Simoa hd 1

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.

NfL was included as a secondary outcome if available. Follow-up values under treatment were assessed at the earliest time point available, but not before 3 months after treatment start. In total, follow-up values were available for 55 patients (ocrelizumab n = 23, natalizumab n = 30) after a mean time to follow-up of 9.9 months. For 30 patients (ocrelizumab n = 17, natalizumab n = 13), both baseline and follow-up values were available (mean time to follow-up = 8.8 months). NfL values were assessed from the patient’s sera using a standardized protocol described in detail previously.^{25 (link)} NfL levels were determined in duplicates by single molecule array with an SiMoA HD-1 (Quanterix, Billerica, MA, USA) using the Nf-Light Advantage Kits (Quanterix) according to the manufacturer’s instructions. All coefficients of variation (CVs) of the two replicates were below 20%, resulting in a mean intra-assay CV of 6.8%. Low and high controls, consisting of recombinant human NfL antigen, were included in each sample run to monitor plate-to-plate variation (low: mean = 3.0 pg/ml, inter-assay CV = 4.0%; high: mean = 132.1 pg/ml, inter-assay CV = 6.9%). The NfL measurements were performed blinded and without information on clinical data.

Fasting blood samples were collected according to the international guidelines for AD biomarker studies.³⁷ Our previously validated proteomic profile was assayed using electrochemiluminescence (ECL) per our published methods on the following biomarkers: fatty acid binding protein 3 (FABP3); beta 2 microglobulin (B2M); C‐reactive protein (CRP); thrombopoietin (TPO); alpha 2 macroglobulin (A2M) eotaxin 3; tumor necrosis factor alpha (TNFa); tenascin C (TNC); interleukin (IL)‐5, IL‐6, IL‐7, IL‐10, IL‐18; I‐309; factor VII (factor 7); soluble intercellular adhesion molecule 1 (sICAM1); circulating vascular cell adhesion molecule 1 (sVCAM1); pancreatic polypeptide (PPY); thymus activation regulated chemokine (TARC); and serum amyloid A (SAA).²⁷, ²⁸, ³⁰ Glucagon‐like peptide 1 (GLP‐1), insulin, glucagon, and peptide YY (PYY) were also assayed weekly via ECL multiplex kit. The ITR Biomarker Core has conducted > 20,000 assays using these specifications and the platform performs excellently (coefficient of variation [CV] < = 10%). The Quanterix Simoa HD‐1 platform was used for assay of plasma amyloid beta (Aβ)₄₀, Aβ₄₂, total tau (3‐plex plate), and neurofilament light (NfL). The ITR Biomarker Core has conducted n > 5000 assays with CVs < = 5%.