Stoichiometric constraints for detection of EV-borne biomarkers in blood

Stochiometric issues, encompassing both the quantity and heterogeneity of extracellular vesicles (EVs) derived from tumour or other tissues in blood, pose important challenges across various stages of biomarker discovery and detection, affecting the integrity of data, introducing losses and artifacts during blood processing, EV purification and analysis. These challenges shape the diagnostic utility of EVs especially within the framework of established and emerging methodologies. By addressing these challenges, we aim to delineate crucial parameters and requirements for tumour-specific EV detection, or more precisely, for tumour identification via EV based assays. Our endeavour involves a comprehensive examination of the layers that mask or confound the traceability of EV markers such as nucleic acids and proteins, and focus on ‘low prevalence—low concentration’ scenario. Finally, we evaluate the advantages versus limitations of single-particle analysers over more conventional bulk assays, suggesting that the combined use of both to capture and interpret the EV signals, in particular the EV surface displayed proteins, may ultimately provide quantitative information on their absolute abundance and distribution.