Marlies Illi

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.

Unattended endotoxin (ETX) contamination in biological samples constitute a major challenge for in vitro and in vivo applications. Besides being potentially life‐threatening, ETX contamination is especially relevant in global transcriptome analyses, where competing ETX stimulation can significantly skew the final gene expression profile. Our studies in mice and cultured skin epithelial cells (epidermal keratinocytes) aiming to characterize the effect of antibodies such as AK23 immunoglobulins (IgG directed against the cell‐cell adhesion molecule desmoglein [DSG] 3) in the autoimmune disease pemphigus vulgaris (PV) revealed that laboratory‐produced and even commercial control antibodies can exhibit non‐negligible ETX contaminations. Moreover, these contaminants are extremely difficult to remove. To overcome these challenges, we have devised a simple yet nontoxic and scalable two‐step protocol to efficiently reduce ETX levels during or after the IgG purification process. It consists firstly of 0.5 M NaOH pre‐treatment of all devices, including the protein A resin, used during IgG sanitization and purification, in parallel with meticulous in‐process monitoring of ETX levels. Secondly, before IgG elution from protein A, ETX is stripped from IgG by ion‐exchange with the common amino acid arginine. This two‐step approach successfully reduces ETX by >95% from hybridoma‐derived, laboratory‐produced AK23 IgG, as well as patient PV and control IgG, resulting in an 85% IgG recovery rate and ETX levels compatible with U.S. Pharmacopeia guidelines. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Sanitization of devices and protein A resin with 0.5 M NaOH Basic Protocol 2 : On‐column stripping of ETX from AK23 IgG Basic Protocol 3 : Quality control of sanitized AK23 IgG