(T1330-09-51) Laboratory Validation of a Neurofilament Light Chain (NfL) Clinical Trial Assay for Applications in Neurological Diseases Testing Services

Purpose: The identification and quantification of axonal damage in neurological diseases management could be crucial for improved prognosis and diagnostic accuracy. Neurofilament light chain (NfL) is a neuron specific structural protein which can be detected in blood and Cerebrospinal fluid (CSF) as a biomarker associated with axonal injury or degeneration.¹ The role of NfL as a biomarker has been reported in Alzheimer’s disease (AD), traumatic brain injury (TBI), and multiple sclerosis (MS).² An NfL assay was validated in a CLIA CAP accredited laboratory on an Atellica™ Immunoassay system for use in clinical trials.

Methods: The NfL Clinical Trial Assay (CTA) is an automated 2-site sandwich immunoassay using direct chemiluminometric technology. This NfL assay uses two specific non-competing monoclonal antibodies against the conserved rod domain of NfL, without cross-reactivity for neurofilament medium chain (NfM), neurofilament heavy chain (NfH), or glial fibrillary acid protein (GFAP). In the assay Lite Reagent, the detection antibody is labeled with a high yield acridinium ester. The NfL CTA was analytically validated in a CLIA-CAP accredited laboratory for use in clinical trials. Validation studies were performed using serum, plasma, and cerebrospinal fluid.

Results: Analytical performance studies were performed using serum, CSF, lithium heparin, and EDTA plasma. NfL levels were detected reproducibly across the assay reporting range for all these sample types. The assay range for CSF was 85.5 to 25,700 pg/mL, serum was 3.9 to 500 pg/mL, lithium heparin was 2.4 to 549 pg/mL and EDTA plasma was 4.9 to 477 pg/mL. Precision studies for each sample type were performed with a minimum of five samples spanning the assay range. Percent CVs for all sample types were in the range of 4 to 18%. The lower limit of quantitation (LLoQ) was determined as the lowest concentration tested that met acceptance criteria for precision and was in the linear range of the assay. Specimen handling studies were performed to evaluate limits for freeze/thaw cycling and storage times at different temperatures. All sample types yielded acceptable results when frozen and thawed up to 6 times. All sample types also yielded acceptable results when stored at room temperature and refrigerated for one week. Long term stability for samples frozen at -60 to -90 degrees Celsius is ongoing. Current long-term frozen data supports -60 to -90 °C storage for two 2 years. Assay interference was assessed using endogenous substances known to affect assay results, common over-the-counter drugs, and drugs commonly used to treat Alzheimer’s disease and multiple sclerosis. The highest observed result bias in the study was 10%. A method comparison was performed with another widely used NfL assay that is run on the SIMOA platform. 50 serum and 51 CSF samples were used in the comparison. Both sample types exhibited good correlation yielding Deming regression correlation coefficients > 0.99.

Conclusion: Using the NfL CTA, NfL levels in CSF, serum, and plasma samples were detected reproducibly across the reporting range. The assay produced reliable results in the presence of endogenous substances known to cause assay interference, common over-the-counter drugs, and drugs used to treat two neurological conditions. Our assay also exhibited good correlation to the SIMOA NfL assay. The stability of NfL in samples exposed to different storage conditions was shown to be practical for the use of NfL CTA (for NfL detection) in clinical trials.

References: 1. Gaetani L, Blennow K, Calabresi P, Di Filippo M, Parnetti L, Zetterberg H. Neurofilament light chain as a biomarker in neurological disorders. J Neurol Neurosurg Psychiatry. 2019;90(8):870-881.
2. Barro C, Chitnis T, Weiner HL. Blood neurofilament light: a critical review of its application to neurologic disease. Ann Clin Transl Neurol. 2020;7(12):2508-2523.

Analytical Performance



Method Comparison



Sample Stability