Neurology 3 plex a

Plasma and EV Levels of IL-6, IL-10 and TNFα were measured using an ultrasensitive assay (Neurology 3-Plex A, item 101995, Quanterix Corporation, Lexington, MA) and single-molecule technology (SIMOA™) on a HD-1 analyzer (Quanterix Corporation) and reported in pg/mL. Samples were randomized over plates and tested in duplicate with laboratory scientists blinded to participant groups. All samples underwent quality control analysis and samples with reported coefficients of variation over 20% were not used for analysis. Samples with high coefficients of variation and concentrations below lower limits of quantification were replaced by values equal to half of the limit of concentration respective to the assay.

We performed CSF biomarker assays using the Simoa SR-X platform^{1 (link),20 (link)} and plasma biomarker assays using the Simoa HD-X platform (both Quanterix). Samples were assayed in duplicate per the package insert instructions using the following Quanterix kits: Neurology 3-Plex A (catalog No. 101995) for Aβ42, Aβ40, and T-tau; pTau-181 V2 Advantage (catalog No. 103714) for P-tau181; and Neurology 2-Plex B (catalog No. 103520) for GFAP and NfL. Ratios of Aβ42/Aβ40, T-tau/Aβ42, and P-tau181/Aβ42 were calculated. Cerebrospinal fluid positivity for AD was determined using the CSF P-tau181/Aβ42 optimal cut point of 0.223 established in our laboratory. This CSF cut point is derived from receiver operating characteristic (ROC) analysis of a validation group of combined autopsy cases (n = 20) and amyloid PET cases (n = 59) with CSF biomarkers, including Aβ40, Aβ42, T-tau, P-tau181, and NfL (using Quanterix kit 103400), measured on the SR-X system. The area under the curve (AUC) was best for P-tau181/Aβ42, at 0.88 (0.79-0.97) with a Youden index of 0.82. At the P-tau181/Aβ42 cut point of 0.22, sensitivity was 0.95 and specificity was 0.87.

Blood samples were drawn from the peripheral vein of each participant and collected in tubes with and without EDTA, within 24 hours since admission for patients with CAA and during follow-up period for healthy elder people. After centrifugation at 3,000rpm for 15 minutes, the plasma and serum were kept at −80° C until measurement.
All measures were performed blinded to the clinical data. Serum NfL and plasma Aβ40, Aβ42 and total tau were measured using the SIMOA NF-light assay (Cat No: 102258) and Neurology 3-Plex A (Cat No: 101995) respectively, per manufacturer instructions (Quanterix, MA, USA) on a HD-1 platform at GBIO (Hangzhou, China) with dilution at 1:4 ratio. All samples were duplicated for detection and intra- and inter-assay concentration variabilities were <15%.