Development of an LC-MRM-MS-Based Candidate Reference Measurement Procedure for Standardization of Serum Apolipoprotein (a) Tests

Abstract

Background Medical results generated by European CE Marking for In Vitro Diagnostic or in-house tests should be traceable to higher order reference measurement systems (RMS), such as International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-endorsed reference measurement procedures (RMPs) and reference materials. Currently, serum apolipoprotein (a) [apo(a)] is recognized as a novel risk factor for cardiovascular risk assessment and patient management. The former RMS for serum apo(a) is no longer available; consequently, an International System of Units (SI)-traceable, ideally multiplexed, and sustainable RMS for apo(a) is needed. Methods A mass spectrometry (MS)-based candidate RMP (cRMP) for apo(a) was developed using quantitative bottom-up proteomics targeting 3 proteotypic peptides. The method was provisionally validated according to ISO 15193 using a single human serum based calibrator traceable to the former WHO-IFCC RMS. Results The quantitation of serum apo(a) was by design independent of its size polymorphism, was linear from 3.8 to 456 nmol/L, and had a lower limit of quantitation for apo(a) of 3.8 nmol/L using peptide LFLEPTQADIALLK. Interpeptide agreement showed Pearson Rs of 0.987 and 0.984 for peptides GISSTVTGR and TPENYPNAGLTR, and method comparison indicated good correspondence (slopes 0.977, 1.033, and 1.085 for LFLEPTQADIALLK, GISSTVTGR, and TPENYPNAGLTR). Average within-laboratory imprecision of the cRMP was 8.9%, 11.9%, and 12.8% for the 3 peptides. Conclusions A robust, antibody-independent, MS-based cRMP was developed as higher order RMP and an essential part of the apo(a) traceability chain and future RMS. The cRMP fulfils predefined analytical performance specifications, making it a promising RMP candidate in an SI-traceable MS-based RMS for apo(a).

An LC–MS-based designated comparison method with similar performance to the Lp(a) reference measurement procedure to guide molar Lp(a) standardization

“…The long-term imprecision has also previously been monitored for both the RMP and the DCM. For the RMP, a native human serum IQC sample containing 44 nmol/L apo(a) yielded CVs of 9.0% and 10.0% for peptides GISSTVTGR and LFLEPTQADIALLK, respectively over a 2-year period, 84 measurements and 34 96-well batches [ 10 ]. Similar results have been obtained for the DCM, where a native human serum sample containing 102 nmol/L apo(a) yielded a CV of 7.3% for peptide LFLEPTQADIALLK over a 2-year period [ 16 , 17 ].…”

“…Apolipoprotein quantification was conducted according to the standard operating procedure, as outlined previously [ 10 ]. In short, samples were 20 × diluted in 100 mmol/L ABC.…”

“…A prerequisite for this transition is the availability of an Lp(a) Reference Measurement Procedure (RMP) that allows unequivocal molecular detection and quantification of apolipoprotein(a) (apo(a)) in Lp(a). The International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) working group for standardization of apolipoproteins by mass spectrometry (IFCC WG APO-MS) set out a conceptual approach for such an RMP [ 8 ], and recently reported on an ISO 17511:2020 compliant liquid chromatography – mass spectrometry (LC–MS) based and IFCC-endorsed RMP [ 10 ]. The RMP quantifies proteotypic peptides of apo (a) which is the specific apolipoprotein and a major structural component of lipoprotein(a) [ 11 ].…”

“…The RMP quantifies proteotypic peptides of apo (a) which is the specific apolipoprotein and a major structural component of lipoprotein(a) [ 11 ]. Importantly, apo(a) is a highly heterogenic protein that manifests in various proteoforms, due to the KIV 2 polymorphisms and post translational modifications [ 10 , 12 ]. Moreover, it is present in serum in a wide concentration range.…”

Recommendations of the Experts of the Polish Cardiac Society (PCS) and the Polish Lipid Association (PoLA) on the diagnosis and management of elevated lipoprotein(a) levels

“…Recently, a mass spectrometric method has been developed and validated for the standardisation of Lp(a) measurements, which addresses the problem of size polymorphisms by selecting specific peptides for quantification that are not present in the KIV2 region [ 117 ]. Research is also currently underway on the global standardisation of Lp concentration measurements, which will improve diagnostic interpretation and clinical decision-making regarding Lp(a)-dependent CVD risk [ 117 , 118 ].…”