Are Gamers Prone to eThrombosis during Long Gaming Sessions?

During the last two decades, several cases of venous thrombosis (VTE) after a prolonged period at a computer have been described, denominated as "eThrombosis". Video gaming on a computer has become very popular and can be a social activity where several players gather to play against each other or in a virtual environment for several days ("LAN (i.e., Local Area Network) parties") where the participants are sedentary and consuming calorie-rich food items. The aim of this study was to investigate potential coagulation activation during a 42 h LAN party. Nine male gamers volunteered for the LAN party. Citrated blood was sampled before and every 6 h, and plasma was analyzed for thrombin generation, thrombin-antithrombin complexes (TAT), prothrombin fragment 1 + 2 (F1 + 2), and D-dimer. Thrombin generation increased slightly but not significantly during the LAN party, whereas the coagulation activation markers were unchanged. These results do not indicate that the coagulation system is activated significantly during 42 h of gaming with minimal physical activity. Although increased activity cannot be excluded, it does not directly indicate a risk of VTE in general.

Monocyte recruitment in venous pulmonary embolism at time of cancer diagnosis in upper gastrointestinal cancer patients.

Upper gastrointestinal cancer is frequently complicated by venous thromboembolisms (VTE), especially pulmonary embolisms (PE) increase the mortality rate. Monocytes are a part of the innate immune system and up-regulation may indicate an ongoing inflammatory response or infectious disease and has lately been associated with a moderate risk of suffering from VTE. This prospectively study aims to compare the incidence of pulmonary embolism with markers of coagulation and compare it to the absolute monocyte count. A consecutive cohort of 250 patients with biopsy proven upper gastrointestinal cancer (i.e. pancreas, biliary tract, esophagus and gastric cancer) where included at the time of cancer diagnosis and before treatment. All patients underwent bilateral compression ultrasonography for detection of deep vein thrombosis (DVT). Of these 143 had an additionally pulmonary angiografi (CTPA) with the staging computer tomography. 13 of 250 patients (5.2%) had a DVT and 11 of 143 (7.7%) had CTPA proven PE. PE was significantly more common among patients with elevated D-dimer (OR 11.62, 95%CI: 1.13-119, P = 0.039) and elevated absolute monocyte count (OR 7.59, 95%CI: 1.37-41.98, P = 0.020). Only patients with pancreatic cancer had a significantly higher risk of DVT (OR 11.03, 95%CI: 1.25-97.43, P = 0.031). The sensitivity of absolute monocyte count was 63.6 (95%CI: 30.8-89.1) and specificity 80.3 (95%CI: 72.5-86.7), with a negative predictive value of 96.4 (95%CI: 91-99) in PE. An increased absolute monocyte count was detected in patients suffering from PE but not DVT, suggesting a possible interaction with the innate immune system.

Survival after cancer-related venous thrombosis: the Scandinavian Thrombosis and Cancer Study.

Patients with cancer have an increased risk of developing venous thromboembolism (VTE), and this combination is reported to result in poorer survival compared with cancer alone. This study aimed to investigate the impact of VTE on the survival of patients with cancer in a general population. The Scandinavian Thrombosis and Cancer (STAC) cohort, a population-based cohort including 144 952 participants without previous VTE or cancer, was used. During follow-up, cancer and VTE incidences were registered. "Cancer-related VTE" was defined as VTE diagnosed in patients with overt or occult cancer. The survival of participants without cancer and/or VTE ("disease-free") was compared with the survival of participants with cancer and cancer-related VTE. Cox regression models with cancer and VTE as time-varying exposures were performed to calculate hazard ratios for death. Subanalyses were performed across cancer types and stages and VTE type (deep vein thrombosis or pulmonary embolism). During follow-up (mean, 11.7 years), 14 621 participants developed cancer, and 2444 developed VTE, of which 1241 were cancer-related. The mortality rates (per 100 person years) for disease-free participants, VTE only, cancer only, and cancer-related VTE were 0.63, 5.0, 9.2, and 45.3, respectively. Compared with patients with cancer only, the risk of death for patients with cancer-related VTE was increased 3.4-fold. Within all cancer types, the occurrence of VTE increased the mortality risk 2.8- to 14.7-fold. In a general population, patients with cancer with VTE had a 3.4-fold higher mortality risk than patients with cancer without VTE, independent of cancer type.

Atrial fibrillation, liver cirrhosis, thrombosis, and bleeding: A Danish population-based cohort study.

OBJECTIVES: We examined the impact of liver cirrhosis on the risk of thromboembolic events and bleeding complications in patients with atrial fibrillation or flutter (AFF). METHODS: This population-based cohort study used data from Danish health registries. We identified all patients with a first-time diagnosis of AFF during 1995 to 2015, and followed them from their AFF diagnosis until the end of 2016. Patients were categorized according to the presence or absence of liver cirrhosis. We computed incidence rates per 1000 person-years and hazard ratios (HRs) with 95% confidence intervals (CIs) based on Cox regression analyses, adjusting for age, CHA2DS2VASc score, and Charlson Comorbidity Index score. RESULTS: We identified 273 225 patients with AFF. Of these, 1463 (0.54%) had liver cirrhosis. During 0 to 5 years of follow-up, compared to patients without liver cirrhosis, patients with liver cirrhosis had higher incidence rates and hazards of ischemic stroke (29.7 vs 21.6; HR, 1.3; 95% CI, 1.1-1.6), venous thromboembolism (9.2 vs 5.5; HR, 1.5; 95% CI, 1.2-2.3), but not myocardial infarction (10.2 vs 11.2; HR, 0.9; 95% CI, 0.7-1.2). Patients with liver cirrhosis also had higher rates of hemorrhagic stroke (5.8 vs 3.3; HR, 1.7; 95% CI, 1.1-2.6), subdural hemorrhage (5.3 vs 1.6; HR, 3.2; 95% CI, 2.1-4.9), hemorrhage of the lung or urinary tract (24.6 vs 15.2; HR, 1.6; 95% CI, 1.3-2.0), and gastrointestinal hemorrhage (34.5 vs 10.4; HR, 3.3; 95% CI, 2.7-3.9). CONCLUSION: In patients with AFF, liver cirrhosis was associated with an elevated risk of ischemic stroke, venous thromboembolism, and all evaluated bleeding complications.

Framework for identification and measurement of spillover effects in policy implementation: intended non-intended targeted non-targeted spillovers (INTENTS).

BACKGROUND: There is increasing awareness among researchers and policymakers of the potential for healthcare interventions to have consequences beyond those initially intended. These unintended consequences or "spillover effects" result from the complex features of healthcare organisation and delivery and can either increase or decrease overall effectiveness. Their potential influence has important consequences for the design and evaluation of implementation strategies and for decision-making. However, consideration of spillovers remains partial and unsystematic. We develop a comprehensive framework for the identification and measurement of spillover effects resulting from changes to the way in which healthcare services are organised and delivered. METHODS: We conducted a scoping review to map the existing literature on spillover effects in health and healthcare interventions and used the findings of this review to develop a comprehensive framework to identify and measure spillover effects. RESULTS: The scoping review identified a wide range of different spillover effects, either experienced by agents not intentionally targeted by an intervention or representing unintended effects for targeted agents. Our scoping review revealed that spillover effects tend to be discussed in papers only when they are found to be statistically significant or might account for unexpected findings, rather than as a pre-specified feature of evaluation studies. This hinders the ability to assess all potential implications of a given policy or intervention. We propose a taxonomy of spillover effects, classified based on the outcome and the unit experiencing the effect: within-unit, between-unit, and diagonal spillover effects. We then present the INTENTS framework: Intended Non-intended TargEted Non-Targeted Spillovers. The INTENTS framework considers the units and outcomes which may be affected by an intervention and the mechanisms by which spillover effects are generated. CONCLUSIONS: The INTENTS framework provides a structured guide for researchers and policymakers when considering the potential effects that implementation strategies may generate, and the steps to take when designing and evaluating such interventions. Application of the INTENTS framework will enable spillover effects to be addressed appropriately in future evaluations and decision-making, ensuring that the full range of costs and benefits of interventions are correctly identified.

Who is paid in pay-for-performance? Inequalities in the distribution of financial bonuses amongst health centres in Zimbabwe.

Although pay-for-performance (P4P) schemes have been implemented across low- and middle-income countries (LMICs), little is known about their distributional consequences. A key concern is that financial bonuses are primarily captured by providers who are already better able to perform (for example, those in wealthier areas), P4P could exacerbate existing inequalities within the health system. We examine inequalities in the distribution of pay-outs in Zimbabwe's national P4P scheme (2014-2016) using quantitative data on bonus payments and facility characteristics and findings from a thematic policy review and 28 semi-structured interviews with stakeholders at all system levels. We found that in Zimbabwe, facilities with better baseline access to guidelines, more staff, higher consultation volumes and wealthier and less remote target populations earned significantly higher P4P bonuses throughout the programme. For instance, facilities that were 1 SD above the mean in terms of access to guidelines, earned 90 USD more per quarter than those that were 1 SD below the mean. Differences in bonus pay-outs for facilities that were 1 SD above and below the mean in terms of the number of staff and consultation volumes are even more pronounced at 348 USD and 445 USD per quarter. Similarly, facilities with villages in the poorest wealth quintile in their vicinity earned less than all others-and 752 USD less per quarter than those serving villages in the richest quintile. Qualitative data confirm these findings. Respondents identified facility baseline structural quality, leadership, catchment population size and remoteness as affecting performance in the scheme. Unequal distribution of P4P pay-outs was identified as having negative consequences on staff retention, absenteeism and motivation. Based on our findings and previous work, we provide some guidance to policymakers on how to design more equitable P4P schemes.

Association of smoking and cancer with the risk of venous thromboembolism: the Scandinavian Thrombosis and Cancer cohort.

Smoking is a well-established risk factor for cancer, and cancer patients have a high risk of venous thromboembolism (VTE). Conflicting results have been reported on the association between smoking and risk of VTE, and the effect of smoking on VTE-risk in subjects with cancer is scarcely studied. We aimed to investigate the association between smoking and VTE in subjects with and without cancer in a large population-based cohort. The Scandinavian Thrombosis and Cancer (STAC) cohort included 144,952 participants followed from 1993-1997 to 2008-2012. Information on smoking habits was derived from self-administered questionnaires. Active cancer was defined as the first two years following the date of cancer diagnosis. Former smokers (n = 35,890) and those with missing information on smoking status (n = 3680) at baseline were excluded. During a mean follow up of 11 years, 10,181 participants were diagnosed with cancer, and 1611 developed incident VTE, of which 214 were cancer-related. Smoking was associated with a 50% increased risk of VTE (HR 1.49, 95% CI 1.12-1.98) in cancer patients, whereas no association was found in cancer-free subjects (HR 1.07, 95% CI 0.96-1.20). In cancer patients, the risk of VTE among smokers remained unchanged after adjustment for cancer site and metastasis. Stratified analyses showed that smoking was a risk factor for VTE among those with smoking-related and advanced cancers. In conclusion, smoking was associated with increased VTE risk in subjects with active cancer, but not in those without cancer. Our findings imply a biological interaction between cancer and smoking on the risk of VTE.

Prothrombotic genotypes and risk of venous thromboembolism in occult cancer.

BACKGROUND: Studies have reported that the combination of some prothrombotic genotypes and overt cancer yields a synergistic effect on VTE risk. Whether individual prothrombotic genotypes or number of risk alleles in a genetic risk score (GRS) affect VTE risk in occult cancer have not been addressed. The aim of this study was to investigate the joint effect of five prothrombotic genotypes and occult cancer on VTE risk. METHODS: Cases with incident VTE (n = 1566) and a subcohort (n = 14,537) were sampled from the Scandinavian Thrombosis and Cancer Cohort (1993-2012). Five single nucleotide polymorphisms previously reported in a GRS were genotyped: ABO (rs8176719), F5 (rs6025), F2 (rs1799963), FGG (rs2066865) and F11 (rs2036914). Hazard ratios (HRs) for VTE by individual SNPs and GRS were estimated according to non-cancer and occult cancer (one year preceding a cancer diagnosis) exposure. RESULTS: Occult cancer occurred in 1817 subjects, and of these, 93 experienced a VTE. The VTE risk was 4-fold higher (HR 4.05, 95% CI 3.28-5.00) in subjects with occult cancer compared with those without cancer. Among subjects with occult cancer, those with VTE had a higher proportion of prothrombotic and advanced cancers than those without VTE. The VTE risk increased according to individual prothrombotic genotypes and GRS in cancer-free subjects, while no such effect was observed in subjects with occult cancer (HR for ≥4 versus ≤1 risk alleles in GRS: 1.14, 95% CI 0.61-2.11). CONCLUSIONS: Five well-established prothrombotic genotypes, individually or combined, were not associated with increased risk of VTE in individuals with occult cancer.

Venous thromboembolism after lower extremity orthopedic surgery: A population-based nationwide cohort study.

BACKGROUND: Venous thromboembolism (VTE) causes morbidity and mortality in the general population. Several events occur after lower limb orthopedic surgery, but the contribution from various types of lower limb surgery is not well known. OBJECTIVE: To investigate the postoperative incidence of VTE for all types of lower extremity orthopedic surgery compared with the background population. METHODS: Individual-level linkage of Danish nationwide register data for all Danish residents with first-time orthopedic surgery of the lower limb (1996-2017) and, for each of these, four controls from the general population matched on age, sex, and history of VTE. Adjusted hazard ratios (HR) compared the postoperative risk of VTE to the matched controls. RESULTS: In total 7203 of the 1 012 823 patients with a first orthopedic procedure had a VTE within 180 days after surgery, corresponding to a postoperative cumulative incidence of 0.71% (95% confidence interval [CI], 0.70-0.73). The cumulative incidence of VTE among controls was 0.11% (95% CI, 0.11-0.12). The HR of VTE within the first 30 days after surgery below knee level was 20.5 (95% CI, 17.9-23.5) compared with matched controls. The HRs of VTE after minor distal procedures (eg, meniscectomy and arthroscopies) were 2.9 (95% CI, 1.9-4.4) to 7.1 (95% CI, 6.4-8.0). CONCLUSION: All types of lower limb orthopedic surgery including minor distal procedures were associated with higher rates of VTE compared with matched controls, in particular within the first 30 days after surgery.

A realist review to assess for whom, under what conditions and how pay for performance programmes work in low- and middle-income countries.

Pay for performance (P4P) programmes are popular health system-focused interventions aiming to improve health outcomes in low-and middle-income countries (LMICs). This realist review aims to understand how, why and under what circumstance P4P works in LMICs.We systematically searched peer-reviewed and grey literature databases, and examined the mechanisms underpinning P4P effects on: utilisation of services, patient satisfaction, provider productivity and broader health system, and contextual factors moderating these. This evidence was then used to construct a causal loop diagram.We included 112 records (19 grey literature; 93 peer-reviewed articles) assessing P4P schemes in 36 countries. Although we found mixed evidence of P4P's effects on identified outcomes, common pathways to improved outcomes include: community outreach; adherence to clinical guidelines, patient-provider interactions, patient trust, facility improvements, access to drugs and equipment, facility autonomy, and lower user fees. Contextual factors shaping the system response to P4P include: degree of facility autonomy, efficiency of banking, role of user charges in financing public services; staffing levels; staff training and motivation, quality of facility infrastructure and community social norms. Programme design features supporting or impeding health system effects of P4P included: scope of incentivised indicators, fairness and reach of incentives, timely payments and a supportive, robust verification system that does not overburden staff. Facility bonuses are a key element of P4P, but rely on provider autonomy for maximum effect. If health system inputs are vastly underperforming pre-P4P, they are unlikely to improve only due to P4P. This is the first realist review describing how and why P4P initiatives work (or fail) in different LMIC contexts by exploring the underlying mechanisms and contextual and programme design moderators. Future studies should systematically examine health system pathways to outcomes for P4P and other health system strengthening initiatives, and offer more understanding of how programme design shapes mechanisms and effects.

Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation.

Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells' biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusion chromatography (SEC), and peptide-affinity precipitation (PAP/ME kit) for subsequent MS-based proteome analysis. Successful EV isolation was evaluated by nanoparticle-tracking analysis, immunoblotting, and transmission electron microscopy, while EV abundance, EV subtypes, and contamination was evaluated by label-free tandem MS. High-speed centrifugation and SEC isolates showed high EV abundance at the expense of contamination by non-EV proteins and lipoproteins, respectively. These two methods also resulted in EVs of a similar type, however, with smaller EVs in SEC isolates. PAP isolates had a relatively low EV abundance and high contamination. We consider high-speed centrifugation and SEC suitable as EV isolation for MS biomarker studies, where the choice between the two should depend on the scientific questions and whether the focus is on larger or smaller EVs or a combination of both.

How are pay-for-performance schemes in healthcare designed in low- and middle-income countries? Typology and systematic literature review.

BACKGROUND: Pay for performance (P4P) schemes provide financial incentives to health workers or facilities based on the achievement of pre-specified performance targets and have been widely implemented in health systems across low and middle-income countries (LMICs). The growing evidence base on P4P highlights that (i) there is substantial variation in the effect of P4P schemes on outcomes and (ii) there appears to be heterogeneity in incentive design. Even though scheme design is likely a key determinant of scheme effectiveness, we currently lack systematic evidence on how P4P schemes are designed in LMICs. METHODS: We develop a typology to classify the design of P4P schemes in LMICs, which highlights different design features that are a priori likely to affect the behaviour of incentivised actors. We then use results from a systematic literature review to classify and describe the design of P4P schemes that have been evaluated in LMICs. To capture academic publications, Medline, Embase, and EconLit databases were searched. To include relevant grey literature, Google Scholar, Emerald Insight, and websites of the World Bank, WHO, Cordaid, Norad, DfID, USAID and PEPFAR were searched. RESULTS: We identify 41 different P4P schemes implemented in 29 LMICs. We find that there is substantial heterogeneity in the design of P4P schemes in LMICs and pinpoint precisely how scheme design varies across settings. Our results also highlight that incentive design is not adequately being reported on in the literature - with many studies failing to report key design features. CONCLUSIONS: We encourage authors to make a greater effort to report information on P4P scheme design in the future and suggest using the typology laid out in this paper as a starting point.

MicroRNAs and Neutrophil Activation Markers Predict Venous Thrombosis in Pancreatic Ductal Adenocarcinoma and Distal Extrahepatic Cholangiocarcinoma.

Cancer-associated venous thrombosis (VTE) increases mortality and morbidity. However, limited tools are available to identify high risk patients. Upon activation, neutrophils release their content through different mechanisms, thereby prompting thrombosis. We explored plasma microRNAs (miRNAs) and neutrophil activation markers to predict VTE in pancreatic ductal adenocarcinoma (PDAC) and distal extrahepatic cholangiocarcinoma (DECC). Twenty-six PDAC and 6 DECC patients recruited at cancer diagnosis, were examined for deep vein thrombosis and pulmonary embolisms, and were then followed-up with clinical examinations, blood collections, and biCUS. Ten patients developed VTE and were compared with 22 age- and sex-matched controls. miRNA expression levels were measured at diagnosis and right before VTE, and neutrophil activation markers (cell-free DNA, nucleosomes, calprotectin, and myeloperoxidase) were measured in every sample obtained during follow-up. We obtained a profile of 7 miRNAs able to estimate the risk of future VTE at diagnosis (AUC = 0.95; 95% Confidence Interval (CI) (0.987, 1)) with targets involved in the pancreatic cancer and complement and coagulation cascades pathways. Seven miRNAs were up- or down-regulated before VTE compared with diagnosis. We obtained a predictive model of VTE with calprotectin as predictor (AUC = 0.77; 95% CI (0.57, 0.95)). This is the first study that addresses the ability of plasma miRNAs and neutrophil activation markers to predict VTE in PDAC and DECC.

Real-Time Interferometric Refractive Index Change Measurement for the Direct Detection of Enzymatic Reactions and the Determination of Enzyme Kinetics.

Back scatter interferometry (BSI) is a sensitive method for detecting changes in the bulk refractive index of a solution in a microfluidic system. Here we demonstrate that BSI can be used to directly detect enzymatic reactions and, for the first time, derive kinetic parameters. While many methods in biomedical assays rely on detectable biproducts to produce a signal, direct detection is possible if the substrate or the product exert distinct differences in their specific refractive index so that the total refractive index changes during the enzymatic reaction. In this study, both the conversion of glucose to glucose-6-phosphate, catalyzed by hexokinase, and the conversion of adenosine-triphosphate to adenosine di-phosphate and mono-phosphate, catalyzed by apyrase, were monitored by BSI. When adding hexokinase to glucose solutions containing adenosine-triphosphate, the conversion can be directly followed by BSI, which shows the increasing refractive index and a final plateau corresponding to the particular concentration. From the initial reaction velocities, KM was found to be 0.33 mM using Michaelis⁻Menten kinetics. The experiments with apyrase indicate that the refractive index also depends on the presence of various ions that must be taken into account when using this technique. This study clearly demonstrates that measuring changes in the refractive index can be used for the direct determination of substrate concentrations and enzyme kinetics.

Elevated blood plasma levels of tissue factor-bearing extracellular vesicles in patients with atrial fibrillation.

BACKGROUND: The risk of thrombus formation in the left atrial appendage (LAA) in patients with atrial fibrillation (AF) may result from blood stasis, local endocardial changes, and/or changed blood composition. Extracellular vesicles (EVs), especially subtypes exposing tissue factor (TF), have procoagulant capacity. We hypothesized that blood concentrations of TF-bearing EVs and other procoagulant biomarkers are elevated in AF patients, particularly in the LAA lumen. METHODS: From 13 AF patients and 12 controls a venous blood sample was drawn prior to cardiac surgery. Intraoperatively, venous blood and blood directly from the LAA was drawn. Plasma levels of EVs, including TF- and cell type specific antigen-bearing EVs, were measured using a protein microarray platform. Plasma levels of TF, von Willebrand factor (vWF), cell free deoxyribonucleic acid (cf-DNA), procoagulant phospholipids (PPLs), and total submicron particles were also evaluated. RESULTS: Significantly higher EV levels, including a several-fold higher median level of TF-bearing EVs were measured in AF patients compared with controls. Median concentrations of TF and vWF were approximately 40% and 30% higher, respectively, in the AF group than in the control group, while no significant differences in levels of cf-DNA, PPLs, or total submicron particles were observed. No significant differences in levels of any of the measured analytes were observed between intraoperative venous and LAA samples. CONCLUSIONS: Increased plasma concentrations of TF in AF patients are accompanied and probably at least partly explained by increased levels of TF-bearing EVs, which may be mechanistically involved in increased thrombogenicity in AF patients.

The smartphone camera as a potential method for transcutaneous bilirubin measurement.

BACKGROUND: Hyperbilirubinemia is a common problem in neonates that can progress into kernicterus. Suspected neonatal hyperbilirubinemia is a common reason for contact with the healthcare system. The severity and management of jaundice are determined based on estimated bilirubin levels. However, no easy and accessible tool for self-assessing neonatal jaundice is currently available. Smartphones could potentially be transformed into a medical device that could be used by both patients and practitioners. OBJECTIVE: To investigate whether a digital image produced by a camera embedded on a smartphone can be a used as a screening tool for neonatal hyperbilirubinemia. STUDY DESIGN: A total of 64 randomly selected newborns were enrolled. The inclusion criteria were healthy Caucasians, gestational age >35 weeks, age >24 hours and ≤14 days old, and parental informed consent. The exclusion criteria were facial skin lesions and light treatment. Images of the glabella were obtained with an iPhone 6 via i) directly applied pressure, ii) a dermatoscope, or iii) a dermatoscope equipped with a Wratten No. 11 filter. The red, green and blue colour intensities of each image were compared to bilirubin levels. RESULTS: Only the dermatoscope-acquired intensities of the green and blue channels were significantly correlated (p < 0.001) with bilirubin measurements (Pearson's r: 0.59 and 0.48, respectively). For the green and blue channels, discrimination limits of 212 and 190, respectively, revealed a sensitivity and specificity of 100% and 62.5%, respectively, for green and 90.9% and 60%, respectively, for blue for a plasma bilirubin above 205 µmol/L. CONCLUSIONS: The results of this study indicate that a smartphone equipped with a consistent light source in the form of a dermatoscope may be a simple screening tool for neonatal hyperbilirubinemia. However, the method requires some improvement before clinical application.

Analytical and between-subject variation of thrombin generation measured by calibrated automated thrombography on plasma samples.

BACKGROUND: The Calibrated Automated Thrombography (CAT) is an in vitro thrombin generation (TG) assay that holds promise as a valuable tool within clinical diagnostics. However, the technique has a considerable analytical variation, and we therefore, investigated the analytical and between-subject variation of CAT systematically. Moreover, we assess the application of an internal standard for normalization to diminish variation. METHODS: 20 healthy volunteers donated one blood sample which was subsequently centrifuged, aliquoted and stored at -80 °C prior to analysis. The analytical variation was determined on eight runs, where plasma from the same seven volunteers was processed in triplicates, and for the between-subject variation, TG analysis was performed on plasma from all 20 volunteers. The trigger reagents used for the TG assays included both PPP reagent containing 5 pM tissue factor (TF) and PPPlow with 1 pM TF. Plasma, drawn from a single donor, was applied to all plates as an internal standard for each TG analysis, which subsequently was used for normalization. RESULTS: The total analytical variation for TG analysis performed with PPPlow reagent is 3-14% and 9-13% for PPP reagent. This variation can be minimally reduced by using an internal standard but mainly for ETP (endogenous thrombin potential). The between-subject variation is higher when using PPPlow than PPP and this variation is considerable higher than the analytical variation. CONCLUSION: TG has a rather high inherent analytical variation but considerable lower than the between-subject variation when using PPPlow as reagent.

Long-Term Incidence of Venous Thromboembolism in Cancer: The Scandinavian Thrombosis and Cancer Cohort.

The risk of venous thromboembolism (VTE) in patients who survive the first years after a cancer diagnosis after the acute effects of disease and treatment in comparison to a similar background population has been sparsely investigated. The aim of the study was to investigate if incidence rates (IRs) of VTE differed in patients who were alive at least 2 years after a cancer diagnosis without VTE compared with cancer-free references in a population-based cohort study. The study entry was 2 years after a first cancer diagnosis. For each cancer-exposed subject, five reference subjects were identified within the cohort. The IRs were calculated as number of VTEs per 1,000 person years (×10 -3 p-y) in total and in distinct cancer types and corresponding reference subjects. Incidence rate ratios (IRRs) were calculated by Poisson's regression. During a mean follow-up of 5.3 years, 110 VTEs occurred among the 7,288 cancer-exposed subjects and 321 VTEs occurred among the 36,297 identified reference subjects. The IR of VTE was higher for cancer-exposed subjects compared with reference subjects, IRs 3.7 × 10 -3 p-y, 95% CI: 3.1 to 4.5 and 1.9 × 10 -3 p-y, 95% CI: 1.7 to 2.2, respectively. IRs of VTE in most solid cancer types declined to almost the same level as in the reference subjects 2 years after cancer diagnosis, but remained higher in hematological cancers, IRR 4.0, 95% CI: 2.0 to 7.8.

Postprandial Increase in Blood Plasma Levels of Tissue Factor-Bearing (and Other) Microvesicles Measured by Flow Cytometry: Fact or Artifact?

Tissue factor (TF)-bearing microvesicles (MVs) and exosomes may play a role in hemostasis and thrombosis. MVs may be quantified by flow cytometry (FC)-based detection of phosphatidylserine (PS)-positive submicron particles carrying specific antigens, although interference from lipoproteins complicates this approach. In this study, we evaluated the effect of food intake on blood levels of TF-bearing particles measured by FC and small extracellular vesicles (EVs) measured by a protein microarray-based test termed EV Array. Platelet-free plasma (PFP) was obtained from 20 healthy persons in the fasting state and 75 minutes after consumption of a meal. Postprandial changes in the concentration of PS-positive particles, including subgroups binding labeled antibodies against TF, CD41, CD146, and CD62E, respectively (FC), small EVs (EV Array), and TF antigen and procoagulant phospholipids (PPLs) were measured. Furthermore, we tested the effect on FC results of in vitro addition of lipoproteins to fasting PFP. We found significantly increased plasma concentrations of PS-positive particles and all examined subgroups postprandially, while no changes in small EVs, PPL, or TF antigen levels were found. Levels of all types of particles measured by FC were also elevated by lipoprotein spiking. In conclusion, meal consumption as well as in vitro addition of lipoproteins to fasting plasma induces increased levels of PS-positive particles as measured by FC, including TF-positive subtypes and subtypes exposing other antigens. While the observed postprandial increase may to some extent reflect elevated MV levels, our results indicate a substantial interference from lipoproteins.

Prospects and limitations of antibody-mediated clearing of lipoproteins from blood plasma prior to nanoparticle tracking analysis of extracellular vesicles.

Introduction: Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA). Methods: Ten platelet-free plasma (PFP) samples from healthy fasting subjects were incubated with magnetic beads coated with antibodies against apolipoprotein B-48 and B-100 (ApoB). Plasma samples were analysed with NTA before and after application of the bead procedure. Four fasting PFP samples were analysed with an ELISA specific for human ApoB to estimate the degree of removal of lipoproteins and EV array analysis was used for identification of possible EV loss. Results: The magnetic bead separation procedure resulted in a median reduction of the particle concentration in plasma by 62% (interquartile range 32-72%). The mean size of the remaining particles generally increased. ApoB concentration was reduced to a level close to the background signal, whereas a median reduction of the EV content by 21% (range 8-43%) was observed. Conclusion: Anti-ApoB antibody coated magnetic beads may hold potential for removal of lipoproteins from human PFP prior to EV measurement by NTA but some artefactual effect and EV loss may have to be endured.