2017 Working Group Study

ISEV-ISAC-ISTH EV FC Working Group Summary



Spring 2017 Fluorescence Calibration study – John Nolan

This year’s study aims to demonstrate a consensus approach to calibration and reporting of EV fluorescence measurements. The study has three tasks:

1) Calibrate instrument fluorescence response using commercial reagents,

2) Measure a common set of EV reference materials using a diverse set of flow cytometry methods and instruments, and report the data in a uniform manner such that results can be compared across methods and instruments, and

3) Evaluate a prototype set of EV-scale fluorescence intensity and antibody binding standards that may be more appropriate for calibration and standardization of EV measurements.

All participants will perform a common set of measurements and report their methods and results according to draft Minimum Information about an EV Flow Cytometry measurement (MI EV FlowCyt) guidelines. The methods, results and guidelines that result from the study will be posted here and reported in a manuscript to be submitted to JEV, and a set of protocol units describing the methods used by study participants will be commissioned for Current Protocols in Cytometry.


Standardize EV Measurements by Light Scatter.

Rienk Nieuwland, also on behalf of Frank Coumans, Ton van Leeuwen, Marcell Pálmai, Edwin van der Pol, Guus Sturk, Zoltán Varga (AMC, Amsterdam, Netherlands; MTA TTK, Budapest, Hungary).

Flow cytometers differ in light scattering sensitivity and therefore measure different concentrations of EVs in the same sample. Light scattering, however, is a complex process which depends on the size (diameter) and refractive index (RI) of a particle. Consequently, once the RI is known, the size of EVs can potentially be derived by measuring light scattering.

Previously, the ISTH distributed polystyrene reference beads of known diameter to determine EV scatter gates on different flow cytometers. The idea was to determine the concentration of EV reference samples within the scatter signals corresponding to two sizes of polystyrene beads. However, this approach suffers two shortcomings. Firstly, polystyrene has a higher RI than EVs. Consequently, the diameters of the polystyrene beads do not correspond to the diameter of gated EVs. In fact, the diameter of the gated EVs remains unknown. Secondly, because flow cytometers differ in optical configuration, a bead gate selects different EV diameters on different flow cytometers (J Thromb Haemostas 2014; 12; 1182-92).

To overcome these shortcomings, we tested two approaches. In the first approach (FC, EvdP, TvL), the light scattering of a mixture of polystyrene is measured and modelled by Mie theory to obtain the optical configuration of the flow cytometer and set EV size gates in nm, thereby taking into account the low RI of EVs (exometry.com).  A comparison study applying this approach showed improved detection of measured EV concentration compared to the earlier ISTH approach, but also revealed that 1 in 3 flow cytometers is too insensitive to detect EVs (manuscript in prep).

In the second approach, reference particles resembling the light scattering properties of EVs were developed. Together with the EV Core lab of the University of Helsinki (Eur J Pharm Sci 2017; 98: 4-16), we prepared and characterized biological reference materials. Because the reference materials were based on real EVs (e.g. from erythrocyte ghosts), the light scattering properties should resemble other EV types. However, our biological reference materials lacked stability and monodispersity.

Alternatively, in the second approach, together with MTA TTK (ZV, MP, EvdP) hollow silica particles were produced, i.e. inorganic reference particles with approximate light scattering properties of EVs. These particles are monodisperse and stable, do not sediment, and provide end-users direct insight in the detection limit of their instrument. For example, a flow cytometer that detects 400 nm hollow silica particles, can also detect 400 nm EVs. Taken together, we believe that these hollow silica particles are useful to set EV size gates on different types of flow cytometers. At present (May 2017), characterization of these hollow silica particles is ongoing, and results will be reported at ISTH, ISAC and ISEV.


Biological Reference Materials Overview, by Jennifer Jones (NIH)

As the ultimate purpose of this Working Group is to develop shared methods and metrics for performing and reporting the analysis of extracellular vesicles and other micro- and nano-scale biological materials, it is critical that we begin to test and validate the methods and results with such biological materials. As such, we have obtained biological reference materials, which have been produced in Working Group Participant Labs, and characterized with orthogonal methods, including electron microscopy, nanoparticle tracking analysis, etc.

Among the materials contributed are EVs from glioblastoma (Higgenbotham) and CD63-GFP-expressing THP-1 cell lines (Giebel and Goergens).  In addition to these EV reference preparations, John Nolan contributed beads with two concentrations of immunglobulin binding sites, for use to calibrate fluorescence intensity to measure number of binding sites (epitopes), as compared to the of fixed numbers of directly-conjugated antibodies, as used for staining EVs.

In addition to these biological reference materials, we hope to distribute a test set of concentration standards (http://www.colloidalmetrics.com/products.php) to working group participants to test in the near future.


Summary of Audience Discussion, Led by Joanne Lannigan (UVA)

The audience echoed the group’s desire to establish a set of EV reference materials and tools for evaluating instrument performance and sensitivity. There was also a comment regarding the accuracy of EV counts using standard flow cytometers, with or without volumetric measurements built in. There is currently no concentration standard that exists, even the National Institute of Standards and Technology (NIST) currently does not have such a standard.  This type of standard is very difficult to make.

Someone else brought up the possibility to use viruses as reference material due to their homogeneous nature, with regard to size. The audience member also said she had viruses that express known numbers of a fluorophor; i.e. GFP, with as few as 100 and this would be useful for not only assessing size but fluorescence sensitivity. A discussion of this ensued and there were concerns regarding the stability and biosafety of such standards, however, the topic would need further discussion. There was a general consensus that to continue to move forward using flow cytometry for EV measurements, we needed to work toward having the appropriate materils to assess the existing instrumentation available as well as those currently under development geared more specifically toward the measurement of small particles rather than whole cells. These reference materials and standards need to more closely reflect the size, concentration, and fluorescence intensity of the EVs.


Data may be contributed for analysis for the 2017 Working Group study at the following link: