Age-specific upper reference limits for Simoa-measured cerebrospinal fluid glial fibrillary acidic protein: a cross-sectional DANPAIN biobank study

Glial fibrillary acidic protein (GFAP) is an emerging biomarker of astrocytic activation and central nervous system injury. As GFAP is increasingly investigated in neurological and neurodegenerative disorders, robust upper reference limits (URLs) are needed to support interpretation of cerebrospinal fluid (CSF) measurements and identify concentrations that may represent pathological elevations. This cross-sectional study used CSF samples from the Danish Pain Research Biobank. Participants without known neurological or neurodegenerative disease and without evidence of neuroinflammation or significant CSF abnormalities were included to establish a biologically refined reference population. CSF GFAP concentrations were measured using the Single Molecule Array (Simoa) HD-X platform. Associations between CSF GFAP concentrations and demographic and clinical variables were evaluated using linear regression and restricted cubic spline analyses. URLs were estimated non-parametrically as one-sided 95th percentiles with corresponding 90% confidence intervals (CIs). A total of 325 participants were included (median age 50 years; 72.6% women). Median (Q1-Q3) CSF GFAP concentrations across age groups were 5,988 ng/L (4,773–7,582), 8,406 ng/L (6,867–11,083), and 13,766 ng/L (10,877–18,127, p < 0.001) in 18–39 year-olds ( n = 93), 40–59 year-olds ( n = 143), and 60–79 year-olds ( n = 89), respectively. In multivariable analyses, age was the only independent determinant among the evaluated demographic and clinical variables ( p < 0.001). Age-stratified 95th percentile URLs were 10,580 ng/L (90% CI: 6,576 − 14,584), 15,814 ng/L (90% CI: 12,734 − 18,893), and 29,066 ng/L (90% CI: 24,748 − 33,385) for 18–39, 40–59, and 60–79 year-olds, respectively. CSF GFAP concentrations increase markedly with age, supporting age-specific interpretation of measurements. This study provides age-specific URL estimates across three adult age strata, establishing a reference framework for future clinical and translational applications of CSF GFAP.