Hd x instrument

Nf-L levels were determined in serum obtained from 7 mL of blood withdrawal after centrifugation for 10 min at 1500× g. sNf-L levels were measured by using an MSD (R-PLEX Human Neurofilament L Kit, according to the manufacturer’s instructions, Meso Scale Discovery, Rockville, MD, USA) in the Italian cohort. Analysis was performed using a QuickPlex SQ 120 instrument (MSD, Rockville, MD, USA) and DISCOVERY WORKBENCH^®4.0 software. sNf-L levels of the Amsterdam MS Center cohort were measured using a single-molecule array (Simoa) assay to measure the Nf-L levels on an HD-X instrument (Quanterix, Billerica, MA, USA) following the manufacturer’s instructions.

Blood for plasma biomarker measurement was collected at the time of 3 T MRI scan. Plasma was separated, aliquoted and stored at −80°C using standardized protocols. EDTA plasma was used to measure Aβ₄₂, Aβ₄₀, glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL) using the Quanterix Single Molecule Array (Simoa) Neurology 4‐Plex E assay on the HD‐X Instrument (Quanterix Corporation). Phosphorylated tau‐181 (pTau181) was measured using the Quanterix Simoa p‐Tau181 Assay version 2 on the HD‐X Instrument. All assays were run in duplicate, and the mean value of the duplicate measurements was used for all analyses. Biomarkers derived from BLSA and GESTALT were quantified using the same set of assays (identical lot numbers). The intraassay coefficients of variation (CV) as derived from the parent study were 1.9, 2.8, 5.0, 5.1, and 4.4 for measures of Aβ₄₂, Aβ₄₀, GFAP, NfL, and pTau181, respectively. One NfL measurement was considered an outlier based on visual inspection and was removed from subsequent analyses.

The single molecule array (Simoa) platform was used to measure protein biomarker concentrations in EDTA plasma specimens. GFAP, t-tau and NFL concentrations were measured using the Neurology 4-Plex A kit (QTX-102153, Quanterix, MA, USA), while p-tau181 and p-tau231 concentrations were measured with in-house assays developed at the University of Gothenburg, Sweden^{15 (link),17 (link)}. Calibrators and samples were run in duplicates in all assays. Two internal controls provided with the Simoa kits were included in duplicate at the beginning of each experiment. Plasma Aβ concentrations were measured using the Amyblood test that was developed at Amsterdam University Medical Center in collaboration with ADx NeuroSciences (Ghent, Belgium), on the Simoa platform (HDx instrument, Quanterix)^{27 (link),28 (link)}.