Abstract
Essentials Standardization of extracellular vesicle (EV) measurements by flow cytometry needs improvement. Hollow organosilica beads were prepared, characterized, and tested as reference particles. Light scattering properties of hollow beads resemble that of platelet-derived EVs. Hollow beads are ideal reference particles to standardize scatter flow cytometry research on EVs. Summary: 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 interlaboratory 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, owing 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 with 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 with HOBs and platelet-derived EVs. Results HOBs proved to be monodisperse with a homogeneous shell thickness. Two-angle light-scattering measurements by flow cytometry confirmed that HOBs have light-scattering properties similar to those of platelet-derived EVs. Conclusions Because the structure and light-scattering properties HOBs resemble those of EVs, HOBs with a given size will gate EVs of the same size. Therefore, HOBs are ideal reference beads with which to standardize optical measurements of the EV concentration within a predefined size range.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1646-1655 |
| Number of pages | 10 |
| Journal | Journal of Thrombosis and Haemostasis |
| Volume | 16 |
| Issue number | 8 |
| DOIs | |
| State | Published - Aug 2018 |
| Externally published | Yes |
Bibliographical note
Funding Information:We thank C. Hau and L. Rikkert (Laboratory of Experimental Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands) for the TEM investigation of the cell-depleted plasma sample. This work was supported by the National Research, Development and Innovation Office (Hungary) under grant numbers PD 121326 and NVKP_16-1-2016-0007. Z. Varga was supported by the J?nos Bolyai Research Fellowship of the Hungarian Academy of Sciences. Part of this work was supported by the Cancer-ID program (www.utwente.nl/tnw/cancer-id), the MEMPHISII program of the Netherlands Technology Foundation STW, and the VENI program (15924, E. van der Pol) of the Netherlands Organization for Scientific Research ? Domain Applied and Engineering Sciences (NWO-TTW).
Funding Information:
We thank C. Hau and L. Rikkert (Laboratory of Experimental Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands) for the TEM investigation of the cell-depleted plasma sample. This work was supported by the National Research, Development and Innovation Office (Hungary) under grant numbers PD 121326 and NVKP_16-1-2016-0007. Z. Varga was supported by the János Bolyai Research Fellowship of the Hungarian Academy of Sciences. Part of this work was supported by the Cancer-ID program (www. utwente.nl/tnw/cancer-id), the MEMPHISII program of the Netherlands Technology Foundation STW, and the VENI program (15924, E. van der Pol) of the Netherlands Organization for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW).
Publisher Copyright:
© 2018 International Society on Thrombosis and Haemostasis
Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.
Keywords
- cell-derived microparticles
- exosomes
- extracellular vesicles
- flow cytometry
- microspheres