From beads to bedside Standardizing extracellular vesicle concentration measurements

Extracellular vesicles (EVs) are cell-derived particles with a phospholipid bilayer that are present in body fluids, such as blood and urine.
Concentrations of EVs in body fluids are potential disease biomarkers and can be measured with flow cytometers (FCMs). To explore the biomarker potential of EV concentrations, reliable and reproducible measurements of EVs are needed. At present, however, measured EV concentrations are incomparable between instruments and institutes. The aim of this thesis is to improve reliability and comparability of EV concentration measurements with flow cytometry by 1) identifying current challenges faced in EV flow cytometry, 2) developing a robust methodology for reliable and comparable EV concentration measurements, and 3) evaluating the developed methodology in an interlaboratory comparison study.
In this thesis, we present a filtration procedure to remove residual platelets from blood plasma, thereby improving the quality of plasma for EV research. Furthermore, we introduce a novel methodology to generate reliable and comparable EV concentration measurements by flow cytometry. The newly developed methodology includes a plasma EV test sample and reference materials to calibrate all parameters of an FCM influencing measured EV concentrations. The methodology was evaluated in a global interlaboratory comparison study including 38 FCMs from 24 laboratories. Emphazising the clinical relevance of the developed methodology, we applied the methodology to determine the first-ever reference ranges of cell-type specific EV concentrations in human blood plasma of healthy volunteers. The methodology presented in this thesis opens the door to explore EV concentrations as potential biomarkers for disease.