Moritz Saxenhofer

BACKGROUND: After aneurysmal subarachnoid hemorrhage (aSAH), erythrocyte lysis within cerebrospinal fluid (CSF) releases cell-free hemoglobin, a potential driver of secondary brain injury (SBI). Whether hemolysis precedes SBI and how its temporal associations compare with those of inflammation and neuroglial injury remain unclear. METHODS: In this biomarker profiling study of the prospective multicenter HeMoVal cohort, 259 patients with aSAH requiring external ventricular drainage contributed 2,411 serial CSF samples over days 1–14 after hemorrhage. We quantified 22 analytes using spectrophotometry, immunoassay, and mass spectrometry; 19 were assigned to five prespecified disease pathway composites. Day- and site-adjusted generalized estimating equations estimated associations with same-day SBI and future first SBI (angiographic vasospasm, delayed cerebral ischemia, or delayed ischemic neurological deficit); a day-wise landmark analysis assessed robustness. RESULTS: On the day of sampling, SBI was associated with four pathways, with the largest estimates for inflammation and neuroglial injury. Within the prespecified +72-hour horizon, only hemolysis (OR 1.40 per SD; 95% CI, 1.17–1.68) and the erythrophagocyte response (OR 1.28; 1.07–1.54) had FDR-significant forward associations with first SBI. Hemolysis had the largest forward estimate at every horizon; landmark analysis was concordant (pooled OR 1.86; 95% bootstrap CI, 1.37–2.65). The protective scavenger proteins haptoglobin and hemopexin declined to fitted plateaus of 2.4% and 12.5% of day-1 concentrations, versus ∼36% for plasma-protein references. CONCLUSION: In a scavenger-depleted CSF compartment, hemolysis and the erythrophagocyte response were associated with subsequent SBI. These findings identify hemolysis as a candidate upstream process and support testing sustained CSF hemoglobin scavenging to prevent SBI. TRIAL REGISTRATION: ClinicalTrials.gov NCT04998370 . FUNDING: CSL Behring; Swiss National Science Foundation; University of Zurich

Heme toxicity plays a central role in the pathophysiology of Sickle Cell Disease (SCD), contributing to severe complications such as vaso-occlusion and acute chest syndrome. The continuous release of hemoglobin and heme from increased intravascular hemolysis can exceed the capacity of protective scavenger proteins, leading to heme accumulation in plasma. Interactions with various binding partners result in the formation of different plasma heme species and the compartmentalization of the plasma heme pool. In an observational biomarker study, we used novel bioanalytical assays to quantify plasma heme species in 36 stable-state SCD patients and 36 age, sex, and ethnicity-matched controls. Our results revealed substantially different compartmentalization of plasma heme, despite similar levels of total plasma heme in SCD patients (50 µmol/L) and controls (43 µmol/L). Using a correlation analysis across 85 biomarkers, we examined the association of specific heme species with SCD pathophysiology. Hemopexin-accessible heme (HAH) emerged as a refined indicator of heme burden linked to pathways driving severe SCD complications. A strong inverse correlation was observed between HAH and hemopexin (R = –0.73, p < 0.001), suggesting that hemopexin deficiency contributes to elevated HAH levels. Accurate characterization of clinically relevant plasma heme species and understanding their effects on SCD pathophysiology is essential for the development of new targeted therapies.

Objectives To validate whether cerebrospinal fluid oxyhaemoglobin (CSF-Hb), measured from external ventricular or lumbar drains, is associated with secondary brain injury (SAH-SBI) after aneurysmal subarachnoid haemorrhage (aSAH), and to assess its value as a real-time monitoring biomarker. Design Preregistered multicentre prospective observational cohort study. Setting Eight neurosurgical tertiary centres in Switzerland, Germany, and Austria, between August 2021 and June 2024. Participants 366 patients with aSAH (mean age 58 years; 65% women). Of these, 260 provided cerebrospinal fluid (CSF) samples via external ventricular drain (EVD; 2,467 samples, median 10 days per patient) and 66 via lumbar drain (LD; 379 samples, median 6 days). Interventions Daily CSF samples were collected via EVD or LD from day 1 to day 14 after haemorrhage; no therapeutic interventions were tested. Main outcome measures CSF-Hb and its metabolites were analysed post hoc in a blinded manner. The primary outcome was SAH-SBI, defined as a composite of angiographic vasospasm (aVSP), delayed cerebral ischaemia (DCI), and delayed ischaemic neurological deficits (DIND), assessed daily over 14 days. Secondary outcomes included temporal CSF-Hb profiles and associations with aneurysm location, haematoma volume, intraventricular haemorrhage, chronic hydrocephalus, and 3-month functional outcome. Results CSF-Hb showed a delayed peak pattern: concentrations were low after aSAH, rose to a maximum on day 10 (EVD-derived CSF-Hb median 11.3 μM, IQR 2.64 to 25.90), and then declined. Larger haematoma volume (pConclusions In this preregistered multicentre validation study, EVD-derived CSF-Hb did not perform as a robust real-time monitoring biomarker for SAH-SBI, showing limited same-day discrimination and substantial between-centre heterogeneity. These findings argue against clinical implementation of CSF-Hb point-measurement as a single-parameter biomarker. In contrast, CSF methaemoglobin remained consistently associated with SAH-SBI, supporting the mechanistic relevance of haemolysis-related pathways. Future work using the HeMoVal biobank will apply multi-marker, pathway-level analyses to define haemolysis-related biomarker signatures and provide a platform for robust external validation of future candidates. Study registration ClinicalTrials.gov NCT04998370 ; date of registration 10 August 2021.