.2-3.4) vs 4 g/L (IQR 2.5-9.2)) and higher D-dimer (median 4.8 (IQR 2.4-8.1) vs 3.6 g/L (IQR 1.7-6.2)) levels vs the HIV-DIC cohort. Patients with HIV-DIC were more immunocompromised with frequent secondary infections, higher platelet and hemoglobin levels, more deranged coagulation parameters and less hemolysis. Overlap in scoring systems was however observed. CONCLUSION: The laboratory parameter overlap between HIV-DIC and HIV-TTP might reflect a shared pathogenesis including endothelial dysfunction and inflammation and further research is required. Fibrinogen in DIC may be elevated as an acute phase reactant and D-dimers may reflect the extensive hemostatic activation in HIV-TTP. Inclusion of additional parameters in TMA scoring systems such the LDH/upper-limit-of-normal ratio, schistocytes count and wider access to ADAMTS-13 testing may enhance diagnostic accuracy and ensure appropriate utilization of plasma.

Thrombotic thrombocytopenic purpura in HIV-infected patients: new twists on an old disease.

OBJECTIVE: Investigate the presence of inflammation, endothelial dysfunction and complement activation in patients with HIV-associated thrombotic thrombocytopenic purpura (HIV-TTP) to support the hypothesis that these processes probably contribute to the development of this thrombotic microangiopathy. DESIGN: A prospective, investigational cohort study of 35 consecutive patients diagnosed with HIV-associated TTP presenting to three academic, tertiary care hospitals in Johannesburg, South Africa over 2 years. METHODS: The patients with HIV-TTP received therapeutic plasma therapy and supportive treatment. Demographic data, the results of routine investigations and patient outcomes were recorded. Peripheral blood samples were collected prior to and on completion of plasma therapy and the following additional parameters were assessed at both time points: activity of the von Willebrand factor (VWF) cleaving protease, a-disintegrin-and-metalloproteinase-with-thrombospondin-motifs 13 (ADAMTS-13) and the presence of ADAMTS-13 autoantibodies, levels of pro-inflammatory cytokines, interleukin-6 and tumour necrosis factor-alpha, and two endothelial cell adhesion molecules. Complement activation was assessed by sequential measurement of C3 and C4 as well as levels of the complement inhibitor, factor H. RESULTS: The inflammatory and endothelial activation markers were significantly ( P  < 0.001) elevated in the cohort of patients prior to plasma therapy compared with levels on discharge. Complement was activated and normalized with therapy. The ADAMTS-13 levels were reduced with significant auto-antibodies to this protease at presentation. CONCLUSION: Inflammation in HIV mediates endothelial damage and complement activation. This study proposes that these processes are probably contributory to the development of HIV-TTP, which can therefore be characterized in part as a complementopathy, resembling TTP-like syndrome.

Von Willebrand Factor Multimer Patterns in People Living with Human Immunodeficiency Virus Infection.

BACKGROUND: Endothelial dysfunction contributes to hypercoagulability in people with HIV (PWH). A surrogate marker of this is elevated von Willebrand factor (VWF) which has been documented in PWH. This study compared VWF multimer patterns in PWH who were anti-retroviral therapy (ART) naive and immune reconstituted on ART. METHODS: VWF multimer analysis was performed with a semi-quantitative electrophoresis assay on plasma from 79 PWH (39 ART-naive and 40 virally suppressed on ART) and 25 normal control samples. RESULTS: The total optical density mean VWF level was significantly increased in PWH and higher in ART-naive versus ART-exposed, virally suppressed patients (p < 0.0001). No quantitative difference was demonstrated in the multimer distribution pattern between the groups. CONCLUSIONS: Our findings suggest that there is no difference in multimer distribution between ART-naive and virally suppressed PWH. Due to a small sample size, confirmation in a larger study is required.

HIV-associated thrombotic thrombocytopenic purpura (HIV-TTP): A practical guide and review of the literature.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP), a serious thrombotic microangiopathy (TMA), is prevalent in the South African HIV-infected population. The exact pathogenesis of HIV-associated TTP (HIV-TTP) is however still unclear with diagnostic and therapeutic inconsistancies. METHODS: A systematic review of the published literature regarding HIV-TTP was performed. RESULTS: HIV-TTP is still associated with significant morbidity and mortality in Africa despite the availability of anti-retroviral therpy (ART). Diagnosis of HIV-TTP requires the presence of a micro-angiopathic haemolytic anaemia with significant red blood cell schistocytes and thrombocytopenia in the absence of another TMA but background activation of the coagulation system and inflammation in HIV infected people can result in diagnostic anbiguity. Plasma therapy in the form of infusion or exchange is successful but expensive, associated with side-effects and not widely available. Adjuvant immunosuppression therapy may of benefit in patients with HIV-TTP and ART must always be optimised. Endothelial dysfunction caused by chronic inflammation and complement activation most likely contributes to the development of HIV-TTP. CONCLUSION: The role of adjuvant immunomodulating therpy, the therapeutic targets and pathogenic contribution from endothelial dysfunction in HIV-TTP requires further investigation.

Diagnosis of human immunodeficiency virus associated disseminated intravascular coagulation.

INTRODUCTION: Disseminated intravascular Coagulation (DIC) is a thrombotic microangiopathy which may complicate a number of severe disease processes including sepsis. Development of microvascular thromboses results in consumption of coagulation factors and platelets and ultimate bleeding. Patients with HIV infection (PWH) often present with baseline dysregulation of the coagulation system which may increase severity and derangement of DIC presentation. Previously, we have shown that HIV is a significant risk factor for development of DIC. METHODOLOGY: We conducted a retrospective record review of all DIC screens submitted to our tertiary coagulation laboratory in Johannesburg, South Africa, over a one year period and compared the laboratory presentation of DIC in PWH with presentation of DIC in patients without HIV infection. RESULTS: Over the year, 246 patients fulfilled the International Society of Thrombosis and Haemostasis (ISTH) diagnostic criteria for DIC- 108 were confirmed HIV-infected and 77 were confirmed uninfected. PWH and DIC presented at a significantly earlier age (41 vs 46 years respectively, p<0.02). The prothrombin time was significantly more prolonged (30.1s vs 26.s), the d-dimer levels were substantially higher (5.89mg/L vs 4.52mg/L) and the fibrinogen (3.92g/L vs 1.73g/L) and platelet levels (64.8 vs 114.8x109/l) were significantly lower in PWH. PWH also showed significant synthetic liver dysfunction and higher background inflammation. CONCLUSION: PWH who fulfil the diagnostic criteria for DIC show significantly more dysregulation of the haemostatic system. This may reflect baseline abnormalities including endothelial dysfunction in the context of inflammation and liver dysfunction.

Assessment of candidate variants causative of inborn metabolic diseases in SUDI cases in South Africa, and a case report.

Sudden unexpected death in infants (SUDI) is a devastating event, and unfortunately is still a burden in many parts of the world, including in South Africa. Due to the absence of routine testing for inborn metabolic diseases in newborns and in a post-mortem context, little is known about the presence of metabolic diseases in local SUDI cases. The aim of this study was to genotype five candidate variants previously associated with metabolic disorders in a cohort of SUDI cases (n = 169) from Salt River Mortuary, Cape Town. DNA was isolated from blood, and SNaPshot® PCR and Sanger sequencing were used to genotype the following variants: ACADM: c.583G > A, ACADM: c.985A > G, GCDH: c.877G > A/T, GALT: c.404C > G/T and GALT: c.563A > G. Four carriers of GCDH: c.877G > A/T were identified, while one infant was homozygous for the founder mutation GALT: c.404C > G/T; the latter which is causative of galactosaemia and was previously undiagnosed. During the follow-up with the family, it emerged that the affected infant's identical twin had subsequently demised. The findings in this study highlight possible new candidate variants to assess in South African SUDI cases, and these results directly contribute to the development of a molecular autopsy which is locally relevant. It is evident that until newborn screening becomes routine and accessible in South Africa, molecular autopsies should include testing for inherited metabolic disorders, as it holds potential to save lives.

A Rare Prothrombin Gene Mutation C20209T in a South African Patient with Pulmonary Embolism in Pregnancy: a Case Study and Systematic Review.

BACKGROUND: The G202010A prothrombin gene mutation is a documented prothrombotic risk factor in Caucasian patients. Several other mutations have been described within the prothrombin gene, predominantly in non-Caucasians, including the C20209T mutation. The clinical significance of this mutation is uncertain, but it has been associated with thrombotic events and pregnancy complications. METHODS: We describe a 28-year-old black South African woman who presented with pulmonary embolism during pregnancy. She was investigated for underlying prothrombotic biomarkers. RESULTS: Genetic screening for the prothrombin G202010A mutation by real-time polymerase chain reaction and melting curve analysis demonstrated an atypical mutant peak. Sequencing confirmed a variant C20209T prothrombin mutation. CONCLUSIONS: This is the first report of the C20209T mutation in the Southern African population. It remains uncertain whether genetic testing should be offered routinely to non-Caucasian patients in a resource-limited setting.

The Utility of the Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Assay in Detecting Abnormalities in Lipid Metabolism and Cardiovascular Risk in an HIV-Infected South African Cohort.

People with HIV (PWH) have an increased prevalence of cardiovascular disease (CVD) compared to uninfected patients. Lipoprotein-associated phospholipase A2 (Lp-PLA2) catalyzes the synthesis of pro-inflammatory lipids that recruit monocytes. Current guidelines for assessing cardiovascular risk in HIV-infected patients suggest that Lp-PLA2 may be a useful surrogate marker for CVD health in this patient population. Lipoprotein-associated phospholipase A2, lipids, glucose, physical parameters, and carotid intimal-medial thickness (CIMT) were measured in 98 participants (49 HIV-uninfected, 27 antiretroviral therapy [ART]-naive PWH, and 22 ART-treated PWH). HIV viral load (VL) and CD4+ T-cell count were measured in HIV-infected participants. Lipoprotein-associated phospholipase A2 was increased in participants on protease inhibitor (PI) ART (median 50.5 vs 127.0 nmol/mL, P = .05) and correlated with age, body mass index, and cholesterol. Lipoprotein-associated phospholipase A2 was not related to Framingham risk score or CIMT but correlated directly with VL (r = .323, P = .025) and inversely with CD4+ T-cell count (r = -.727, P < .001). Lipoprotein-associated phospholipase A2 was increased in HIV-infected participants on PIs and correlated strongly with VL and CD4+ T-cell count suggesting that HIV-associated inflammation is linked to increased Lp-PLA2, providing a mechanistic link between HIV and CVD.

Good Fences Make Good Neighbors: Human Immunodeficiency Virus and Vascular Disease.

Cardiovascular disease, venous thrombosis, and microvascular disease in people with HIV (PWH) is predicted to increase in an aging HIV-infected population. Endothelial damage and dysfunction is a risk factor for cardiovascular events in PWH and is characterized by impaired vascular relaxation and decreased nitric oxide availability. Vascular disease has been attributed to direct viral effects, opportunistic infections, chronic inflammation, effects of antiretroviral therapy, and underlying comorbid conditions, like hypertension and use of tobacco. Although biomarkers have been examined to predict and prognosticate thrombotic and cardiovascular disease in this population, more comprehensive validation of risk factors is necessary to ensure patients are managed appropriately. This review examines the pathogenesis of vascular disease in PWH and summarizes the biomarkers used to predict vascular disease in this population.

Verification of the Performance of a Fully Automated Coagulation Analyzer and Ongoing Assessment of External Quality Assurance Results in an Academic Laboratory in South Africa.

BACKGROUND: Verification of the performance of analytical platforms is indicated prior to adoption of new Technology for patient sample analysis. Acceptance criteria for the performance of coagulation analytical platforms are not always readily available and is complicated by the multiple assays and test principles in this section of the clinical laboratory. Coagulation samples are also prone to pre-analytical, post-sample collection variables potentially interfering with accuracy analysis. METHODS: This verification study assessed the accuracy of the automated STAGO STA-R Max® coagulation analyzers by means of a comparison study of results obtained on the previously validated STAGO STA-R Evolution® analyzer for 22 coagulation parameters on 40 individual patient samples for each parameter. Within- and between- run reproducibility on commercial control material, carry-over from abnormal to normal samples and the interference of bilirubin, hemoglobin and lipids on the chromogenic analytical channel were also assessed. Ongoing evaluation of the analyzer performance was assessed by External Quality Assurance (EQA) scheme participation. RESULTS: The reproducibility (precision) on 2 levels (Normal and Pathological) commercial control material was acceptable with co-efficient of variance (CV) results below the manufacturer target % CVs. The correlation study demonstrated accuracy of results obtained on the analyzers for all parameters except for D-dimers and coagulation Factor VII. Subsequent EQA performance for these two parameters were however satisfactory. Interference from bilirubin, hemoglobin and lipids did occur in the chromogenic channel. No clinically significant carry over from abnormal to normal samples were observed. CONCLUSIONS: The performance of the STAGO STA-R Max® analyzer is acceptable across the full coagulation test repertoire with the exception of the von Willebrand activity assay. Participation in EQA scheme assessments will be an integral part of ongoing monitoring of the performance of this automated analyzer.

The Hemostatic Conundrum of Thrombotic Thrombocytopenic Purpura: a Case Report.

BACKGROUND: In thrombotic thrombocytopenic purpura (TTP), von Willebrand factor (VWF)-platelet rich thrombi form in the microvasculature with ischemia in vital organs. The pathogenesis relates to deficiency of the VWF cleaving protease, ADAMTS-13. METHODS: A case study of a 58-year-old female with previously undiagnosed HIV (human immunodeficiency virus) infection, left hemiparesis, and soft tissue hemorrhages. RESULTS: Investigations revealed microangiopathic hemolytic anemia and a left basal ganglia infarct on magnetic resonance imaging (MRI). A diagnosis of acquired, HIV-related TTP was made and therapeutic plasma exchange and antiretroviral therapy were initiated. CONCLUSIONS: The combined thrombotic and hemorrhagic manifestations of TTP pose therapeutic challenges.

Analytical Performance of a New Immunoturbidimetric Assay for von Willebrand Factor (VWF) Activity Testing.

BACKGROUND: Von Willebrand disease requires laboratory confirmation with quantitative and qualitative measurements of von Willebrand factor (VWF). Qualitative VWF-activity (VWF-Ac) tests have poor inter- and intra-laboratory reproducibility with coefficients of variation (CVs) as high as 64%, often lacking accuracy at low VWF-Ac levels. METHODS: This study evaluated the recently launched immunoturbidometric STAGO® STA-VWF:RCo® reagent for VWF-Ac. Accuracy was evaluated on 32 samples by comparing results using the Siemens® Innovance® reagent. An intra-run reproducibility study was performed on controls. Linearity and lower limit of detection was studied on external-quality-assurance (EQA) material with a known VWF-Ac level. RESULTS: STA-VWF:RCo® reagent results were within clinical interpretation agreement with Siemens® Innovance®. The reproducibility study yielded % CVs of 8.41 for normal and 11.46 for abnormal controls and the assay was linear between 73 and 14.6% and remained linear to 2% with extrapolation. CONCLUSIONS: The STAGO® STA-VWF:RCo® reagent showed clinically meaningful accuracy and acceptable precision.