(M1130-06-34) Comparison of Platform Sensitivities: Correlation of Endogenous Neurofilament-Light Chain Concentration Determination in Human Serum and K₂EDTA Plasma across Immunoassay Platforms

Purpose: Biomarker detection has expanded tremendously in the last decade and has especially changed the landscape for rare neurological disorders due to the increased sensitivity and availability of early detection of biomarkers. Neurofilament-Light chain (Nf-L) is a crucial biomarker for multiple neurodegenerative diseases and traumatic brain injury. Nf-L can be released into cerebrospinal fluid (CSF) and the bloodstream due to neuroaxonal injury. Multiple platforms have claimed different sensitivities for Nf-L. Here we report the challenges in direct comparison of their sensitivities and propose using matrix panels as a standard tool for these assessments.

Methods: Quanterix SiMoA Nf-Light V2 Advantage Kit (product # 104073), MSD S-Plex Neurology Panel 1 (human) ultrasensitive immunoassay kit (product #K12639S), Bio-Techne Ella Simple Plex NF-L Assay (product #SPCKB-PS-002448) and Millipore Sigma SMC Human NF-L High Sensitivity Immunoassay Kit (product# 03-0202-00) platforms were used to quantitate Nf-L concentrations. A summary of the kit specified sensitivities are listed in Table 2. A non-matched matrix panel of 17 apparently healthy individual human serum samples and a non-matched matrix panel of 20 apparently heathy individual human K₂EDTA samples were obtained from BioIVT.

Results: The concentrations measured varied across the different platforms and reported in Table 1. Several errors were encountered in the Nf-L assay performance on SMCxPro and the results were inconclusive due to lack of acceptable inter-well precision values. The Ella platform resulted in three serum samples below the limit of quantitation (< LLOQ) and one sample < LLOQ for K₂EDTA Plasma.

Conclusion: Comparing blood levels of Nf-L between Quanterix and MSD, as seen in Graph 1, a multiplicative conversion factor from Quanterix to MSD of 18 for serum and 25 for K₂EDTA plasma. This is established by taking the reciprocal of their fitted slopes. This was consistent with the ratio of their kit-specified sensitivities (5.94pg/mL (MSD)/0.34pg/mL (Quanterix)=17.47). Thus, while Quanterix claims to measure to a much lower concentration of Nf-L, their sensitivities are very similar. Based on Ella’s product information and literature, similar antibodies seem to be in use in the Quanterix and Ella platforms.¹ Accordingly, both platform results were within the same scale and their kit-specified sensitivities were directly comparable. MSD and Ella platforms show no relation to one another, and no conclusions can be drawn about their comparable sensitivities from these data. No data was obtained from the SMC platform due to several precision errors in the calibration standards. While Quanterix Nf-L assay offers high throughput and minimum assay processing times, even the latest versions of these assays are still undergoing several modifications impacting future lots, bridging and shelf-life. While MSD S-plex Nf-L assay shows reliability, comparable sensitivity, and extended shelf-life, it involves longer assay times and very stringent assay conditions. Based on our results, using same matrix panels spanning the range of endogenous expression of these biomarkers gives a better perspective on the platform sensitivities. This practice can serve as guide in choosing the right platform of choice for a program weighing in the sample size and length of the study.

References: 1. Gauthier A, Viel S, Perret M, Brocard G, Casey R, Lombard C, Laurent-Chabalier S, Debouverie M, Edan G, Vukusic S, Lebrun-Frénay C, De Sèze J, Laplaud DA, Castelnovo G, Gout O, Ruet A, Moreau T, Casez O, Clavelou P, Berger E, Zephir H, Trouillet-Assant S, Thouvenot E; OFSEP Investigators. Comparison of SimoaTM and EllaTM to assess serum neurofilament-light chain in multiple sclerosis. Ann Clin Transl Neurol. 2021 May;8(5):1141-1150. doi: 10.1002/acn3.51355. Epub 2021 Apr 8. PMID: 33830650; PMCID: PMC8108418.


Graph 1. Measured results of Quanterix vs MSD


Table 1. Results measured across three different platforms in serum and K2EDTA plasma


Table 2. Kit specified sensitivities for different platforms.