Analytical validation of an IFCC-endorsed mass spectrometry-based semi-automated reference measurement procedure for global standardization of serum apolipoprotein(a)

Objectives An LC-MRM-MS-based Reference Measurement Procedure (RMP) with manual sample preparation for standardization of commercial Lp(a)/apo(a) test kits in molar units was previously developed by three calibration laboratories of the IFCC Working Group on Apolipoprotein Standardization. Semi-automation of the sample preparation was introduced in the Leiden Apolipoprotein Reference Laboratory to improve the precision of the RMP. Methods The RMP was adapted for semi-automation on an Agilent BRAVO Automated Liquid Handling Platform using VWorks software. Measurement of apo(a) is based on three proteotypic peptides and five serum calibrators traceable to the former WHO-IFCC reference material SRM2B, and is independent of the apo(a) size polymorphism. Concentration-dependent performance criteria were established according to the EFLM Biological Variation Database, and precision was assessed per CLSI EP05-A3. Results Compared to manual sample preparation, semi-automation improved analytical precision, resulting in within-run imprecision estimates for quantifying peptide LFLEPTQADIALLK of 5.0, 4.7, and 4.5 % at 146.8, 197.4 and 238.7 nmol/L, respectively. Furthermore, compared to the concentration-dependent maximum allowable uncertainty, and following correction for repeated measurements, the measurement uncertainty of the RMP was deemed acceptable over the entire measurement range of apo(a) (3.8–456.0 nmol/L). Conclusions Semi-automation improved the analytical precision of the RMP for the quantitation of apo(a), meeting concentration-dependent analytical performance criteria and facilitating longitudinal accuracy of Lp(a)/apo(a) test results produced by CE-IVD Lp(a)/apo(a) kits. With semi-automation of the sample preparation, a more precise RMP has been developed, making it easier to meet the maximum allowable measurement uncertainty in the Lp(a)/apo(a) calibration hierarchy.