Xmap luminex platform

CSF was collected by using standardized lumbar puncture procedures. CSF was collected into siliconized polypropylene tubes and the first 1–2 mL of CSF of sent to the site's local laboratory for routine testing for cell count, total protein level, and glucose level. An additional 15–20 mL of CSF was transferred into 15-mL conical propylene tubes at room temperature, mixed gently, centrifuged at 2,000 g for 10 min at room temperature, and transferred into 1.5-mL pre-cooled siliconized polypropylene aliquot tubes followed by immediate freezing on dry ice. The frozen aliquots of CSF were shipped overnight to the PPMI Biorepository Core Laboratories on dry ice and then thawed, aliquoted into 0.5-mL siliconized polypropylene tubes, refrozen once, and stored at −80°C. The coded frozen aliquots of CSF were transferred from the PPMI Biorepository Core laboratories to the University of Pennsylvania and to Covance for analyses. CSF Aβ1-42, t-tau and p-tau were measured using the xMAP-Luminex platform with INNOBIA AlzBio3 immunoassay kit-based reagents (Fujirebio-Innogenetics, Ghent, Belgium). CSF α-syn and CSF hemoglobin levels were analyzed using appropriate commercially available sandwich type ELISA kits (Covance, Dedham, MA) [2011; (19 (link), 20 (link))].

The detailed approaches were as described previously [20 (link)]. Cerebrospinal fluid (CSF) amyloid-β_1-42 (Aβ_1-42), total-Tau (T-tau), and phosphorylated tau (P-tau) were measured using the xMAP-Luminex platform with INNOBIA AlzBio3 immunoassay kit-based reagents (Fujirebio-Innogenetics, Ghent, Belgium), while Total α-synuclein and NfL levels were measured by enzyme-linked immunosorbent assay kit (Covance, Dedham, MA). Serum neurofilament light (NfL) levels were measured by the Simoa Human NF-light Advantage kit (UmanDiagnostics, Umeå, Sweden) using Single Molecule Array (Simoa) technology. The impacts of possible extreme outliers on the results were weakened through additional quality control.

Levels of Aβ42, t-tau, and p-tau in CSF were determined by the ADNI Core Biomarkers Team using xMAP Luminex platform and Innogenetics/Fujirebio AlzBio3 immunoassay kits. The detailed procedure for this determination is described elsewhere (Shaw et al., 2009 (link)). Using a previously proposed CSF Aβ42 cutoff < 192 pg/ml (Shaw et al., 2009 (link)), participants were classified as abnormal or normal. The ROC analysis found that this cutoff value can detect the autopsy-confirmed AD cases with a high sensitivity (96.4%) (Shaw et al., 2009 (link)). Tau phosphorylated at the threonine 18 was measured.

Biomarker analyses have been previously described and based on the PPMI biologics manual (Marek et al., 2018 (link)). CSF was collected by standardized lumbar puncture procedures. Measurements of Aβ₄₂ and t-tau were analyzed by using the xMAP-Luminex platform with INNOBIA AlzBio3 immunoassay kit–based reagents (Fujirebio-Innogenetics, Ghent, Belgium) at Penn (Kang et al., 2013 (link)). Additionally, CSF total α-syn levels were measured by BioLegend (San Diego, CA, United States) by means of a commercially accessible and previously described sandwich immunoassay. Serum NFL was quantified by the Simoa Human NF-light Advantage Kit (Quanterix, Lexington, MA, United States) using the Single Molecule Array technology in a fully automated SIMOA HD-1 analyzer. Biochemical analyses of uric acid have been carried out in Covance laboratories in a uniform fashion, as per the study protocol.

Cerebrospinal fluid collection procedures can be obtained from the ADNI database and prior publications (ADNI_Methods_UPENN_Biomarker_20120710.pdf) (Shaw et al., 2011 (link)). The xMAP Luminex platform (flow cytometric method) and Innogenetics/Fujirebio AlzBio3 immunoassay kits (monoclonal assay) were used following procedures in place at the UPenn/ADNI Biomarker Laboratory, according to the kit manufacturer’s instructions and as described in previous publications (Shaw et al., 2011 (link)). These assays produced measurements for Aβ_1-42, total tau, and p-tau₁₈₁. In this study, we used total CSF Aβ_1-42 as a variable of interest.