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

Varga, Zoltán and Van der Pol, Edwin and Pálmai, Marcell and Garcia-Diez, Raul and Gollwitzer, Christian and Krumrey, Michael and Fraikin, Jean-Luc and Gasecka, Aleksandra and Hajji, Najat and van Leeuwen, Ton G. and Nieuwland, Rienk (2018) Hollow organosilica beads as reference particles for optical detection of extracellular vesicles. Journal of Thrombosis and Haemostasis, 16 (8). pp. 1646-1655. ISSN 1538-7933

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Abstract

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 inter-laboratory 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 due 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 by 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 using HOBs and platelet-derived EVs. Results: HOBs proved monodisperse with homogeneous shell thickness. Two angle light scattering measurements by flow cytometry confirmed that HOBs have light scattering properties similar to platelet-derived EVs. Conclusions: Because HOBs resemble the structure and light scattering properties of EVs, HOBs with a given size will gate EVs of the same size. Therefore, HOBs are ideal reference beads to standardize optical measurements of the EV concentration within a predefined size range.

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