Abstract
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 steric hindrance-mediated EV analysis and size fractionation (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.
Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD075777. The main data supporting the results in this study are available within the paper and its Supplementary Information. Source data are provided with this paper for Figs. 2 - 5 and Supplementary Figs. 1, 5 − 7, 9 − 14, and 18. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China: Grant No. 22064007 (X.W.), 52375577 (H.Z.), 82560244 (G.W.), and 82260244 (G.W.). X.W. acknowledges support from the Guizhou Provincial Natural Science Foundation (Grant No. ZK[2021]482). G.W. acknowledges support from the Leading Discipline Program of The Affiliated Hospital of Guizhou Medical University (Grant No. gyfyxkrc-2023-05), the Key Lab of Acute Brain Injury and Function Repair in Guizhou Medical University (Grant No. [2024]fy007), and the Key Advantageous Discipline Construction Project of Guizhou Provincial Health Commission in 2023 in Emergency Department.
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X.W., H.Z., G.W., and X.S. conceived the project, designed the experiments and wrote the manuscript. X.W., S.X., and L.Z. conducted the experiments and performed data analysis. Y.Z. performed SEC- and DGUC-based EV size fractionation. Q.G. and Y.E.W. established cellular inflammation models. W. Long conducted the Western blotting experiments and proteomic analyses. Z.S., L.W., W.L., and G.W. collected clinical samples and performed clinical diagnosis. Y.C. and L.T. coded the programs.
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Wu, X., Xiong, S., Zhang, L. et al. Steric hindrance-mediated extracellular vesicle size fractionation for rapid prehospital diagnosis of intracerebral hemorrhage. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71751-y
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DOI: https://doi.org/10.1038/s41467-026-71751-y
