OR 15 - AF4 Profiling of Cerebrospinal Fluid Extracellular Vesicles: Towards Prognostic Monitoring in Traumatic Brain Injury

Anna Placcia,b,c,Valentina Marassia,b,c, Mladenka Malenicad, Hrvoje Križand, Vedrana Krušić Aliće, Stefano Giordania,b,c, Virginia Rondininia, Barbara Rodaa,b,c, Andrea Zattonia,b,c, Pierluigi Reschigliana,b,c

aDepartment of Chemistry “G. Ciamician”, University of Bologna, 40129 Bologna, Italy,
bbyFlow s.r.l., 40129 Bologna, Italy,
cBiostructures and Biosystems National Institute (INBB), 00165 Rome, Italy,
dDepartment of Medical Chemistry, Biochemistry and Clinical Chemistry, Faculty of Medicine, University of Rijeka, 51000 Rijeka, Croatia,
eDepartment of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, 51000 Rijeka, Croatia

Extracellular vesicles (EVs) are emerging as promising biomarkers for neurological disorders due to their ability to reflect the state of the central nervous system through their concentration and molecular cargo. Cerebrospinal fluid (CSF) is a valuable yet challenging matrix for EV analysis due to its complexity and limited availability. This work presents robust methodologies for the isolation and characterization of EVs directly from untreated human CSF, using Asymmetric Flow Field-Flow Fractionation (AF4) coupled with a multi-detection system (UV-Vis/DAD and Multi Angle Light Scattering). Unlike conventional ultracentrifugation, AF4 provides a gentle, size-based separation that preserves vesicle integrity and reduces common drawbacks such as long processing times, poor selectivity, and sample damage. 

CSF samples were obtained from multiple patients with traumatic brain injury (TBI) at different time points post-trauma. These included individual patient samples, collected on specific days after injury, and a pooled sample generated by combining aliquots from different patients and time points. All samples were analysed by AF4, which enabled EV-enriched fractions to be collected for downstream Western blot analysis and Atomic Force Microscopy (AFM). The pooled CSF sample enabled the development of a robust separation method, the identification of the EV elution zone, and an initial molecular and morphological characterization. Analysis of the individual patient samples revealed a bell-shaped trend in EV/protein peak intensity: a progressive increase up to the fourth day post-injury, followed by a decline. This is consistent with a transient neuroinflammatory response, followed by stabilization. 

These findings support the use of CSF-derived EVs in monitoring post-injury processes and defining biomarker panels. AF4 fractograms captured temporal patterns that were aligned with the pathophysiology of TBI, highlighting their potential for integration into clinical workflows for monitoring and prognosis.

Acknowledgements: Financed by UNIRI-INOVA, project title: “AF4 Profiles for Monitoring Recovery from Traumatic Brain Injury in Humans by Artificial Intelligence (AF4-AI)”

Keywords: Extracellular Vesicles, Asymmetrical Flow Field-Flow Fractionation, Cerebrospinal fluid, Traumatic Brain Injury