by Dr L. R. Ruhaak and Prof. C. M. Cobbaert

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.

Introduction

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) [1], 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) [2], 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 [5], 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 [1]. 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 [6]. 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 [7]. 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 [8]. 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 [9]. 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 [10]. 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 [11]. The certified secondary reference materials are expected to be available in Q2 2024.

Figure 1. Metrological traceability of apolipoprotein tests
We will aid IVD manufacturers in the transition from the former ELISA-based RMS to the new MS-based RMS. The transition will be accomplished in a two-step process: first to molar units and the MS-based RMP, and subsequently to SI units. Dedicated protocols for method comparisons with commutable materials over a 2- to 3-fold extended measurement range will be available, that provide advice on traceability to SRM2B, transition to nmol/L and kit design in relation to measurement range.

Standard taking for Lp(a) by MS

The availability of a RMS and calibration laboratories in itself is not sufficient to achieve standardization. This should be an active process in which all stakeholders of the traceability chain (calibration labs, metrology institutes, IVD manufacturers and end-users) collaborate. The transition from the previous ELISA-based RMP to the next-generation MS-based RMP currently in development with peptide-based calibration in nmol/L, traceable to SI units will have implications for IVD manufacturers. Therefore, a 2-step approach for aiding IVD manufacturers in this transition phase is developed:
1. transitioning from mass to molar units (nmol/L) and keeping intact the traceability of test results to SRM2B reference material; and
2. transition to SI units with the peptide-based calibration and the SI-traceable RMS.

Most manufacturers provide Lp(a) results in mass units, even though traceability to the former RMS in nmol/L is claimed. Oftentimes a conversion factor between mass and molar units is applied. However, due to the large heterogeneity in Lp(a) composition, this cannot be correct [6]. The candidate RMP (cRMP) currently generates test results in nmol/L that are traceable to the former WHO/IFCC SRM2B reference material and RMP. It is anticipated that for IVD manu-facturers the change to molar units will have the largest impact on IVD test results, while the transition from ELISA-based RMS to MS-based RMS will be minor, as the results generated by the IFCC-endorsed MS-based cRMP are (on average) in the measuring range equivalent to SRM2B traceable test results. Outside the measuring range we encounter problems with all immunoassays. As peptide-based calibration traceable to SI units is still in development, its impact cannot yet be estimated. However, as MS relies on linear calibration, only a linear shift towards SI-traceability, compared to the current value assignment based on SRM2B, is envisioned. In the first step, the MS-based cRMP with provisional calibration is now available to aid IVD manufacturers to investigate the transition to nmol/L via alignment with the LC-MS-based RMS (Fig. 1). Specifically, manufacturers can assess the traceability of Lp(a) test results to SRM2B, but also assess implementation of multiple independent calibrators, extension of the Lp(a) measuring range, and implementation of molar reporting units. Once these steps have been taken, better harmonized Lp(a) results between IVD manufacturers are anticipated, which will simplify standardization to SI units, once the peptide-based calibration is available.

Concluding remarks

Overall, there is an urgent need for a next-generation RMS for apo(a), so that IVD manufacturers can certify their commercial tests via IFCC-endorsed apo(a) calibration labs and end-users can rely on accurate Lp(a) test results. The latter is essential as Lp(a)-lowering medications are on the horizon and risk stratification of patients as well as assessment of the Lp(a)-lowering therapeutic effects are needed in the near future. Therefore, a sustainable RMS with traceability to SI units, implemented in a network of calibration labs working according to harmonized procedures is in development by the IFCC WG APO-MS. The apo(a) RMS is anticipated to be completed and operational by Q2 2024, with Joint Committee for Traceability in Laboratory Medicine (JCTLM) database listing anticipated by 2025. Meanwhile, IVD manufacturers are invited to re-establish metrological traceability of their Lp(a) test results, assess whether their test results are fit for purpose, pre-assess the implications of the new RMS and improve persisting inadequacies in their tests to guarantee safe and accurate test results for patients, and enable precision medicine for patients at risk for CVD.

The author

L. Renee Ruhaak and Christa M. Cobbaert*
Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, the Netherlands

*Corresponding author
E-mail: c.m.cobbaert@lumc.nl

References

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