2.2. Laboratory measurements of clusterin, aβ, and CRP

Whole blood was collected in fasting state using a 21-gage needle. For serum preparation, blood was collected into red top tubes and was allowed to clot in a vertical position at room temperature for 30 minutes before centrifugation. For plasma preparation, blood was collected to EDTA-treated tubes and gently inverted 5–10 times. All tubes underwent centrifugation at 3000 rpm/1850 g for 30 minutes at 4°C. Serum and plasma were then apportioned into 0.5-mL aliquots and stored at −80°C.

All samples were analyzed for beta-amyloid levels at the Department of Molecular Pharmacology and Experimental Therapeutics of the Mayo Clinic, Jacksonville, FL. Quantification of aβ in plasma was performed using INNO-BIA assays (Innogenetics, Ghent, Belgium), which is a multiplex microsphere-based Luminex xMAP technique. Intra-assay coefficients of variations (CVs) for aβ_1–40 and aβ_1–42 were 3.2% and 2.6%, and inter-assay CVs were 10.5% and 7.6%, respectively. Plasma clusterin levels were assessed as part of the Systems Approach to Biomarker Research (SABRe) project 2 panel 1 using a Luminex xMAP assay. The Multiplex assay had five protein targets (Clusterin, Apo-A1, Apo-B100, Lp(a), and CRP) and used a plasma dilution of 1/10,000. The intra-assay %CV for the clusterin measurements was 3.95%, and Inter-assay %CV of 8.4%. The assay had a lower limit of quantitation of 1.5 and upper limit of quantitation (ULOQ) of 2.67 × 10ˆ4, with a seven-point calibration curve. Two quality control (QC) samples were prepared to include five protein targets in the panel, one a high concentration (QC1) and the other a low concentration (QC2). To determine the plate-to-plate and day-to-day performance of each assay plate, each 96 well assay plate included (1) the standards in triplicate, (2) the QC1 and QC2 in quadruplicate, and (3) 32 individual plasma samples in duplicate. The acceptable range for each FHS assay plate was determined to be the mean +/− 3SD. If the QC1 or QC2 value of any assay plate was out of the range, the plate was repeated.

High-sensitivity CRP levels were determined in serum by Dade Behring BN100 nephelometer, and the average interassay coefficients of variation was 2.2%.