Simoa sr x platform

Handling and biobanking of serum samples were performed according to international recommendations (Teunissen et al., 2009 (link)). After withdrawal, blood was kept at room temperature for 15–30 min to allow clot formation, then refrigerated at 4°C and finally centrifuged at a speed of 2,000 × g for 10 min. Serum was then stored (within a maximum time of 4 h from initial blood draw) in 0.5- or 1-mL aliquots in polypropylene vials at −80°C until analysis. NFL measurement was performed on the Simoa SR-X platform (Quanterix, Lexington, MA, United States) using a commercial kit (catalog number, 103400). All samples were measured in duplicates [coefficient of variation (CV) <20%].

CSF samples were obtained by LP following a standard procedure, centrifuged in case of blood contamination, divided into aliquots, and stored in polypropylene tubes at − 80 °C until analysis. CSF total tau (t-tau) and p-tau were measured by chemiluminescent enzyme immunoassays on the automated Lumipulse G600II platform (Fujirebio), as described [28 (link)]. The inter-assay coefficients of variation (CVs) were < 8% for both biomarkers. We used commercially available ELISA kits to measure CSF NfL and 14–3-3 gamma isoform, as described [14 (link), 29 (link)]. SNAP-25 concentrations were determined by running the commercially available SNAP-25 Advantage Kit (Quanterix) on the SIMOA SR-X platform (Quanterix). Ng concentrations were assessed with ELISA technology using the Human Neurogranin (Trunc P75) ELISA Kit (EUROIMMUN). The intra-assay and the inter-assay CVs were < 8% and < 15%, respectively, for all four biomarkers. As previously reported, all CSF samples from patients without autopsy examination, classified as probable sCJD or np-RPD, were tested by second-generation prion CSF RT-QuIC [9 (link)].

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.

EDTA plasma samples were collected, aliquoted, and stored at −80 °C according to standard procedures [33 (link)]. The pl-NfL, pl-tau, and pl-GFAP were measured with the SiMOA NF-light advantage, SiMOA Human t-tau, and SiMOA GFAP Discovery Kits (i.e., the same used for CSF GFAP quantification) on the SiMOA SR-X platform (Quanterix). The mean intra-assay and inter-assay CVs were 4% and 12% for pl-NfL, 5% and 10% for pl-tau, and 5% and 11% for pl-GFAP.

CSF samples were obtained by LP at the L3/L4 or L4/L5 intervertebral level, centrifuged in case of blood contamination, divided into aliquots, and stored in polypropylene tubes at −80 °C until analysis. For the AD core biomarkers measurements, t-tau, p-tau, Aβ₄₂, and Aβ₄₀ were measured by automated chemiluminescent enzyme immunoassay on the Lumipulse G600II platform (Fujirebio, Gent, Belgium). The inter-assay coefficients of variation (CVs) were <8% for all biomarkers. Pathological values for defining the A/T status were determined using validated cutoff values [33 (link)]. More specifically, an Aβ₄₂/Aβ₄₀ ratio < 0.65 and a p-tau > 62 pg/mL supported the A+ and T+ statuses, respectively.
Commercially available ELISA kits were used to measure the NfL and 14-3-3 gamma isoform, as described [34 (link),35 (link)]. The GFAP concentrations were determined by running the commercially available GFAP Discovery Kit (Quanterix) on the SiMOA SR-X platform (Quanterix, Billerica, MA, USA). The intra-assay and the inter-assay CVs were respectively 7% and 15% for NfL, 6% and 13% for 14-3-3, and 8% for GFAP (only one plate was used). Eventually, all CSF samples from patients without autopsy examination, classified as probable sCJD or np-RPD, were tested by the second-generation prion RT-QuIC, as described [6 (link)].

BDNF measurements in serum samples were performed using the BDNF Discovery Kit single-molecule array assay (Simoa^®) from Quanterix (Cat no. 102039). Calibrators, participant samples, and two quality control samples per plate were measured in duplicate using a 2-step assay on a Simoa SR-X platform (Quanterix) according to manufactures protocols. Serum samples were diluted 1/1,000. The intra-assay concentration coefficient of variation was calculated for the duplicates of each participant sample and if they were more than 20% the sample was remeasured as per De Wolf et al. (2020 (link)). The intra-assay coefficient of variation for included sample measures was 7.2%. The median values for bi-participant replicates were then calculated and if the absolute difference between a replicate and the median value exceeded 10% of the median, the replicate was treated as an outlier and deleted from the statistical analysis. The mean of each biomarker concentration was then calculated for each participant sample.

Serum NfL levels were measured using the NF-LIGHT™ (SR-X VERSION) single molecule array assay (SIMOA®) from Quanterix™ (cat number 103400). Serum samples were measured in duplicate using a 2-step assay on the SIMOA® SR-X™ platform (Quanterix™), according to manufacturer’s protocols. Serum samples were diluted 4 times in sample diluent by adding 25 μL of serum to 75 μL of sample diluent. High- and low- quality control samples provided by Quanterix™ were run on each plate. If the intra-assay coefficient of variation between two duplicates was more than 20%, the sample was measured again. The average intra-assay coefficient of variation for included samples was 5.77%.