Nf light advantage kit

sNfL levels were determined using the highly sensitive single molecule array (SiMoA) technology. Samples were measured in duplicates in several rounds by SiMoA HD-1 (Quanterix, USA) using the NF-Light Advantage Kits (Quanterix) according to manufacturer’s instructions. Resorufin-β-D-galactopyranoside (RGP) was incubated at 33°C for 60 min prior to running the assay. The coefficient of variation (CV, as a percentage) of the two replicates was obtained by dividing the standard deviation of both replicates by the mean of both replicates multiplied by 100. CVs above 20% (or missing replicate result) were measured twice. Finally, the mean intra-assay CV of 5.9% was obtained by averaging all individual sample CVs. Two low and high controls, consisting of recombinant human NfL antigen, were included in each sample run to monitor plate-to-plate variation (low: mean 8.8 pg/ml, interassay CV 13.6%; high: mean 192.7 pg/ml, interassay CV 13.3%). sNfL measurements were performed in a blinded fashion without information about clinical data.

All measurements were performed in duplicate using Single Molecule Array technology (Quanterix) with the commercial NF‐Light advantage Kit and GFAP Discovery Kit on a HD‐X platform according to the manufacturer’s instructions (www.quanterix.com/products-technology/assays). The inter-assay variation for NfL was 8%, based on three levels of internal quality control pools, measured in 45 runs. The inter-assay variation for GFAP was 15%, based on three levels of internal quality control pools, measured in 40 runs. The mean intra‐assay coefficients of variation (duplicate measurements) were <10% for both NfL and GFAP. Therefore, samples with too low volume for a duplicate measurement (n = 4) were also included in the analysis. Based on an in-house quality study, NfL values in heparin plasma, GFAP values in serum and GFAP values in heparin plasma were adjusted by −29, −13 and −18%, respectively, to allow comparison between measurements in heparin plasma, serum and EDTA plasma.³¹ (link)^,32 Finally, the GFAP values of the adult reference cohort were multiplied by a factor of 1.3, based on internal quality control values, to correct for inter-batch variability. All measurements were performed by certified technicians at the Neurochemistry laboratory of the Amsterdam UMC location VUmc blinded to clinical information.

sNfL of 112 patients with CIS and MS and of 62 healthy donors was measured by SiMoA technology as previously described.^{7 (link)} Briefly, blood samples were spun at 2000g at room temperature for 10 minutes within 2 hours after withdrawal and stored in polypropylene tubes at −80°C. Serum NfL was measured by SiMoA HD-1 (Quanterix) using the NF-Light Advantage Kit (Quanterix) according to the manufacturer's instructions. Samples were measured in duplicates, and the intra-assay coefficient of variation of all samples was 4.5%. sNfL measurements were performed in a blinded fashion without information about clinical data.