Lumipulse g600ii platform

Blood samples were collected into EDTA tubes on the same day as the lumbar puncture. These samples were centrifuged at 1800 g (10 min at 4 °C), aliquoted into polypropylene tubes, and stored at − 80 °C until analysis.
Plasma concentrations of the analytes of interest were determined in the LUMIPULSE G600II platform (Fujirebio, Japan). For the exploratory study, the plasma samples were analyzed between October and November 2022, while for the validation cohort, the analysis took place between February and March 2023.
Plasma concentrations of p-Tau181, Aβ42, and Aβ40 were assessed simultaneously using the LUMIPULSE G pTau 181 plasma, LUMIPULSE G β-amyloid 1–40 plasma, and LUMIPULSE G β-amyloid 1–42 plasma research use only (RUO) assays, following the manufacturer’s instructions. Concentrations were determined via a lot-specific calibration curve, assayed in duplicate, and quality control procedures were performed at the beginning of each test day to ensure that control values (low and high) fitted the target ranges.

EDTA plasma samples were collected, aliquoted, and stored at −80 °C, according to standard procedures. CSF samples were obtained by lumbar puncture following a routine procedure, centrifuged in case of blood contamination (even minimal), divided into aliquots, and stored in polypropylene tubes at −80 °C until analysis.
From CSF routine analysis, we extrapolated CSF albumin to calculate the albumin index.
Plasma GFAP, Plasma NfL, and CSF NfL levels were determined with the Single molecule array (Simoa) technology [41 (link)] on a Simoa SR-X instrument using the commercially available GFAP Discovery and NF-light Advantage Kits (Quanterix, Billerica, MA, USA). The mean intra- and inter-assay coefficients of variation (CVs) were below 15% for all analyses.
CSF Aβ42, Aβ40, p-tau, and t-tau were measured by automated chemiluminescent enzyme immunoassay on the Lumipulse G600II platform (Fujirebio, Gent, Belgium). The inter-assay CVs were <8% for all biomarkers. The Aβ42/Aβ40 was calculated as described [42 (link)]. We used in-house validated cutoffs to determine pathological values for the AD core markers. In particular, a CSF Aβ42/Aβ40 ratio < 0.68 was considered supportive of amyloid deposition (i.e., A+ according to the ATN classification [43 (link)]), while a CSF phosphorylated tau at site 181 (p-tau181) > 62 pg/mL was considered indicative of p-tau deposition (i.e., T+).

In CSF samples from the Bologna PRO-HYDRO study, we measured total tau (t-tau), p-tau, Aβ42, and amyloid-beta 1–40 (Aβ40) by automated chemiluminescent enzyme-immunoassay (CLEIA) on the Lumipulse G600II platform (Fujirebio Europe NV, Gent, Belgium). The mean interassay coefficients of variation (CVs) were ˂ 8% for all biomarkers. We calculated the Aβ42/40 ratio according to a previously published formula [26 (link)]. In the same CSF samples, we measured NfL concentration by a validated commercial enzyme-linked immunosorbent assay (ELISA) (NfL ELISA kit, IBL, Hamburg, Germany) [27 (link)]. The mean intra- and interassay CVs for NfL analyses were 2 and 10%, respectively. In the Finnish cohort, the CSF levels of Aβ42, t-tau, and p-tau were measured by commercial ELISA kits (Innotest β-amyloid1–42, Innotest Tau-Ag, Innotest Phosphotau (181P), Fujirebio, Ghent, Belgium) using the manufacturer's protocol. The cut-off values for Aβ + status were as follows: Aβ42/40 ratio < 0.65 in the Bologna cohort [12 (link)] and Aβ42 < 500 mg/mL in the Kuopio cohort [28 (link)].