Neurofilament light chain (NfL) is a sensitive biomarker of neuroaxonal injury detectable in blood and cerebrospinal fluid. Its concentrations are strongly influenced by age, yet no age-specific reference values have been established for the fully automated Lumipulse® platform, and no direct comparison between serum and plasma has been reported for this assay.

Matched serum and plasma samples from 49 participants were analyzed to assess matrix comparability using correlation, paired comparison, and equivalence testing within predefined ±15% bounds. Reference values were determined in 211 control adults aged 18–87 years from two international centers. Age groups were derived using K-means clustering, and multivariable linear regression was applied to examine the effects of recruitment center, age, and sex.

Serum and plasma NfL levels were highly correlated (Spearman's ρ = 0.969, P < 0.001). Although serum values were slightly higher than plasma (+3.07 pg/mL, P = 0.042), the difference was within equivalence margins ( P = 0.019), indicating that either matrix can be used without compromising clinical interpretation. NfL concentrations increased markedly with age (Spearman's ρ = 0.721, c < 0.001), with median values rising from 8.45 pg/mL in individuals aged 18–34 years to 23.4 pg/mL in those aged ≥ 69 years. Reference values were defined as the 97.5th percentile and were 18.0 pg/mL for patients aged 18–50, 25.4 pg/mL for those aged 51–68, and 43.5 pg/mL for patients aged 69 years or older. No significant effect of sex was observed after adjusting for age.

This is the first study to establish serum–plasma comparability for NfL on the Lumipulse® platform and to provide age-stratified reference values in control adults, emphasizing the need for age-adjusted interpretation when distinguishing pathological elevations from normal age-related changes.