Simoa human neurology 3 plex a assay kit

Blood for preparation of EDTA plasma was drawn through venipuncture at the Amsterdam UMC, VU University Medical Center. Plasma tubes were centrifuged at 1800×g for 10 min within 1 h of collection, and plasma was aliquoted in 0.5-mL portions in polypropylene tubes and stored at − 80 °C. Samples were first thawed at the Amsterdam UMC for the quantitative determination of plasma concentrations of Aβ_1–40 and Aβ_1–42 with the SIMOA Human Neurology 3-Plex A assay kit (Quanterix, Lexington, MA) using the SIMOA HD-1 analyzer [16 (link)]. Thereafter, 200 μl of plasma per subject was shipped from Amsterdam to Bochum on dry ice.

Plasma samples for both younger and older adults were analyzed using the automated Simoa SR-X analyzer with the commercially available Simoa Human Neurology 3-Plex A assay kit (Quanterix, Billerica, MA, USA) for Aβ42, Aβ40, and tTau. Plasma concentrations of pTau-181 were measured using the automated Simoa HD-X analyzer and the Simoa pTau-181 Advantage V2 kit (Quanterix, Billerica, MA, USA). Analyses were performed in duplicates (mean % coefficient of variation [%CV]: Aβ42: 6.33, Aβ40: 4.55, tTau: 8.81, pTau-181: 6.01) using a 1:4 dilution protocol according to the manufacturer's instructions. Prior to analysis, plasma samples were thawed at room temperature for 1 h and centrifuged at 10,000×g for 5 min to prevent transfer of debris. All assays were conducted by the same operator with the same respective instrument.

EDTA plasma samples were obtained through venipuncture (fasted). Samples were centrifuged at 2000 × g, aliquoted in polypropylene tubes, and stored at −80°C in our biobank within 60 min of collection. Plasma was analyzed using ultra-sensitive single-molecule array technology of the automated Simoa HD-1 analyzer with the Simoa Human Neurology 3-Plex A assay kit (Quanterix, Lexington, KY, USA) that simultaneously measures plasma concentrations of Aβ₄₀, Aβ₄₂, and total tau. Analyses were performed in duplicates (mean % coefficient of variation [%CV]: Aβ₄₀ 3.8%CV, Aβ₄₂ 4.1%CV, total tau 6.7%CV) using a 1:4 automated dilution protocol. The levels of tau phosphorylated at threonine 181 (p-tau₁₈₁) were measured using the Simoa Human tau immunoassay kits on the Simoa HD-1 analyzer (7.1%CV). APOE genotyping was further performed using polymerase chain reaction based on blood sample DNA extraction. Participants’ APOE status was defined as ‘ε4 carrier’ if they carry at least one ε4 allele.

Fibrinogen levels were measured using standard laboratory methods in the Clinical Laboratory, Daping Hospital, Chongqing, China. Fibrinogen−C is the test to measure fibrinogen by the Clasus method and is carried out with the commercial kit HemosIL Fibrinogen assay (Instrumentation Laboratory Company, United States) on ACL-TOP (Instrumentation Laboratory Company, United States). The kit uses an excess of thrombin to convert fibrinogen to fibrin in diluted plasma. Plasma levels of Aβ42, Aβ40 were measured using the commercially available single-molecule array (SIMOA) Human Neurology 3-Plex A assay kit (Quanterix, United States) on-board of the automated SIMOA HD-1 analyzer (Quanterix, United States). CSF levels of Aβ40, Aβ42, total tau (t-tau), and phosphorylated tau-181 (p-tau) were measured using the human Aβ and tau enzyme-linked immunosorbent assay (ELISA) kits (Innotest, United States). All of the measurements were performed according to the manufacturer’s instructions (Wilke et al., 2018 (link)).

According to global measurement standardization from the Alzheimer’s Association Global Biomarkers Consortium [23 (link)], CSF Aβ42, Aβ40, P-tau181 and T-tau levels had previously been measured using enzyme-linked immunosorbent assays (ELISA) kits (INNOTEST, Fujirebio, Belgium) according to the manufacturer’s protocol in both CADS and CABLE cohorts. (Supplementary Table 3). Due to the significant influences of pre-analytical and analytical factors on AD biomarker in different laboratories [24 (link), 25 (link)], the cutoff values of ATN biomarkers were determined in CADS and CABLE cohorts, respectively. The cutoff values to define abnormal ATN biomarkers were CSF Aβ42 ≤ 930.35 pg/mL (A⁺), CSF P-tau181 > 48.56 pg/mL (T⁺), CSF T-tau > 284.53 pg/mL (N⁺) in CADS cohort, and Aβ42/40 ratio < 0.022 (A+), CSF P-tau181 > 45 pg/mL (T+), CSF T-tau > 236 pg/mL (N+) in CABLE cohort.
In CADS cohort, plasma Aβ42, Aβ40 and T-tau were simultaneously measured using the commercially available single-molecule array (SIMOA) Human Neurology 3-Plex A assay kit (Quanterix, Massachusetts, USA) on-board of the automated SIMOA HD-1 analyzer. ELISA data and SIMOA data were corrected with the mean values of two age- and sex-matched cognitively normal populations from the same sample. CSF to plasma ratios were calculated with the original concentration detected by SIMOA assay.