Simoa hd 1 analyser

EDTA plasma samples were collected from the participants, processed and stored at −80°C following standardised procedures. Plasma level concentrations were measured using the Human Total Tau kit (Quanterix, Boston, Massachusetts, USA) with the Simoa HD-1 Analyser (Quanterix, Boston, Massachusetts, USA). Briefly, samples, magnetic beads coated with Tau5 monoclonal capture antibody, and HT7 and BT2 monoclonal biotinylated detector antibodies were combined. Antibody epitopes in the mid-region of tau make the assay sensitive to normal and phosphorylated tau, including most of the known protein isoforms. Thereafter, the capture beads were resuspended with streptavidin-β-galactosidase (SBG) and resorufin β-D-galactopyranoside (RPG) and transferred to the Simoa disk. Each bead fits into a microwell in the disk and if tau has been captured the β-galactosidase hydrolyses the RGP substrate which generates a fluorescent signal whose concentration can be measured against a standard curve derived from known concentrations of recombinant tau. The lower limit of detection for the assay is 0.019 pg/mL and the intra-assay coefficient of variation was 4.7% (all samples were analysed in duplicate on one occasion using one batch of reagents).

The blood was sampled and sent for haematologic examination upon patient arrival at the hospital. Blood samples were immediately collected upon hospital arrival in the emergency department. They were separated and stored within 2 hours after sampling at −80°C for future analysis. The concentrations of sNfL were measured using the commercially available single-molecule array (SIMOA) Human Neurology 3-Plex A assay kit on the automated SIMOA HD-1 analyser (Quanterix, Lexington, Massachusetts). The parameters of SIMOA were set as follows: the limit of detection (LOD) was set to 0.038 pg/mL (range: 0.003–0.079 pg/mL), and the lower limit of quantification (LLOQ) was set to 0.174 pg/mL (pooled CV: 15.3%; mean recovery: 105%).