FCMPASS Software Aids Extracellular Vesicle Light Scatter Standardization.

The study of extracellular vesicles (EVs) is a rapidly growing field due to their great potential in many areas of clinical medicine including diagnostics, prognostics, theranostics, and therapeutics. Flow cytometry is currently one of the most popular methods of analyzing EVs due to it being a high-throughput, multiparametric technique, that is readily available in the majority of research labs. Despite its wide use, few commercial flow cytometers are designed specifically for the detection of EVs. Many flow cytometers used for EV analysis are working at their detection limits and are unable to detect the majority of EVs. Currently, very little standardization exists for EV flow cytometry, which is an issue because flow cytometers vary considerably in the way they collect scattered or fluorescent light from particles being interrogated. This makes published research hard to interpret, compare, and in some cases, impossible to reproduce. Here we demonstrate a method of flow cytometer light scatter standardization, utilizing flow cytometer postacquisition analysis software (FCMPASS ). FCMPASS is built upon Mie theory and enables the approximation of flow cytometer geometric parameters either by analyzing beads of known diameter and refractive index or by inputting the collection angle if known. The software is then able to create a scatter-diameter curve and scatter-refractive index curve that enables researchers to convert scattering data and instrument sensitivity into standardized units. Furthermore, with the correct controls, light scatter data can be converted to diameter distributions or refractive index distributions. FCMPASS therefore offers a freely available and ergonomic method of standardizing and further extending EV characterization using flow cytometry.

Droplet Microfluidics with Reagent Micromixing for Investigating Intrinsic Platelet Functionality.

INTRODUCTION: Precision mapping of the functional structure of platelet populations holds great promise for the identification of hyper-reactive subtypes that are likely to be disease drivers, having value in prognostics and as therapeutic targets. However, the ability to measure the intrinsic functional capacity of individual platelets is confounded by potent paracrine cross-talk, resulting in phenotypic remodeling of the entire platelet population, and in doing so obscuring the identity of hyper-reactive platelets. METHODS: To address this we have developed a droplet microfluidics strategy for single platelet confinement to exclude paracrine signaling. Consideration of the Poisson distribution was used for high throughput single platelet encapsulation and the preparation of minimal platelet collectives serving as digital models for understanding the role of hyper-reactive platelets coordinating system-level behavior by paracrine signaling. Platelets are retrieved from the droplets for phenotyping using standard flow cytometry. In addition, we have incorporated a staggered herringbone micromixing element for accurate agonist and antibody dispensing in droplets. RESULTS: The methodology was used for characterizing sensitivity distributions from healthy blood donors in response to convulxin (agonist of the GPVI receptor, the major platelet receptor for collagen). P-selectin exposure and α IIb ß 3 integrin activation were used as analytical end-points to demonstrate the existence of hyper-reactive platelets that direct 20-fold gains in system level sensitivity. CONCLUSIONS: The analytical workflow represents an enabling tool for the accurate classification of platelet subtypes and description of their underlying biology. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12195-020-00665-6) contains supplementary material, which is available to authorized users.

Single platelet variability governs population sensitivity and initiates intrinsic heterotypic responses.

Investigations into the nature of platelet functional variety and consequences for homeostasis require new methods for resolving single platelet phenotypes. Here we combine droplet microfluidics with flow cytometry for high throughput single platelet function analysis. A large-scale sensitivity continuum was shown to be a general feature of human platelets from individual donors, with hypersensitive platelets coordinating significant sensitivity gains in bulk platelet populations and shown to direct aggregation in droplet-confined minimal platelet systems. Sensitivity gains scaled with agonist potency (convulxin > TRAP-14>ADP) and reduced the collagen and thrombin activation threshold required for platelet population polarization into pro-aggregatory and pro-coagulant states. The heterotypic platelet response results from an intrinsic behavioural program. The method and findings invite future discoveries into the nature of hypersensitive platelets and how community effects produce population level responses in health and disease.

Monolithically-integrated cytometer for measuring particle diameter in the extracellular vesicle size range using multi-angle scattering.

Size measurement of extracellular vesicles is hampered by the high cost and measurement uncertainty of conventional flow cytometers which is mainly due to the use of non-specialised free space optics. Integrated cytometry, where the optics and fluidics are embedded in a monolithic chip shows promise for the production of low cost, micro-flow cytometers dedicated for extracellular vesicle (EV) analysis with improved size measurement accuracy and precision. This research demonstrates a unique integrated cytometer for sub-micron particle size measurement using multi-angle scattering analysis. A combination of three technologies is used: (i) Dean-based hydrodynamic focussing to deliver a tight sample core stream to the analysis region, (ii) integrated waveguides with multimode interference devices to focus a narrow excitation beam onto the sample stream, and (iii) an angular array of collection waveguides to measure particle scattering distribution and calculate diameter. Low index 200 nm liposomes could be detected and polystyrene size standards as small as 400 nm diameter could be measured with an uncertainty of ±21 nm (1/2 IQR) demonstrating a first step on the path to high performance integrated cytometry of EVs.

Aspirin resistance is more common in lacunar strokes than embolic strokes and is related to stroke severity.

The aim of this study was to investigate the relationship between aspirin resistance, ischaemic stroke subtype, stroke severity, and inflammatory cytokines. Aspirin resistance was assessed by thrombelastography in 45 people with ischaemic stroke and 25 controls. Plasma interleukin (IL)-6 was measured. Stroke severity was assessed using the modified Rankin scale and National Institute of Health Stroke Score within 72 h of stroke. Aspirin resistance was more common in the stroke than the control group (67% versus 40%, P=0.028), and within the stroke group the aspirin-resistant group had a higher Rankin score (4.0 versus 2.0, P=0.013). Aspirin resistance was greater in lacunar than embolic strokes (platelet activation 79% versus 59%, P=0.020). The stroke aspirin-resistant group had higher levels of IL-6 than the stroke aspirin-sensitive group (2.4+/-1 versus 1.8+/-0.9 ng/mL, P=0.037). Using multivariate analysis, we examined the interrelationships between aspirin resistance, IL-6, and stroke severity. These analyses showed that IL-6 was independently associated with stroke severity as the outcome (B=3.738, P=0.036), and aspirin resistance was independently associated with IL-6 (B=0.765, P=0.005) as the outcome. In conclusion, aspirin resistance is related to stroke severity and aspirin resistance is more common in lacunar strokes than embolic strokes.

Leukocyte extracellular vesicle concentration is inversely associated with liver fibrosis severity in NAFLD.

The enhanced liver fibrosis (LFS) score and the nonalcoholic fatty liver disease (NAFLD) fibrosis score (NFS) are algorithmic-derived scores for diagnosing severe (F3/F4) liver fibrosis. In a pilot, substudy of the Wessex Evaluation of fatty Liver and Cardiovascular markers in NAFLD with OMacor thErapy (WELCOME) trial, we tested whether measurements of plasma platelet-, endothelial-, and leukocyte-derived extracellular vesicles (EVs) counts are (a) associated with, and predict, F3/F4 fibrosis and (b) able to improve risk prediction of F3/F4 fibrosis in NAFLD, building upon LFS or NFS algorithms. Twenty-six individuals with NAFLD had liver fibrosis severity determined by Kleiner scoring after liver biopsy. Plasma samples stained with CD41a, CD42b, CD31, CD105, CD14, CD16, and CD284 antibodies were analyzed using flow cytometry to measure platelet-, endothelial-, and leukocyte-derived EVs counts. The independence of associations between EVs and F3/F4 fibrosis were tested using logistic regression. Receiver operator characteristic (ROC) curves were used to evaluate F3/F4 fibrosis prediction models. LFS was more strongly associated with F3/F4 fibrosis than NFS (χ2= 15.403, P < 0.0001, and χ2= 6.300, P = 0.012, respectively). The association between LFS and F3/F4 fibrosis was further improved by addition of CD14+ EVs (χ2=20.847,P = 0.016 vs. χ2=12.803,P = 0.015, respectively) or CD16+ EVs (χ2=22.205,P = 0.009 vs. χ2=17.559,P = 0.001, respectively), and the area under the ROC for LFS (AUC = 0.915, se = 0.055, P = 0.001) was increased by the addition of CD14+ or CD16+ EVs (AUC = 0.948, se = 0.042, and P < 0.001 and AUC = 0.967, se = 0.055, P < 0.001, respectively) as predictor variables. In this small preliminary study, CD14+ and CD16+ EV counts show potential to predict liver fibrosis severity with either marker improving the ability of the LFS to identify F3/F4 fibrosis in this small preliminary cohort study.

Long-chain n-3 fatty acid supplementation in men increases resistance to activated protein C.

It has recently and controversially been demonstrated that fish oil supplementation may not be beneficial for everyone, but to date there have been no biological explanations. We suggest that resistance to the anticoagulant, activated protein C (APC), be considered as a potential mechanism, because it has been demonstrated that the type of fatty acids on phospholipids modulates function of the APC pathway. The APC ratio in plasma was decreased by 7% after fish oil supplementation in healthy men (P<.005; n=35). The decrease in APC ratio equates to an increase in APC resistance. Fish oil lowered the APC ratio by (1) increasing low-density lipoprotein (LDL) cholesterol (P<.01) and apolipoprotein B (P<.05) and (2) increasing platelet microparticles (P<.05). In vitro, purified LDL decreased the APC ratio and increased microparticle formation. These changes affecting the anticoagulant APC could contribute toward a prothrombotic state, potentially explaining the recent observation that fish oil supplementation may not always be of benefit. These findings will need to be repeated in different disease states.

Extracellular Vesicle Flow Cytometry Analysis and Standardization.

The term extracellular vesicles (EVs) describes membranous vesicles derived from cells, ranging in diameter from 30 to 1,000 nm with the majority thought to be in the region of 100-150 nm. Due to their small diameter and complex and variable composition, conventional techniques have struggled to accurately count and phenotype EVs. Currently, EV characterization using high-resolution flow cytometry is the most promising method when compared to other currently available techniques, due to it being a high-throughput, single particle, multi-parameter analysis technique capable of analyzing a large range of particle diameters. Whilst high resolution flow cytometry promises detection of the full EV diameter range, standardization of light scattering and fluorescence data between different flow cytometers remains an problem. In this mini review, we will discuss the advances in high-resolution flow cytometry development and future direction of EV scatter and fluorescence standardization. Standardization and therefore reproducibility between research groups and instrumentation is lacking, hindering the validation of EVs use as diagnostic biomarkers and therapeutics.

Percentage of body fat and plasma glucose predict plasma sialic acid concentration in type 2 diabetes mellitus.

Circulating sialic acid is an independent risk factor for cardiovascular disease and is higher in people with type 2 diabetes mellitus. Sialic acid is associated with body mass index, but it is uncertain whether body fat contributes to the higher levels of sialic acid in type 2 diabetes mellitus. Therefore, we have investigated whether the higher levels of sialic acid observed in type 2 diabetes mellitus persist when controlling for fatness. Fasting plasma samples were collected from 24 individuals with type 2 diabetes mellitus and 24 controls. Percentage of body fat was measured by bioelectrical impedance. Plasma sialic acid was quantified by an enzymatic method. Plasma sialic acid was higher in the group with type 2 diabetes mellitus than controls (602 +/- 14 vs 545 +/- 14 mg/L, P = .007). Percentage of body fat was associated with plasma sialic acid concentration in both the control group (r = 0.481, P = .020) and the group with type 2 diabetes mellitus (r = 0.527, P = .007). Fasting glucose was also associated with plasma sialic acid in the group with type 2 diabetes mellitus (r = 0.700, P < .001). Adjustment for percentage of body fat accounted for the higher levels of sialic acid in type 2 diabetes mellitus. Using linear regression, 54.3% of the variation of plasma sialic acid was explained by percentage of body fat and glucose concentrations in the whole group. Seventy-four percent of sialic acid variation was explained by the same model in type 2 diabetes mellitus. In conclusion, this is the first study to show that percentage of body fat predicts plasma sialic acid concentration and contributes toward higher levels of sialic acid in type 2 diabetes mellitus.

A novel role for CD36 in VLDL-enhanced platelet activation.

Type 2 diabetes is characterized by increased plasma triglyceride levels and a fourfold increase in ischemic heart disease, but the mechanism is unclear. CD36 is a receptor/transporter that binds fatty acids of lipoproteins. CD36 deficiency has been linked with insulin resistance. There is strong evidence of in vivo interaction between platelets and atherogenic lipoproteins suggesting that atherogenic triglyceride-rich lipoproteins, such as VLDL, that are increased in diabetic dyslipidemia are important in this process. This study demonstrates that VLDL binds to the platelet receptor CD36, enhances platelet thromboxane A2 production, and causes increased collagen-mediated platelet aggregation. VLDL enhanced collagen-induced platelet aggregation by 1) shortening the time taken for aggregation to begin (lag time) to 70% of control (P = 0.001); 2) increasing maximum aggregation to 170% of control (P = 0.008); and 3) increasing thromboxane production to 3,318% of control (P = 0.004), where control represents platelets stimulated with collagen (100%). A monoclonal antibody against CD36 attenuated VLDL-enhanced collagen-induced platelet aggregation by 1) inhibiting binding of VLDL to platelets by 75% (P = 0.041); 2) lengthening lag time to 190% (P < 0.001); and 3) decreasing thromboxane production to 8% of control (P < 0.001). In support of this finding, platelets from Cd36-deficient rats showed no increase in aggregation, thromboxane production, and VLDL binding in contrast to platelets from rats expressing CD36. These data suggest that platelet Cd36 has a key role in VLDL-induced collagen-mediated platelet aggregation, possibly contributing to atherothrombosis associated with increased VLDL levels.

IL-6 is a predictive biomarker for stroke associated infection and future mortality in the elderly after an ischemic stroke.

BACKGROUND AND PURPOSE: Stroke associated infection (within the first seven days) occurs in approximately half of stroke patients and is associated with a worse prognosis, especially in the elderly. It is uncertain what factors predict stroke associated infection, yet identification of a suitable biomarker for infection may allow early and appropriate intervention with antibiotics. The aims of this study were to: a) identify independent risk factors for stroke associated infection, and b) test relationships between these risk factors and mortality at 2 years. METHODS: Eight-two elderly patients were assessed within 72 h of stroke. Data on stroke severity (Barthel Index), stroke associated infection and mortality at 2 years were collected. Inflammatory biomarkers at baseline and 6 months were measured by ELISA. Logistic regression was used to identify risk factors for stroke associated infection and death. RESULTS: Patients with stroke associated infection, especially pneumonia, had increased IL-6, more severe strokes, and higher mortality. IL-6 was independently associated with stroke associated infection (OR = 19.2, [95%CI 3.68, 100], p < 0.001), after adjustment for other risk factors and cytokines. IL-6 was also independently associated with 2 year mortality (OR = 9.2, [1.0, 85.1], p = 0.031). CONCLUSIONS: These data suggest that IL-6 may be a key biomarker for predicting stroke associated infection and mortality in the first two years post stroke.