Validation of a plasma GFAP immunoassay and establishment of age-related reference values: bridging analytical performance and routine implementation.

Abstract

Objectives: Glial fibrillary acidic protein (GFAP) is a well-established biomarker of astrocytic activation associated with neurodegenerative diseases, neuroinflammatory disorders, and traumatic brain injury. With increasing interest in blood-based biomarkers, the need for analytically validated assays and reliable reference intervals is critical for routine clinical implementation. This study aimed to analytically validate the MSD S-Plex^® GFAP immunoassay for plasma and to establish age-stratified reference intervals in an apparently healthy population.

Methods: This study was conducted in two phases. First, key analytical validation parameters - including repeatability, intermediate precision, measurement range, interferences, and sample stability - were evaluated following Clinical and Laboratory Standards Institute (CLSI) and published protocol guidelines. Second, reference intervals were derived from 579 apparently healthy individuals aged 17-91 years using a right-sided non-parametric percentile method. Age-specific upper reference limits were calculated for three predefined age groups, and a continuous age-dependent centile model was applied.

Results: MSD S-Plex^® GFAP assay demonstrated strong analytical performance, with coefficients of variation for repeatability and intermediate precision below 12 %. After accounting for the 1:2 dilution ratio, the validated measurement range was 0.425-1760 ng/L, with all calibration residuals remaining within ±15 %. GFAP concentrations were unaffected by hemolysis (p=0.85) and remained stable for up to 7 days at 4 °C and under frozen storage conditions. Age-stratified upper reference limits for plasma GFAP were established as 38 pg/mL (18-<50 years), 73 pg/mL (≥50-<70 years), and 156 pg/mL (≥70 years). Additionally, sex-related differences were observed after age 50, with females showing higher absolute GFAP levels than males. A strong positive correlation between age and plasma GFAP levels was observed (Spearman's r=0.832, p<0.0001).

Conclusions: This study demonstrates the robust analytical performance of the MSD S-Plex^® GFAP assay and establishes age-related reference values for plasma GFAP. These findings support its suitability for routine clinical use and enhance its applicability in the diagnosis and monitoring of central nervous system (CNS) pathologies, such as neurodegenerative diseases, neuroinflammatory disorders, and acute brain injuries, within biomarker-supported clinical algorithms.