Fresh-to-frozen comparability of serum neurofilament light chain across four immunoassays

Serum neurofilament light chain (sNfL) is an increasingly used biomarker of neuroaxonal injury in multiple sclerosis (MS), but differences between immunoassays and sample handling may limit comparability of results. We compared paired fresh and frozen serum samples from 20 patients with MS using four immunoassays: Simoa, Elecsys, Atellica, and ELISA. Matched aliquots were stored at − 70 °C for 10 days and subjected to a single freeze–thaw cycle. Fresh-to-frozen differences varied significantly among platforms (Friedman χ² = 24.06, p < 0.001; Kendall’s W = 0.401). Median concentrations decreased after freezing with Simoa (–12.8%), Elecsys (–5.4%), and ELISA (–9.7%), whereas Atellica showed minimal change (+ 0.9%). Between-assay correlations were moderate to strong in fresh (Spearman ρ = 0.663–0.877) and frozen samples (ρ = 0.677–0.931), despite substantial differences in absolute concentrations. Assay-specific Simoa and Elecsys Z-scores were strongly correlated in fresh (ρ = 0.886) and frozen samples (ρ = 0.923; both p < 0.001), but systematic differences remained. These findings indicate assay-dependent fresh-to-frozen differences and support maintaining consistent analytical immunoassays and sample-processing conditions for longitudinal sNfL monitoring.