From Plasma to Whole Blood: Validation of Glial Fibrillary Acidic Protein (GFAP) and Neurofilament Light Chain (NfL) As Accessible Biomarkers in Stroke

BACKGROUND: Glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL) are critical blood-based biomarkers for the diagnosis and monitoring of a wide range of central nervous system (CNS) injuries. Current plasma-based assays require centrifugation, which prolongs turnaround time (TAT) and limits utility in emergency settings. This study aimed to develop and validate a rapid, automated chemiluminescence immunoassay for the direct quantification of GFAP and NfL in whole blood. METHODS: We used a dual-antibody sandwich chemiluminescence immunoassay to measure GFAP and NfL. The assay was optimized for whole blood through adjustments of reagent formulation and implementation of a hematocrit (HCT) correction algorithm. Performance was evaluated in terms of precision, linearity, limit of quantification, and interference. In total, 115 paired whole blood and plasma samples from 78 acute stroke patients were analyzed to assess diagnostic accuracy. RESULTS: The HCT correction algorithm effectively minimized measurement bias across a wide HCT range (35%-55%). Precision of GFAP and NfL in whole blood and plasma showed coefficients of variation below 5% across a broad concentration range. Excellent correlations were observed between whole blood (HCT-corrected) and plasma measurements for both GFAP (R2 = 0.990) and NfL (R2 = 0.955). Diagnostic accuracy for distinguishing hemorrhagic from ischemic stroke was comparable between whole blood and plasma (AUC 0.86 vs 0.84, P = 0.58) in this population. CONCLUSIONS: The novel GFAP and NfL whole-blood assay, due to its shorter TAT, high consistency with plasma, and broad analytical range, demonstrated the potential to accelerate stroke triage in acute care settings.