Investigating the Significance of Protein Biomarkers During the Hyperacute Phase of Traumatic Brain Injury and Other CNS Conditions With Hypoxia/Hypoperfusion in Emergency and Prehospital Settings (CHARON)

Background

Traumatic brain injury (TBI) and cardiac arrest (CA) are among the leading causes of mortality and long-term disability worldwide. Together, these conditions affect an estimated 60 million people globally each year. TBI alone accounts for 27-69 million new cases annually, while out-of-hospital cardiac arrest affects approximately 275,000 individuals per year in Europe. Both conditions share a common pathobiological pathway: acute cerebral hypoxia and hypoperfusion trigger proteolysis-associated systemic cascades among others an inflammatory response in central nervous system (CNS), resulting in the release of neuronal and glial injury markers - including S100 calcium-binding protein B (S100B), neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) - into the systemic circulation.

Six blood-based protein neurobiomarkers have been extensively studied in acute brain injury: S100B and GFAP - indicators of astroglial damage; neuron-NSE and UCH-L1 - reflecting neuronal cell body injury; neurofilament light chain (NFL) - associated with axonal damage; and Tau - signaling post-injury neurodegeneration. Beyond these proteins, microRNAs (particularly miR-124), polar metabolites, and lipid metabolites (lysophosphatidylcholines, ether phosphatidylcholines, and sphingomyelins) are also under investigation as potential biomarkers of acute brain injury.

A critical and unresolved challenge is the behavior of these biomarkers during the hyperacute phase - the first minutes to hours following injury - particularly in prehospital and emergency settings. It remains unclear whether biomarker release is instantaneous at the moment of impact or gradual and continuous over time. Serial sampling studies are scarce (especially the hyperacute prehospital phase), and significant uncertainty exists regarding the influence of secondary injuries, extracranial trauma, prehospital interventions, and hemodilution on biomarker kinetics.

Sample Collection and Processing Arterial or venous blood samples are collected at study arm-specific time points detailed below. At each time point, two blood samples are collected: one serum gel tube (8 ml) and one ethylenediaminetetraacetic acid (EDTA) plasma tube (3.5 ml). Serum tubes are allowed to coagulate at ambient temperature for 45 ± 15 minutes prior to centrifugation. EDTA plasma tubes are kept on ice immediately after collection and centrifuged as soon as possible. Both tube types are centrifuged at 1,500g (4,000 rpm) for 10 minutes. Both tube types must be centrifuged within 120 minutes after sample collection. Following centrifugation, 5 × 0.5 ml serum and 4 × 0.5 ml plasma aliquoted to labelled cryovials and stored at -80°C (temporary storage at -20°C is acceptable). In Arm 1, an additional 2.7 mL 3.2% sodium-citrate tube is collected at the first prehospital contact for point-of-care rotational thromboelastometry (ClotPro®); Citrated whole blood samples for ClotPro® rotational thromboelastometry analysis are maintained at ambient temperature and analysed on-site.