Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non‐interest

Essentials

• Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease.
• Fluorescence‐based flow cytometric analysis is suitable to detect low abundant EV subsets.
• Particles of non‐interest can induce false‐positive light scatter and fluorescent signals.
• Interference of particles of non‐interest can be monitored by analyzing serial dilutions.

Summary

Background

Extracellular vesicles (EVs) in plasma are increasingly being recognized as potential biomarkers. EV analysis for diagnostic purposes should be robust and should allow analysis of EV subsets with a wide range of abundance and in a large number of patient samples. Flow cytometry offers possibilities to meet these criteria, as it allows multiparameter analysis of individual EVs. However, analysis of plasma EVs is challenging, because of their size and heterogeneity, and the presence of other submicrometer‐sized particles in plasma that could interfere with EV analysis.

Objectives

To explore whether fluorescence‐based flow cytometric analysis of EV subsets is suitable when the EVs of interest are present in low abundance in a background of non‐labeled or differently labeled EVs and particles.

Methods

Fluorescently labeled EVs of interest were spiked at different ratios in full plasma, purified plasma components, or (non‐)fluorescent polystyrene beads, and subsequently analyzed by flow cytometry with fluorescence threshold triggering.

Results

We found that light scatter detection of low‐abundance or rare EV subsets during fluorescence threshold triggering was severely affected by particles of non‐interest, owing to coincidence and swarming. Importantly, we show that interfering particles labeled with different fluorophores induced false‐positive fluorescent signals on the particles of interest. These unwanted effects could only be discerned and controlled by performing serial dilutions and analyzing light scatter and fluorescence parameters.

Conclusions

We demonstrate how particles of non‐interest in plasma can impact on the light scatter and fluorescence detection of low‐abundance EVs of interest during fluorescence‐based flow cytometric analysis, and provide a means to prevent erroneous data interpretation.     

Hollow organosilica beads as reference particles for optical detection of extracellular vesicles

Essentials

• Standardization of extracellular vesicle (EV) measurements by flow cytometry needs improvement.
• Hollow organosilica beads were prepared, characterized, and tested as reference particles.
• Light scattering properties of hollow beads resemble that of platelet‐derived EVs.
• Hollow beads are ideal reference particles to standardize scatter flow cytometry research on EVs.

Summary

Background

The concentration of extracellular vesicles (EVs) in body fluids is a promising biomarker for disease, and flow cytometry remains the clinically most applicable method to identify the cellular origin of single EVs in suspension. To compare concentration measurements of EVs between flow cytometers, solid polystyrene reference beads and EVs were distributed in the first ISTH‐organized interlaboratory comparison studies. The beads were used to set size gates based on light scatter, and the concentration of EVs was measured within the size gates. However, polystyrene beads lead to false size determination of EVs, owing to the mismatch in refractive index between beads and EVs. Moreover, polystyrene beads gate different EV sizes on different flow cytometers.

Objective

To prepare, characterize and test hollow organosilica beads (HOBs) as reference beads to set EV size gates in flow cytometry investigations.

Methods

HOBs were prepared with a hard template sol‐gel method, and extensively characterized for morphology, size, and colloidal stability. The applicability of HOBs as reference particles was investigated by flow cytometry with HOBs and platelet‐derived EVs.

Results

HOBs proved to be monodisperse with a homogeneous shell thickness. Two‐angle light‐scattering measurements by flow cytometry confirmed that HOBs have light‐scattering properties similar to those of platelet‐derived EVs.

Conclusions

Because the structure and light‐scattering properties HOBs resemble those of EVs, HOBs with a given size will gate EVs of the same size. Therefore, HOBs are ideal reference beads with which to standardize optical measurements of the EV concentration within a predefined size range.

     

Single vs. swarm detection of microparticles and exosomes by flow cytometry

See also Harrison P, Gardiner C. Invisible vesicles swarm within the iceberg. This issue, pp 916‐8.

Summary.

Background:  Microparticles and exosomes are cell‐derived vesicles and potential biomarkers for disease. Recently, the Scientific Standardization Committee collaborative workshop of the ISTH initiated standardization of vesicle detection by flow cytometry with polystyrene beads. Because polystyrene beads have different optical properties from biological vesicles, and because the mechanisms causing the detection signal are incompletely understood, there are contradictions between expected and observed results. Objectives:  To develop a model with which to relate the detection signal of a flow cytometer to the diameter of vesicles and clarify observed discrepancies. Methods:  We combined measurements of polystyrene and silica beads with an estimated refractive index of vesicles and performed Mie calculations of light scattering. Results:  We established the relationship between measured light scattering and the diameter of vesicles. The Megamix gating strategy proposed by the Scientific Standardization Committee selects single vesicles and cells with diameters between 800 and 2400 nm when applied on the forward‐scattering detector of regular flow cytometers. Nevertheless, we demonstrated that, irrespective of the applied gating, multiple vesicles smaller than 220 nm or multiple 89‐nm silica beads were counted as a single event signal at sufficiently high concentrations. Conclusions:  Vesicle detection by flow cytometry is attributed to large single vesicles and swarm detection of smaller vesicles; that is, multiple vesicles are simultaneously illuminated by the laser beam and counted as a single event signal. Swarm detection allows the detection of smaller vesicles than previously thought possible, and explains the finding that flow cytometry underestimates the concentration of vesicles.     

Recent developments in the nomenclature,presence, isolation,detection and clinical impact of extracellular vesicles

The research field of extracellular vesicles (EVs), such as microparticles and exosomes, is growing exponentially. The goal of this review is to provide an overview of recent developments relevant to the readers of the Journal of Thrombosis and Haemostasis. We will discuss nomenclature, the presence of EVs in fluids, methods of isolation and detection, and emerging clinical implications. Although research on EVs has been performed within the ISTH for over a decade, most of the recent research on EVs has been brought together by the International Society on Extracellular Vesicles (ISEV). To achieve an overview of recent developments, the information provided in this review comes not only from publications, but also from latest meetings of the ISEV (April 2015, Washington, DC, USA), the International Society on Advancement of Cytometry (June 2015, Glasgow, UK), and the ISTH (June 2015, Toronto, Canada).     

利用流式监控高质量小鼠脾细胞的制备

目的 流式技术监控小鼠脾细胞裂解过程,在确保脾淋巴细胞高活力的同时,充分裂解其中的红细胞.方法 以流式细胞术监控ACK和Tris-NH4Cl两种红细胞裂解液的裂解效果,通过细胞融合率判断淋巴细胞活力.结果 结合FSC和PI荧光强度分析,在确保淋巴细胞相对存活率大于或等于90%的前提下,ACK处理0.5～1 min即可去除90%以上的红细胞,Tris-NH4Cl处理10 min也可去除82%的红细胞,获得的淋巴细胞的融合效率为85%～90%.结论 流式细胞术能够实时监控ACK和Tris-NH4Cl的裂解过程,获得高质量的脾淋巴细胞以适用于杂交瘤细胞的制备.     

Validation of flow cytometric detection of platelet microparticles and liposomes by atomic force microscopy

Summary. Background: Platelet microparticles (PMPs) are a promising prognostic marker for thrombotic disorders because of their release during platelet activation. The use of flow cytometry for the enumeration of PMPs in plasma has generated controversy due to their size, which is below the stated detection limits of conventional flow cytometry instruments. The potential impact of this is an underestimation of PMP counts. Objectives/Methods: To address this possibility, we used a combination of fluorescence‐activated cell sorting (FACS) and atomic force microscopy (AFM) to determine the size distribution of PMPs present in plasma from acute myocardial infarction (AMI) patients and normal volunteers, and PMPs generated by expired platelet concentrates and washed platelets treated with agonists such as thrombin and calcium ionophore (A23187). Results: According to AFM image analysis, there was no statistically significant difference in height or volume distributions in PMPs from thrombin‐activated, calcium ionophore‐activated, expired platelet concentrates and plasma from healthy volunteers and AMI patients. Based on volume, expired platelets released the greatest proportion of exosomes (< 1.0 × 10^?22 L³ in volume) in relation to the entire PMP population (29.7%) and the smallest proportion of exosomes was observed in AMI patient plasma (1.8%). Moreover, AFM imaging revealed that PMPs from expired platelets exhibited smooth surfaces compared with other PMP types but this was not statistically significant. Conclusions: We confirm that flow cytometry is capable of analyzing PMPs from plasma by using AFM to perform nanoscale measurements of individual PMP events isolated by FACS. This method also provided the first quantitative nanoscale images of PMP ultrastructure.     

Recent advancements of flow cytometry: new applications in hematology and oncology

Flow cytometry offers great diagnostic opportunities in the vast majority of hematologic and oncologic diseases with multiple cellular and molecular information within an individual cell. We will discuss various applications of flow cytometry, particularly in hematology and oncology, in addition to general principles and limitations of flow cytometry. They include nucleic acid analyses in cancer cells, new methods for assessing rare circulating tumor cells and disease-specific applications in malignancy with emphasis on diagnosis and treatment of hematologic malignancy, including minimal residual disease. With improvement of monoclonal antibodies, fluorescence and laser technology, flow cytometry now offers new avenues of assessing cellular functionality through examination of intracellular compartments. High-throughput quantitative analysis, advancements of in vivo flow cytometry and assessment of minimal residual diseases, as exampled in patient stratification and prediction of leukemia therapeutic response, will further make flow cytometry indispensable in medicine.     

不断规范血液淋巴系肿瘤的流式细胞免疫表型分析

血液淋巴系肿瘤的流式细胞免疫表型分析(FCI)属于一种高度复杂的试验,有其特有的医学适应证;对实验中抗体及其组合的选择、数据分析与报告有较高要求;从事血液淋巴系肿瘤的FCI人员需要经过专门的培训与教育.