Critical Hyperacute Assessment Research On Neurobiomarker Kinetics in Acute Brain Injury

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.

Arm 1 - Prehospital TBI/Polytrauma:

Prehospital phase:

Sample 1: immediately after intravenous access is secured Sample 2: immediately before transport initiation

In-hospital phase:

Sample 3: upon emergency department admission Sample 4: 1 hour after ED admission Sample 5: 6 hours after ED admission Sample 6: 24 hours after ED admission

Arm 2 - Prehospital Cardiac Arrest:

Prehospital phase:

Sample 1: immediately after the first adrenaline dose (asystole/pulseless electrical activity [PEA]) or first defibrillation (ventricular fibrillation [VF]/ pulseless ventricular tachycardia [pVT]) Sample 2: at 20 minutes of ongoing resuscitation Sample 3: immediately after ROSC or prior to declaration of death

In-hospital phase:

Sample 4: upon emergency department admission Sample 5: 1 hour after ED admission Sample 6: 6 hours after ED admission Sample 7: 24 hours after ED admission

Arm 3 - In-hospital TBI:

Sample 1: upon emergency department admission Sample 2: 1 hour after ED admission Sample 3: 6 hours after ED admission Sample 4: 24 hours after ED admission

Biomarker Measurements S100B and NSE: Electrochemiluminescence immunoassay (ECLIA; Elecsys S100 and Elecsys NSE assays) on the Cobas 8000 modular analyzer (Roche Diagnostics, Mannheim, Germany) at the Department of Laboratory Medicine, University of Pécs.

Brain-derived Tau (BD-Tau) and phosphorylated Tau-217 (p-Tau-217): Immunoassay on the Beckman Coulter DXi 9000 analyzer at the Department of Laboratory Medicine, University of Pécs.

GFAP and UCH-L1: VIDAS® TBI assay on the VIDAS®3 instrument (bioMérieux SA., Marcy-l'Étoile, France) at Örebro University, Sweden.

NFL, Tau, ionized calcium-binding adaptor molecule 1 (IBA-1), IL-1β, IL-6, TNF-α: Commercially available ELISA kits using the BMG LabTech CLARIOstar multimode microplate reader (BMG Labtech GmbH, Ortenberg, Germany) at the Szentágothai Research Centre, University of Pécs.

Metabolomics and lipidomics: Polar metabolites detected via Agilent 7890B GC coupled with a 7200 Q-TOF MS; lipids quantified by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) at Örebro University, Sweden.

MicroRNA: Cell-free total RNA isolated from 500 µl serum using the miRNeasy serum/plasma kit (Qiagen, Hilden, Germany). RNA integrity confirmed by NanoDrop 2000. Reverse transcription performed using the miRCury LNA Universal RT microRNA PCR Kit (Qiagen). Droplet digital PCR (ddPCR) targeting miR-30a, miR-487b, let-7b, miR-363, and miR-126 performed at the Department of Laboratory Medicine, University of Pécs.

Cytokine and chemokine profiling: Simultaneous detection of multiple inflammatory mediators using the Human Cytokine Array (ARY022B, R\&D Systems, Minneapolis, MN, USA). Chemiluminescent signals are captured at multiple exposure times for optimal detection.

Rotational thromboelastometry is performed on citrated whole blood samples (sodium citrate 3.2%) using the ClotPro® analyser (Haemonetics Corporation, Boston, MA, USA). Three assays are used: the EX-test (extrinsic pathway activation with recombinant tissue factor), the IN-test (intrinsic pathway activation with ellagic acid), and the FIB-test (EX-test with the platelet inhibitor cytochalasin D, isolating the fibrinogen contribution to clot firmness). The analysis is performed at the ambulance base stations and at the participating hospitals.

Statistical Analysis

Statistical significance threshold: p < 0.05. Given the expected non-normal distribution of biomarker data, non-parametric methods are applied as primary statistical tests, including the Mann-Whitney U test and the Kruskal-Wallis test. Diagnostic and prognostic performance is evaluated using receiver operating characteristic (ROC) curve analysis with area under the curve (AUC); AUC comparisons are performed using the DeLong test. The association with mortality and neurological outcome is examined by binary logistic regression (univariable and multivariable, with pre-defined covariates).

Sample size was determined by power analysis for binary logistic regression - the most statistically demanding analysis - using a medium effect size (Cohen's d = 0.5), α = 0.05, and a power of 0.80. A Mann-Whitney equivalent correction of +10% was applied to account for non-normality of biomarker distributions. This yields a required sample size of 159 participants per arm, for a total target enrollment of 477 participants across all three arms. No single center may contribute more than 40% of the total sample to mitigate center effects. All analyses will be performed using R software (RStudio environment), with a biostatistician involved throughout.