Background and Aims : Reference measurement systems (RMS) are essential to ensure accurate test results for IVD-manufacturers, clinicians and laboratory professionals . Apolipoproteins (apos) are increasingly recognized as relevant functional biomarkers for cardiovascular risk assessment, and new insights and therapies increase clinical measurement frequency. As former WHO-IFCC RMS for apos A-I, B and Lp(a) are no longer available, there is an unmet need for a higher order, globally available, RMS for conventional and emerging apolipoproteins.Methods: A mass spectrometry-based candidate reference measurement procedure (cRMP) was developed using bottom-up proteomics. Proteins in serum were denatured, reduced and alkylated prior to digestion. Thirty-one peptides representing seven apolipoproteins were quantified relative to stable isotope labelled synthetic peptides. Calibration is currently done with value-assigned, serum-based commutable calibrators. The method is provisionally validated according to CLSI guidelines.Results: Interpeptide correlation within individual proteins showed Pearson's Rs > 0.975, except for apoC-I (R = 0.953) and one comparison for C-III (R = 0.970). The quantitation of apo(a) is independent of its size polymorphism by design and was linear between 3.4 and 450 nmol/L, the LoQ for apo(a) was 3.4 nmol/L. Total imprecision for apo(a) was 8.5, 10.2, 10.1, 10.4 and 9.7%, at concentrations of 8.4, 15.6, 49.3, 252 and 364 nmol/L. Average total imprecision for other apos ranged between 3.7 and 7.4%.Conclusions: A next generation, 7-plex, proteomics-based cRMP was developed that allows molecular measurement of targeted apos. As serum apolipoproteins are measured directly through their proteotypic peptides, molar and accurate quantitation of apo(a) and the other apolipoproteins is enabled with this cRMP. Background and Aims : Reference measurement systems (RMS) are essential to ensure accurate test results for IVD-manufacturers, clinicians and laboratory professionals . Apolipoproteins (apos) are increasingly recognized as relevant functional biomarkers for cardiovascular risk assessment, and new insights and therapies increase clinical measurement frequency. As former WHO-IFCC RMS for apos A-I, B and Lp(a) are no longer available, there is an unmet need for a higher order, globally available, RMS for conventional and emerging apolipoproteins. Methods: A mass spectrometry-based candidate reference measurement procedure (cRMP) was developed using bottom-up proteomics. Proteins in serum were denatured, reduced and alkylated prior to digestion. Thirty-one peptides representing seven apolipoproteins were quantified relative to stable isotope labelled synthetic peptides. Calibration is currently done with value-assigned, serum-based commutable calibrators. The method is provisionally validated according to CLSI guidelines. Results: Interpeptide correlation within individual proteins showed Pearson's Rs > 0.975, except for apoC-I (R = 0.953) and one comparison for C-III (R = 0.970). The quantitation of apo(a) is independent of its size polymorphism by design and was linear between 3.4 and 450 nmol/L, the LoQ for apo(a) was 3.4 nmol/L. Total imprecision for apo(a) was 8.5, 10.2, 10.1, 10.4 and 9.7%, at concentrations of 8.4, 15.6, 49.3, 252 and 364 nmol/L. Average total imprecision for other apos ranged between 3.7 and 7.4%. Conclusions: A next generation, 7-plex, proteomics-based cRMP was developed that allows molecular measurement of targeted apos. As serum apolipoproteins are measured directly through their proteotypic peptides, molar and accurate quantitation of apo(a) and the other apolipoproteins is enabled with this cRMP.

Background and Aims : Elevated concentrations of lipoprotein(a) (Lp(a)) are directly related to an increased risk of cardiovascular diseases, thus making its determination a crucial factor for clinical diagnosis. However, the lack of global standardisation of current immunoassay-based measuring procedures (MPs) for Lp(a) leads to inconsistent care of patients. The former Lp(a) reference method and the associated WHO-IFCC reference material are no longer available. Recently, it was decided to evolve to a next generation reference measurement system (RMS) with SI-traceability. To accomplish this an IFCC working group on quantitating apolipoproteins by mass spectrometry (MS) was formed. The SI-traceable RMS will consist of a MS-based, peptide-calibrated candidate reference measurement procedure (cRMP) and secondary serum-based reference materials (RMs) certified for their apolipoprotein(a) molar concentration.Methods: A correlation study was performed between the cRMP and immunoassay-based MPs using a panel of 39 clinical samples (CS) that cover the whole Lp(a) concentration range. In addition, the commutability of 14 different candidate RMs was investigated to select a suitable RM format for the future development of the serum-based RM.Results: Comparison of immunoassay-based MPs with the cRMP for measurements of CS in nmol/L demonstrated a good linear correlation, but showed significant measurement bias and sample specific differences.Conclusions: The results of the commutability study show that RMs based on human serum pools with endogenous Lp(a) are good candidates for future matrix-based certified RM, whereas human pools -spiked with recombinant apo(a) show different behaviour compared to CS, making them unsuitable as RMs in most of the currently available routine assays. Background and Aims : Elevated concentrations of lipoprotein(a) (Lp(a)) are directly related to an increased risk of cardiovascular diseases, thus making its determination a crucial factor for clinical diagnosis. However, the lack of global standardisation of current immunoassay-based measuring procedures (MPs) for Lp(a) leads to inconsistent care of patients. The former Lp(a) reference method and the associated WHO-IFCC reference material are no longer available. Recently, it was decided to evolve to a next generation reference measurement system (RMS) with SI-traceability. To accomplish this an IFCC working group on quantitating apolipoproteins by mass spectrometry (MS) was formed. The SI-traceable RMS will consist of a MS-based, peptide-calibrated candidate reference measurement procedure (cRMP) and secondary serum-based reference materials (RMs) certified for their apolipoprotein(a) molar concentration. Methods: A correlation study was performed between the cRMP and immunoassay-based MPs using a panel of 39 clinical samples (CS) that cover the whole Lp(a) concentration range. In addition, the commutability of 14 different candidate RMs was investigated to select a suitable RM format for the future development of the serum-based RM. Results: Comparison of immunoassay-based MPs with the cRMP for measurements of CS in nmol/L demonstrated a good linear correlation, but showed significant measurement bias and sample specific differences. Conclusions: The results of the commutability study show that RMs based on human serum pools with endogenous Lp(a) are good candidates for future matrix-based certified RM, whereas human pools -spiked with recombinant apo(a) show different behaviour compared to CS, making them unsuitable as RMs in most of the currently available routine assays.

Background and Aims: Global standardization of conventional tests and emerging biomarkers is key to enable exchange of test results and to apply universal clinical decision limits across hospitals. Therefore, calibration hierarchies should be established in line with ISO 17511:2020. For apolipoprotein (apo) A-I, apoB and apo(a), the existing WHO-IFCC Reference Materials (RM) are running out of stock and the Reference Measurement Procedure (RMP) is no longer in place. Technological advances enable SI-traceability through direct protein quantitation using mass spectrometry (MS). Consequently, RM and a common MS-based RMP are developed to quantify serum apolipoproteins A-I, B, (a), C-I, C-II, C-III and E.