Transmission Electron Microscopy-based characterization of Extracellular Vesicles from plasma and serum from...

Parkinson's disease is a neurodegenerative disorder with no curative treatment option and objective biomarker profile. Extracellular Vesicles (EVs) are membrane-enclosed biological nanoparticles released from all cells of the human body. In this pilot study we compare plasma- and serum-derived EVs from Parkinson's disease (PD) patients and healthy controls (HC) utilizing a precipitation-based method. Additionally, we employ an L1CAM antibody to selectively enrich for L1CAM-positive EVs from the total plasma-/serum-derived EV fractions. Successful EV enrichment was shown in western blot experiments for CD63 and for L1CAM as well as in metabolomic analysis for a HC sample. In a side-by-side quantification,. which we based on transmission electron microscopic images from negative stain samples, we identify small but significant differences between EV diameter from PD patients and HC. To streamline the quantification process, we introduce an ImageJ-based computer algorithm for (semi-)automated quantification of EVs from negative stain electron micrographs. We observe that this (semi-)automated quantification determines a smaller diameter than manual quantification. However, the difference between PD and HC group is systematic and reveals the same relative differences calculated from manually measured total plasma-derived EV particles. In this pilot study, we introduce a new workflow implemented into an ImageJ plugin enabling to determine differences in EV size within TEM images. For our data set of plasma-derived EVs from PD patients and HC, we find small, yet consistent differences. We feel that this study contributes to the search of a clinical biomarker for PD.