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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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Background: Understanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations. Materials and Methods: A standardized methodology to measure the concentration of extracellular vesicles (EVs) has been developed and tested. The method is based on measuring the EV concentration as a function of a defined size range. Blood plasma EVs are isolated and purified using size exclusion columns (qEV) and consecutively measured with tunable resistive pulse sensing (TRPS). Six independent research groups measured liposome and EV samples with the aim to evaluate the developed methodology. Each group measured identical samples using up to 5 nanopores with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate data analysis with principal component analysis (PCA) were used to evaluate reproducibility across the groups and to explore and visualise possible patterns and outliers in EV and liposome data sets. Results: PCA revealed good reproducibility within and between laboratories, with few minor outlying samples. Measured mean liposome (not filtered with qEV) and EV (filtered with qEV) concentrations had coefficients of variance of 23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements could be attributed to the use of qEVs and the polydisperse nature of EVs. Conclusion: The results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.

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.

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.

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.

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.

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.

Background: Exosomes are one of the several types of cell-derived vesicles with a diameter of 30–100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define their phenotype and determine their concentration in biological fluids. To identify circulating as well as cell culture-derived vesicles, the current standard is immunoblotting or a flow cytometrical analysis for specific proteins, both of which requires large amounts of purified vesicles. Methods: Based on the technology of protein microarray, we hereby present a highly sensitive Extracellular Vesicle (EV) Array capable of detecting and phenotyping exosomes and other extracellular vesicles from unpurified starting material in a high-throughput manner. To only detect the exosomes captured on the EV Array, a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81 was used. These antibodies were selected to ensure that all exosomes captured are detected, and concomitantly excluding the detection of other types of microvesicles. Results: The limit of detection (LOD) was determined on exosomes derived from the colon cancer cell line LS180. It clarified that supernatant from only approximately 104 cells was needed to obtain signals or that only 2.5×104 exosomes were required for each microarray spot (~1 nL). Phenotyping was performed on plasma (1–10 µL) from 7 healthy donors, which were applied to the EV Array with a panel of antibodies against 21 different cellular surface antigens and cancer antigens. For each donor, there was considerable heterogeneity in the expression levels of individual markers. The protein profiles of the exosomes (defined as positive for CD9, CD63 and CD81) revealed that only the expression level of CD9 and CD81 was approximately equal in the 7 donors. This implies questioning the use of CD63 as a standard exosomal marker since the expression level of this tetraspanin was considerably lower.

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.

Background: Patients admitted to hospital outside normal working hours suffer higher complication and mortality rates than patients admitted at times when the hospital is fully operational. This ‘weekend effect’ is well described but poorly understood. It is not clear whether or not the effect extends to other out-of-hours periods, or how far excess mortality for out-of-hours admissions reflects a different presenting population with higher severity of illness and how much is explained by poorer availability and quality of services. Objectives: We aimed to assess (1) the costs and benefits of introducing 7-day services, (2) whether or not mortality rates are elevated during all out-of-hours periods, (3) whether or not selection of more severely ill patients for admission out of hours explains elevated mortality rates and (4) whether or not mortality rates out of hours are related to staffing levels. Methods: We conducted a series of retrospective observational analyses of hospital episode data in England, using both national data and data from a single, large acute NHS trust. For the national studies, we analysed emergency admissions to all 140 non-specialist acute hospital trusts in England between April 2013 and February 2014 (over 12 million accident and emergency attendances and 4.5 million emergency admissions). For the single trust, we analysed emergency admissions between April 2004 and March 2014 (240,000 admissions). Deaths within 30 days of attendance or admission were compared for normal working hours and out-of-hours periods. Results: We found that, in addition to elevated mortality for weekend admissions, mortality rates are also elevated for patients admitted during night-time periods. Elevated mortality was reduced for stroke patients in a large acute trust when more – and more experienced – nursing staff were present during the first hour of admission. Nationally, we found that excess mortality out of hours was largely explained by a sicker population of patients being selected for admission. However, mortality rates were still elevated on Sunday daytimes when we accounted for severity of patient illness. We also found that the estimated cost of implementing 7-day services exceeds the maximum amount that the National Institute for Health and Care Excellence would recommend the NHS should spend on eradicating excess mortality at weekends. Limitations: Our results depend on the accuracy and completeness of data recording by hospital staff. If accuracy of recording is related to time of patient admission, our results may be biased. Results based on data from a single trust should be treated as indicative. Conclusions: In addressing variations in patient outcomes across the week, a more nuanced approach, extending services for key specialties over critical periods – rather than implementing whole-system changes – is likely to be the most cost-effective. Future work: Future research should aim to develop and use appropriate measures of severity of illness to facilitate meaningful analysis of variations in patient outcomes, and to identify candidate specialties and critical periods for which extending services is likely to be cost-effective. Funding: The National Institute for Health Research Health Services and Delivery Research programme.