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

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.

Use of elution marker for the intratypic characterization of poliovirus strains.

Elution marker was used for intratypic characterization of poliovirus strains with Al(OH)3 gel as adsorbent. The virion suspensions to be tested were partially purified by chromatography and labelled with 32P. In the labelled preparations of wild virus strains practically all radioactivity was found in virus-specific bond, whereas in those of the vaccine strains and isolates of vaccine origin a considerable, but variable, proportion of the activity was bound to residual cell components. For this reason, the EC50 value (i.e. the phosphate molarity corresponding to the 50% adsorption equilibrium of virions), for the vaccine and vaccine-like strains showed a wide scattering. The activity bound to cell components was removable from the gel with 0.005 M phosphate buffer, whereas the elution maxima for all the poliovirus strains examined so far were over 0.02 M. The elution marker was calculated for 25 type-2 and 30 type-3 strains, all isolated during or soon after vaccination periods from cases suspect of poliomyelitis, both with and without taking into account the cell-bound activity. In the latter case, the EC50 values agreed with that for the corresponding reference vaccine strain much better than in the former; furthermore, the type-2 reference vaccine strain and the type-2 isolates, indistinguishable from the wild reference strain by the original method, could easily be differentiated on the basis of the corrected EC50 value. The possibility that the lack of an appreciable proportion of cell-bound radioactivity may reflect the pathogenicity of poliovirus strains is discussed.