Work package 2: Dimensional characterisation of MV and reference materials

The aim of WP2 is to develop reliable methods for the dimensional characterisation of MV samples isolated in WP1 and the reference materials developed in WP4.

The most common method used in hospitals to study MV is flow cytometry, a technology based on scattering of visible light. Since the intensity of scattered light decreases drastically with the diameter for particles significantly smaller than the illumination wavelength, the lower detection limit of flow cytometry is about 300 nm for MV1. Also dynamic light scattering, recently used in several studies to detect MV, was shown to have severe limitations2. In general, detection of MV is difficult since MV have a low refractive index, MV populations are polydisperse, and samples contain similar sized contaminant particles such as lipoprotein particles.

Biological samples prepared in WP1 and reference materials selected in WP4 will be analyzed free in suspension using Small Angle X-ray Scattering (SAXS), Nanoparticle Tracking Analysis (NTA), and Resistive Pulse Sensing (RPS). In addition, MV from the same samples will be adhered to a surface and analyzed under dried and wet conditions by Atomic Force Microscopy (AFM) and under dried conditions only by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Transmission Scanning Electron Microscopy (TSEM) to investigate the influence of deposition, fixation and dehydration. In addition, this will provide complementary information on the size of MV and the contamination of other particles. Knowledge obtained will be used to improve isolation protocols (WP1) and reference materials (WP4). A comparison between the size distributions of MV deposited in liquid and in dried state will be made.

References

1.  E. van der Pol, M.J.C. van Gemert, A. Sturk, R. Nieuwland, and T.G. van Leeuwen
Single versus swarm detection of microparticles and exosomes by flow cytometry
J. Thromb. Haemost. 10 (5), 919-30 (2012)
2.  A.S. Lawrie, A. Albanyan, R.A. Cardigan, I.J. Mackie, and P. Harrison
Microparticle sizing by dynamic light scattering in fresh-frozen plasma
Vox Sang 96, 206-12 (2009)