There is a revival of the genetically determined cardiovascular risk factor Lp(a). Yet, it is one of the most complicated clinical chemistry parameters, and up to 2-fold variation in measurement results may be observed between in vitro diagnostic tests. A next-generation reference measurement system based on mass spectrometry – and endorsed by the International Federation of Clinical Chemistry and Laboratory Medicine – is in development, and expected to be finalized in 2024. However, we can already now aid manufacturers in the transition towards molar reporting units, larger dynamic ranges and better calibration strategies to facilitate improved standardization, which is needed to guarantee safe and accurate test results for patients, and enable precision medicine for patients at risk for cardiovascular disease.
The lipoprotein particle lipoprotein (a) [Lp(a)] underwent a remarkable revival as an independent risk factor for cardiovascular risk assessment over the past decade. As a result, there is renewed interest in the selective and accurate quantitation of Lp(a). Although initial reports from the Physicians Health Study (PHS) in the 1990s did not provide evidence for Lp(a) as risk marker, studies in 2004, relative to the then available reference measurement system (RMS) , indicated these findings were flawed by inaccurate measurements. Further evidence from Mendelian randomization trials, indicating an independent, causal association between Lp(a) levels and cardiovascular disease (CVD; i.e. myocardial infarction, stroke and peripheral artery disease), stimulated the adoption of Lp(a) measurement in clinical guidelines. Specifically, current American College of Cardiology (ACC)/American Heart Association (AHA) guidelines recommend Lp(a) measurement in individuals with a family history of premature atherosclerotic cardiovascular disease (ASCVD) , while the Canadian Cardio-vascular Society (CCS) as well as the European Atherosclerosis Society (EAS) and the European Society of Cardiology (ESC) recommend Lp(a) measurements at least once in an individual’s lifetime [3,4]. Besides uptake of Lp(a) in clinical guidelines, the new evidence also spurred the development of Lp(a)-lowering therapies, which are currently in phase II and III clinical trials.
Current Lp(a) measurements and standardization
The recommendations to measure Lp(a), and the need for patient inclusion in clinical trials, has increased the volumes of Lp(a) testing. Nowadays, a multitude of immunoassay-based Lp(a) tests is commercially available with unacceptable inter-method coefficients of variation (CVs) ranging from 16% to 32%. Also, between-method variation can be as large as 2-fold, considerably complicating interpretation of test results. To enable accuracy of medical test results, within allowable levels of measurement uncertainty, each of the elements of the metrological traceability chain as outlined in ISO 17511:2020 , has to be in place and meet predefined requirements. A World Health Organization (WHO)/International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-endorsed RMS, consisting of an ELISA-based reference measurement procedure (RMP) targeting the unique Lp(a) protein apolipoprotein (a) [apo(a)], and the serum-based reference material SRM2B, was developed in the 1990s . Value assignments by this RMS were reported in molar concentrations, and were considered the gold standard. Yet, most commercially available tests, despite their claimed metrological traceability to the RMS, report in mass concentrations with faulty underlying assumptions.
Besides the availability of a complete RMS, a second important prerequisite for test accuracy is the unequivocal definition of the measurand. For the largely heterogeneous Lp(a) particle, this is not a trivial task. Lp(a) is an LDL-like particle, which besides apoB also contains a single apo(a). Particle heterogeneity originates from the apo(a) kringle IV-2 size polymorphism, other apo(a) genetic variations, protein glycosylation [both apo(a) and apoB], other post-translational modifications, the lipid/protein ratio and lipid compositions. The expression of Lp(a) in total mass through immunoassay (IA) measurements can therefore, by definition, not be accurate . Notably, traceability to the previous gold standard WHO/IFCC RMS reasonably improved immunoassay-based Lp(a) tests to a degree that they are minimally apo(a) isoform independent within their specified measuring range. However, the WHO/IFCC RMP is currently no longer operational and the SRM2B reference material is out of stock, bringing in vitro diagnostic (IVD)-manufacturers into trouble. Therefore, there is an urgent need for a new RMS for apo(a).
Standard making for Lp(a) by mass spectrometry (MS)
To solve the problems that have arisen around the discontinuation of the former apo(a) RMS, an IFCC working group has been established for the standardization of apolipoproteins by mass spectrometry (IFCC WG APO-MS; https://www.ifcc.org/ifcc-scientific-division/sd-working-groups/wg-apo-ms/). The working group aims to develop a next-generation multiplexed RMS for seven apolipoproteins in a step-up approach, in which priority is given to apo(a). The conceptual approach for establishing the SI-traceable apo(a) RMS has been published, and includes the development of peptide-based primary reference materials, a quantitative bottom-up proteomics-based RMP, in which peptide concentrations can be transferred to protein concentrations, and at least five native, serum-based and commutable reference materials with values assigned in nmol/L . Mass spectrometry (MS), while requiring high-end equipment and expertise, is a technique that is highly suited for the development of (protein) RMPs, and its advantages over immunoassays have been outlined . In quantitative protein MS, proteins are enzymatically converted into peptides, after which specific peptides are simultaneously qualitatively and quantitatively assessed. This strategy has been proven to be successful for the quantitation of proteins in complex serum, plasma and urine matrices, and enables antibody-independent quantitation of (protein) measurands at the molecular level . For apo(a) this entails that through careful peptide selection, a candidate RMP is designed and described that is inherently kringle IV-2 independent, and provides precision within allowable measurement uncertainty . While development of a peptide-based calibration strategy is ongoing, with expected availability in Q3 2023, a provisional native serum-based calibration is applied.
A major requirement for establishing a metrological traceability chain is the availability of fit-for-purpose primary and secondary reference materials. The primary reference materials will be used for the calibration of the MS-based RMP and consist of peptide solutions with a well-characterized molar concentration, purity and behaviour. Studies to assess the correct value assignment and mode of implementation of the peptide-based calibrators as primary reference materials are ongoing and expected to be finalized by Q3 2023. This will form the basis of the metrological traceability of the final results to the SI units. The peptide-calibrated RMP will then be used to assign the Lp(a) concentration in molar units to the secondary reference materials, which will be made available to IVD manufacturers for internal traceability procedures and value assignment of their product calibrators. It is imperative that the secondary reference materials resemble real clinical specimens measured with the immunoassay-based Lp(a) tests. The complex and polymorphic nature of Lp(a) requires the production of multiple secondary reference materials with concentrations spread across the Lp(a) concentration range and well-selected apo(a) isoforms. Based on a first commutability study, serum-based materials have been selected that are suitable for the production of five to seven secondary reference materials, spread across the clinically relevant measurement range . The certified secondary reference materials are expected to be available in Q2 2024.