Cerebrospinal fluid hemolysis precedes secondary brain injury after aneurysmal subarachnoid hemorrhage

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 1-4 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