A Systematic Approach to Improve Scatter Sensitivity of a Flow Cytometer for Detection of Extracellular Vesicles.

Extracellular vesicles (EVs) are commonly studied by flow cytometry. Due to their small size and low refractive index, the scatter intensity of most EVs is below the detection limit of common flow cytometers. Here, we aim to improve forward scatter (FSC) and side scatter (SSC) sensitivity of a common flow cytometer to detect single 100 nm EVs. The effects of the optical and fluidics configuration on scatter sensitivity of a FACSCanto (Becton Dickinson) were evaluated by the separation index (SI) and robust coefficient of variation (rCV) of polystyrene beads (BioCytex). Improvement is defined as increased SI and/or reduced rCV. Changing the obscuration bar improved the rCV 1.9-fold for FSC. A 10-fold increase in laser power improved the SI 19-fold for FSC and 4.4-fold for SSC, whereas the rCV worsened 0.8-fold and improved 1.5-fold, respectively. Confocalization worsened the SI 1.2-fold for FSC, and improved the SI 5.1-fold for SSC, while the rCV improved 1.1-fold and worsened 1.5-fold, respectively. Replacing the FSC photodiode with a photomultiplier tube improved the SI 66-fold and rCV 4.2-fold. A 2-fold reduction in sample stream width improved both SI and rCV for FSC by 1.8-fold, and for SSC by 1.3- and 2.2-fold, respectively. Decreasing the sample flow velocity worsened rCVs. Decreasing the flow channel dimensions and the pore size of the sheath filter did not substantially change the SI or rCV. Using the optimal optical configuration and fluidics settings, the SI improved 3.8∙104 -fold on FSC and 30-fold on SSC, resulting in estimated detection limits for EVs (assuming a refractive index of 1.40) of 246 and 91 nm on FSC and SSC, respectively. Although a 50-fold improvement on FSC is still necessary, these adaptions have produced an operator-friendly, high-throughput flow cytometer with a high sensitivity on both SSC and FSC. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Comparison of Generic Fluorescent Markers for Detection of Extracellular Vesicles by Flow Cytometry.

BACKGROUND: Extracellular vesicles (EVs) in biofluids are potential biomarkers of disease. To explore the clinical relevance of EVs, a specific generic EV marker would be useful, one that does not require antibodies and binds to all EVs. Here we evaluated 5 commonly used generic markers for flow cytometry. METHODS: Flow cytometry (A60-Micro, Apogee) was used to evaluate the ability of the generic EV markers calcein acetoxymethyl ester, calcein acetoxymethyl ester violet, carboxyfluorescein succinimidyl ester (CFSE), 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)-1-(3-sulfopropyl)pyridinium (di-8-ANEPPS), and lactadherin to stain EVs from MCF7 human breast adenocarcinoma cell line-conditioned culture medium [epithelial cell adhesion molecule positive (EpCAM+)] or platelet EVs from human plasma [integrin ß3 positive (CD61+)]. Side scatter triggering was applied as a reference, and the influence of non-EV components (proteins and lipoproteins) was evaluated. RESULTS: Di-8-ANEPPS, lactadherin, and side scatter detected 100% of EpCAM+ MCF7 EVs. Lactadherin and side scatter detected 33% and 61% of CD61+ EVs, respectively. Di-8-ANEPPS detected platelet EVs only if soluble protein was first removed. Because all generic markers stained proteins, at best 33% of platelet EVs in plasma were detected. The calcein markers and CFSE were either insensitive to EVs in both samples or associated with swarm detection. CONCLUSIONS: None of the generic markers detected all and only EVs in plasma. Side scatter triggering detected the highest concentration of plasma EVs on our A60-Micro, followed by lactadherin. The choice between scatter or lactadherin primarily depends on the analytical sensitivity of the flow cytometer used.

Absolute sizing and label-free identification of extracellular vesicles by flow cytometry.

Blood contains extracellular vesicles (EVs), which are biological nanoparticles with clinical applications. In blood plasma, EVs are outnumbered by similar-sized lipoprotein particles (LPs), leading to controversial data such as non-specific binding of antibodies to LPs. Flow cytometry is a clinically applicable technique to characterize single EVs in body fluids. However, flow cytometry data have arbitrary units, impeding standardization, data comparison, and data interpretation, such as differentiation between EVs and LPs. Here we present a new method, named flow cytometry scatter ratio (Flow-SR), to relate the ambiguous light scattering signals of flow cytometry to the diameter and refractive index (RI) of single nanoparticles between 200-500 nm in diameter. Flow-SR enables label-free differentiation between EVs and LPs and improves data interpretation and comparison. Because Flow-SR is easy to implement, widely applicable, and more accurate and faster than existing techniques to size nanoparticles in suspension, Flow-SR has numerous applications in nanomedicine.

Stability of Monodisperse Phospholipid-Coated Microbubbles Formed by Flow-Focusing at High Production Rates.

Monodisperse microbubble ultrasound contrast agents may dramatically increase the sensitivity and efficiency in ultrasound imaging and therapy. They can be produced directly in a microfluidic flow-focusing device, but questions remain as to the interfacial chemistry, such as the formation and development of the phospholipid monolayer coating over time. Here, we demonstrate the synthesis of monodisperse bubbles with radii of 2-10 µm at production rates ranging from 10(4) to 10(6) bubbles/s. All bubbles were found to dissolve to a stable final radius 2.55 times smaller than their initial radius, independent of the nozzle size and shear rate, indicating that the monolayer self-assembles prior to leaving the nozzle. The corresponding decrease in surface area by a factor 6.6 reveals that lipid molecules are adsorbed to the gas-liquid interface in the disordered expanded state, and they become mechanically compressed by Laplace pressure-driven bubble dissolution to a more ordered condensed state with near zero surface tension. Acoustic characterization of the stabilized microbubbles revealed that their shell stiffness gradually increased from 0.8 to 2.5 N/m with increasing number of insonations through the selective loss of the more soluble lipopolymer molecules. This work therefore demonstrates high-throughput production of clinically relevant monodisperse contrast microbubbles with excellent control over phospholipid monolayer elasticity and microbubble resonance.

A compendium of single extracellular vesicle flow cytometry.

Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV-containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt-EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses.

Detection of extracellular vesicles in plasma and urine of prostate cancer patients by flow cytometry and surface plasmon resonance imaging.

Large (> 1 µm) tumor-derived extracellular vesicles (tdEVs) enriched from the cell fraction of centrifuged whole blood are prognostic in metastatic castration-resistant prostate cancer (mCRPC) patients. However, the highest concentration of tdEVs is expected in the cell-free plasma fraction. In this pilot study, we determine whether mCRPC patients can be discriminated from healthy controls based on detection of tdEVs (< 1µm, EpCAM+) and/or other EVs, in cell-free plasma and/or urine. The presence of marker+ EVs in plasma and urine samples from mCRPC patients (n = 5) and healthy controls (n = 5) was determined by flow cytometry (FCM) and surface plasmon resonance imaging (SPRi) using an antibody panel and lactadherin. For FCM, the concentrations of marker positive (+) particles and EVs (refractive index <1.42) were determined. Only the lactadherin+ particle and EV concentration in plasma measured by FCM differed significantly between patients and controls (p = 0.017). All other markers did not result in signals exceeding the background on both FCM and SPRi, or did not differ significantly between patients and controls. In conclusion, no difference was found between patients and controls based on the detection of tdEVs. For FCM, the measured sample volumes are too small to detect tdEVs. For SPRi, the concentration of tdEVs is probably too low to be detected. Thus, to detect tdEVs in cell-free plasma and/or urine, EV enrichment and/or concentration is required. Furthermore, we recommend testing other markers and/or a combination of markers to discriminate mCRPC patients from healthy controls.

MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments.

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.

Deriving Extracellular Vesicle Size From Scatter Intensities Measured by Flow Cytometry.

Flow cytometry is commonly used to investigate the potential for extracellular vesicles (EVs) to be biomarkers of disease. A typical flow cytometer detects fluorescence and scatter intensities of single EVs in arbitrary units. These arbitrary units complicate data interpretation and data comparison between different flow cytometers. For example, comparison of detected EV concentrations requires knowledge of the detectable EV sizes. Using Mie theory and knowledge of the optical configuration of the flow cytometer, EV size can be derived from the scatter intensity for a given EV refractive index. Here, a protocol is described to derive the size of EVs and other nanoparticles from the scatter intensity. The resulting size distribution allows the comparison of data between flow cytometers, which is a prerequisite for clinical application of EVs as biomarkers and may advance other fields where sizing of nanoparticles is essential. © 2018 by John Wiley & Sons, Inc.

Hand-opening feedback for myoelectric forearm prostheses: performance in virtual grasping tasks influenced by different levels of distraction.

Sensory feedback and the required attentional demands are important aspects in prosthesis acceptance. In this study, hand-opening feedback is provided and the performance in a virtual grasping task is investigated. Simultaneously, a secondary task was performed to investigate the attentional demands. Ten nondisabled subjects performed the tasks with and without feedback about the hand opening through an array of eight vibrotactile stimulators on the forearm. Activation of one stimulator corresponded to one hand-opening position. For the dual-task experiments, subjects simultaneously performed a secondary auditory counting task. The addition of vibrotactile feedback increased the performance (expressed in percentages of correct hand positions, mean absolute errors in position, and percentages of deviations up to one hand-opening position), but the duration of the tasks was also increased. Three levels of distraction (no distraction, counting task, count and subtract task) were applied, which did not influence the performance in the grasping tasks except for the highest level of distraction. We concluded that the proposed method to provide hand-opening feedback through an array of eight vibrotactile stimulators is successful because the performance in a grasping task increases but it is not significantly attention demanding.