Consensus harmonization of brain‐secreted extracellular vesicles isolation and validation protocols for blood biomarker work in Alzheimer’s disease: An international overview

Abstract Background Isolation of brain‐secreted extracellular vesicles (BEVs) from blood, provides a novel, minimally invasive way to sample brain tissue in individuals along the Alzheimer’s disease (AD) continuum. BEVs in blood demonstrate enormous potential to serve as a novel AD biomarker discovery platform and treatment tool. However, at present, BEV isolation and validation protocols vary from center to center. Given the growing popularity of immunoprecipitation (IP)‐based BEV isolation methods, we assessed the brain‐specificity of reported cell‐surface and cargo proteins associated with this category of methods. Method We performed a systematic evaluation of BEV isolation and validation protocols from 34 published articles investigating biomarker potential of blood‐isolated BEVs in AD. Proteins used for polyethylene glycol‐based precipitation followed by IP‐capture BEV isolation, or as BEV biomarker candidates, were compiled. The proteins were categorized by brain specificity, abundance, extracellular accessibility and extracellular vesicles presence by examining their expression from publicly available datasets in over 60 human tissue types, including 13 central nervous system (CNS) regions and 48 peripheral tissues. Result We compiled 68 unique IP or biomarker protein candidates. Analyses demonstrated that of these, 22 had extracellular domains (that could be used for BEV isolation), 7 were membrane proteins lacking extracellular domains, and the remaining were intracellular proteins. Moreover, >50% of the compiled proteins were present in extracellular vesicle datasets, >20% were considered highly CNS specific, and ∼60% demonstrated moderate‐to‐high CNS abundance (Fig.1). Furthermore, in addition to confirming that some proteins widely used for IP‐based BEV isolation are CNS‐enriched (e.g., L1CAM, Fig. 1B), we also identified, at least, 5 additional proteins with higher CNS abundance and specificity, extracellular domains, and known to be present in extracellular vesicles. Conclusion Consensus harmonization of BEV isolation and validation protocols are essential steps in blood‐isolated BEV biomarker work in AD, and is advocated by the Alzheimer’s Association BBB‐PIA. This harmonization of BEV protocols is crucial, given the growing interest in developing minimally‐invasive AD biomarker discovery tools, and the increasing number of researchers worldwide conducting BEV biomarker work. Moreover, the additional proteins identified by the EWG‐BBB‐PIA will lead to better IP‐based BEV isolation from blood.