Comparison of Simoa, high‑sensitivity ELISA, and CLIA for serum neurofilament light chain quantification in multiple sclerosis

Abstract Serum neurofilament light chain (sNfL) reflects neuro‑axonal injury, and is an emerging biomarker in multiple sclerosis (MS). This prospective cross-sectional study compared analytical agreement and clinical applicability among three analytical platforms. Serum samples from adult MS patients were analysed by single molecule array (Simoa) (frozen samples), highsensitivity ELISA (hsELISA) (frozen samples), and fully automated chemiluminescent immunoassay (CLIA) (fresh and frozen samples). Simoa and hsELISA were strongly correlated (rₛ = 0.796) without systematic bias. CLIA with frozen samples showed higher sNfL levels (relative bias: 39.53% vs. Simoa; 29.56% vs. hsELISA). CLIA with fresh samples correlated very strongly with Simoa (rₛ = 0.820) and strongly with hsELISA (rs = 0.764) (relative bias: −7.7% vs. Simoa; −14.3% vs. hsELISA), though broad limits of agreement indicated notable individual variability. Comparison of fresh vs. frozen CLIA values indicated an influence of pre-analytical conditions. Age was positively associated with sNfL determined by Simoa and hsELISA. Levels of sNfL did not significantly differ based on relapse status, MRI activity, or disease-modifying therapy. Simoa and hsELISA yield interchangeable sNfL results with consistent biological correlations, supporting their clinical and research application. CLIA can be automated but is affected by pre-analytical factors. Assay harmonization is essential before routine clinical implementation.