Development of a Highly Sensitive Neurofilament Light Chain Assay on an Automated Immunoassay Platform

Abstract

BackgroundNeurofilament light chain (NfL) is an axonal cytoskeletal protein that is released into the extracellular space following neuronal or axonal injury associated with neurological conditions such as multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and other diseases. NfL is detectable in the cerebrospinal fluid (CSF) and blood. Numerous studies on MS have demonstrated that NfL is correlated with disease activity, predicts disease progression, and is reduced by treatment with MS disease-modifying drugs, making NfL an attractive candidate to supplement existing clinical and imaging measures in MS. However, for NfL to achieve its potential as a clinically useful biomarker for clinical decision-making or drug development, a standardized, practical, and widely accessible assay is needed. Our objective was to develop a novel NfL assay on an automated, globally available immunoassay platform and validate its performance.MethodsA prototype NfL assay was first developed and evaluated on the ADVIA Centaur® XP immunoassay system from Siemens Healthineers. The lower limit of quantitation (LLoQ), within-lab precision, assay range, cross-reactivity with neurofilament medium and heavy chains, and effect of interfering substances were determined. NfL assay values in serum and CSF were compared with radiological and clinical disease activity measures in patients with MS and ALS, respectively. This assay was further optimized to utilize serum, plasma, and CSF sample types on the Atellica® IM system and transferred to Siemens' CLIA laboratory where it was analytically validated as a laboratory-developed test (LDT).ResultsIn this study, an LLoQ of 1.85 pg/mL, within-lab precision <6%, and an assay range of up to 646 pg/mL were demonstrated with the serum prototype assay. Cross-reactivity of <0.7% with the neurofilament medium and heavy chains was observed. Serum and CSF NfL assay values were associated with radiological and clinical disease activity measures in patients with MS and ALS, respectively. The optimized version of the NfL assay demonstrated specimen equivalence with additional plasma tube types and was analytically validated as an LDT.ConclusionThe analytical performance of the NfL assay fulfilled all acceptance criteria; therefore, we suggest that the assay is acceptable for use in both research and clinical practice settings to determine elevated NfL levels in patients.