Standardization of vesicle detection by flow cytometry using traceable beads and optical scattering theory
||E. van der Pol, F.A.W. Coumans, Y. Yuana, A. Sturk, T.G. van Leeuwen, R. Nieuwland|
||International Society for Extracellular Vesicles 2014, Rotterdam, The Netherlands|
||May 2, 2014|
Background: Flow cytometry is currently the most used method for single vesicle detection in the clinic. At present, most laboratories select vesicles by setting an inclusion gate based on the scatter signal from two polystyrene bead sizes, resulting in a reproducibility of 15-91% in a previous multicenter study. Comparison of flow cytometry results of vesicle measurements between laboratories, however, is not straightforward because (1) the refractive index of beads and vesicles are not matched, and (2) the relation between the size ranges of beads and vesicles is affected by the optical configuration of the flow cytometer.
Methods: We analytically describe the relation between the diameter, refractive index, and light scattering of traceable polystyrene beads (www.metves.eu) with Mie theory to find the relationship between scatter and size of vesicles. Assuming a vesicle refractive index of 1.40, this procedure is expected to minimize the impact of the refractive index mismatch and eliminate differences between optical configurations. Therefore, we initiated a study including 20 centers worldwide to determine whether this procedure improves the reproducibility of vesicle concentration measurements between laboratories. The International Society on Thrombosis and Haemostasis is sponsoring this activity, which will take place between February 2014 and June 2014. Each center measures PMP and erythrocyte vesicle samples with lactadherin and CD61-PE or CD235a-PE staining, respectively.
Results: Based on theoretical simulations we expect this procedure to reduce the coefficient of variation on the measured concentrations by 20% compared to the current standard. We will present preliminary data from the study.
Discussion: This method will allow a closer approximation of the size of vesicles, and improve the reproducibility of concentration measurements with different flow cytometers. There may be a difference between assumed and true refractive index, which would result in a difference between the desired and actual vesicle size range. However, this refractive index difference is small compared to the error made by assuming the refractive index of polystyrene to be equal to vesicles.