Steric hindrance-mediated extracellular vesicle size fractionation for rapid prehospital diagnosis of intracerebral hemorrhage

Diagnosing intracerebral hemorrhage (ICH) in prehospital settings remains challenging due to unavailability of immediate neuroimaging, clinical overlap with ischemic stroke, and absence of validated circulating biomarkers for time-critical settings. Extracellular vesicles (EVs), subcellular structures capable of transporting biomolecular payloads (e.g., proteins, nucleic acids) across the blood-brain barrier, have emerged as compelling diagnostic candidates for ICH. Nevertheless, their clinical translation has been impeded by inherent biophysical heterogeneity, particularly polydisperse size distributions. To address this limitation, we engineer a s teric h indrance-mediated E V a nalysis and size f ractionation (SHEAF) platform, integrating steric hindrance-based size fractionation with membrane protein profiling to stratify EVs into three size subtypes within a 45-min workflow. Systematic evaluation using the SHEAF platform reveals that the 90 − 180 nm EV subtype exhibits superior discriminative capacity in differentiating ICH from ischemic stroke plasma specimens. This technology not only advances rapid prehospital ICH diagnostics but also establishes a method for elucidating size-dependent EV functionalities across neurological pathologies.