VFC requires modern flow cytometers with improved sensitivity over previous generations of lymphocyte analyzers. Currently, the Beckman Coulter CytoFlex™, Cytek®; Aurora, Cytek® Northern Lights, Cytek® CellStream, and Cytek® ImageStream provide suitably sensitive optics for VFC. Other modern flow cytometers are likely suitable for vFC™. We are in the process of qualifying additional flow cytometers for our assay and will include performance information including LOD for vesicle size, concentration, and surface marker immunofluorescence.

VFC and NTA are vesicle characterization technologies based on different detection platforms. VFC is a set of reagents including all calibrators and standards designed for analyzing vesicles on modern flow cytometers. NTA is specialized instrumentation for sizing particles that is also used for vesicle analysis.

Because NTA was designed for sizing particles, it has many limitations in vesicle analysis. In general, it is imprecise when estimating vesicle concentration. It also lacks the fluorescence sensitivity to detect antibody labeled vesicles.

VFC solves these problems with concentration estimates that are over 5-fold more precise as well as greatly enhanced fluorescence sensitivity (current limit of detection is 30 molecules/vesicle).

Other advantages of VFC

• Vesicle specificity (NTA measures any particle, not just vesicles)
• Throughput (VFC is capable of processing 100s of samples per day)

For more information see our page on NTA.

RPS estimates size by measuring impedance of an electrical current as a particle passes through a pore.  Unlike other methods that rely upon light scatter such as NTA and conventional flow cytometry, size estimates made by RPS do not depend upon the refractive index of a particle.

Compared to VFC™, RPS lacks EV specificity and is not suitable for surface cargo measurement. It is a useful orthogonal approach for measuring vesicle size.  RPS is one approach to measuring size that Cellarcus uses to provide size estimates for its size standards.

RPS is a much more precise technology compared to NTA for estimating particle size distribution and concentration and can be used for vesicle analysis. It performs equivalently to vFC™ for vesicle size and concentration estimation, although accuracy of RPS is limited by a lack of vesicle specificity. Compared to VFC™, it lacks EV specificity and is not suitable for surface cargo measurement.

Super-resolution microscopy is being commercialized for EV measurement by ONI.  Super res. is similar to EM in that it is an imaging-based approach and is very sensitive, but unlike EM it cannot visualize unlabeled EVs. It is low throughput and lacks standardization, like other methods in the EV field. Super res. relies on immobilization of EVs to a slide which again, will not capture all EVs. EV size determination is based on estimating the diameter of clusters of antibodies which is problematic for EVs expressing markers at low abundance and could be affected by marker expression patterns which differ from EV to EV. It is, however, very sensitive, and it is a useful visualization tool which can be used as a complement to other methods.

SP-IRIS (eg. Leprechaun) is a new entrant in the small particle characterization. It offers improved cargo measurement capabilities to NTA and RPS and is better tailored to small particle measurement than conventional flow cytometry. However, SP-IRIS suffers from a similar low throughput to NTA and RPS and lacks standardization like other approaches. SP-IRIS is also not capable of measuring all EVs in a sample.  Biological EV surface antigen profiles are very heterogeneous both within and between different cell types yet, SP-IRIS relies on immunocapture of EVs which misses all EVs not expressing a given surface marker. Due to its antigen specificity, it is, perhaps, well suited as an orthogonal approach for rare event detection, but not likely useful for characterization and QC of functional/therapeutic EV preparations.

RPS is a much more precise technology compared to NTA for estimating particle size distribution and concentration and can be used for vesicle analysis. It performs equivalently to vFC™ for vesicle size and concentration estimation, although accuracy of RPS is limited by a lack of vesicle specificity. Compared to VFC™, it lacks EV specificity and is not suitable for surface cargo measurement.

No. In the past there were no methods with sufficient sensitivity to measure cargo on individual EVs.  The field relied upon bulk measurements such as western blot or ELISA to confirm the presence of particular markers in enriched fractions of EVs.  However, methods for enriching EVs are variable.  There is no method that results in a “pure” population of EVs.  Bulk measurements of EV cargo, therefore, suffer from specificity issues.  Furthermore, they provide no information on the distribution of cargo among EVs or the co-expression of markers together on the same EV.

While bulk EV methods for -omics measurements still have their place as there is now suitable single EV counterpart, modern approaches to EV characterization should avoid legacy methods such as ELISA and western blot.