Physical interpretation of the size and concentration of extracellular vesicles
||E. van der Pol, C. Gardiner, P. Harrison, A. Sturk, T.G. van Leeuwen, and R. Nieuwland|
||XXIV Congress of the International Society on Thrombosis and Haemostasis, Amsterdam, The Netherlands|
||July 2, 2013|
||Amsterdam 2013 Size and concentration of vesicles.pdf (662 kB)|
Background: Novel advanced techniques are utilized to determine the size and concentration of extracellular vesicles. All techniques provide data, but often the measured physical quantity or units are different, precluding comparison and validation of measurements with established techniques. We have developed a model to enable data comparison between five different techniques that are increasingly used to detect vesicles.
Aims: Facilitate data comparison between different techniques based on the underlying physical parameters of each technique and provide thorough insight in the application and limitations.
Methods: A heterogeneous mixture of beads (Thermo Fisher Scientific, Waltham, MA, USA) of known size and concentration and a standard population of vesicles from human cell-free urine (n = 5) were prepared and analyzed by single particle tracking (SPT; Nanosight NS500), resistive pulse sensing (RPS; Izon qNano), a novel flow cytometer (Apogee A50-Micro), a conventional flow cytometer (BD FACSCalibur), and transmission electron microscopy (TEM). Data interpretation and comparison is enabled by presenting size distributions on the same absolute scale using a physical model, including data from TEM and flow cytometry, which was previously impossible. The size distributions from the well-characterized mixture of beads were used to investigate the capabilities of each technique, thereby facilitating the interpretation of data obtained from the standard population of vesicles.
Results: Using the beads we obtained that, among the techniques capable of measuring in suspension, surprisingly, flow cytometry can resolve the smallest differences in size, whereas SPT detects the smallest vesicles, and RPS is most accurate in determining the concentration. All methods produce different size distributions and concentrations for the standard population of vesicles.
Conclusions: Various detection techniques produce different size distributions and concentrations for a standard population of vesicles. However, most differences can be explained based on the underlying physical parameters of the technique, which we have investigated using a well-characterized mixture of beads. Since the techniques have divergent capabilities, the most suitable technique for detecting vesicles depends on the requested information.