Repeatability and reproducibility of lipoprotein particle profile measurements in plasma samples by ultracentrifugation

Background: Characterization of lipoprotein particle profiles (LPPs) (including main classes and subclasses) by means of ultracentrifugation (UC) is highly requested given its clinical potential. However, rapid methods are required to replace the very labor-intensive UC method and one solution is to calibrate rapid nuclear magnetic resonance (NMR)-based prediction models, but the reliability of the UC-response method required for the NMR calibration has been largely overlooked. Methods: This study provides a comprehensive repeatability and reproducibility study of various UC-based lipid measurements (cholesterol, triglycerides [TGs], free cholesterol, phospholipids, apolipoprotein [apo]A1 and apoB) in different main classes and subclasses of 25 duplicated fresh plasma samples and of 42 quality control (QC) frozen pooled plasma samples of healthy individuals. Results: Cholesterol, apoA1 and apoB measurements were very repeatable in all classes (intraclass correlation coefficient [ICC]: 92.93%-99.54%). Free cholesterol and phospholipid concentrations in main classes and subclasses and TG concentrations in high-density lipoproteins (HDL), HDL subclasses and low-density lipoproteins (LDL) subclasses, showed worse repeatability (ICC: 19.21%-99.08%) attributable to low concentrations, variability introduced during UC and assay limitations. On frozen QC samples, the reproducibility of cholesterol, apoA1 and apoB concentrations was found to be better than for the free cholesterol, phospholipids and TGs concentrations. Conclusions: This study shows that for LPPs measurements near or below the limit of detection (LOD) in some of the subclasses, as well as the use of frozen samples, results in worsened repeatability and reproducibility. Furthermore, we show that the analytical assay coupled to UC for free cholesterol and phospholipids have different repeatability and reproducibility. All of this needs to be taken into account when calibrating future NMR-based models.