Description
Background
Pathophysiological, epidemiological, and genetic studies have provided convincing evidence that Lp(a) contributes to the development of cardiovascular diseases, cerebrovascular disease, peripheral arterial disease, heart failure, and calcific aortic valve disease. Lp(a) consists of a low-density lipoprotein (LDL)-like particle bound to an additional and specific apolipoprotein(a) [apo(a)]. Apo(a) contains a variable number of structural motifs called ‘kringles’, which give rise to more than 40 different Lp(a) isoforms leading to very heterogeneous and complex Lp(a) particles.
Accurate and reliable measurement of Lp(a) is challenging due to its complex and heterogeneous structure, and represents an important gap in the field. A variety of assays are available to measure Lp(A), but they are not well standardized or validated. A previous workshop organized by NHLBI in 2017 identified standardization of Lp(a) measurement and reporting as one of the top priorities. As a follow-up, this workshop was organized to identify research gaps and needs, as well as opportunities for the standardization of Lp(a) measurements and reporting.
Discussions
The workshop presentations and discussions centered around the following key issues:
- The current state of Lp(a) measurements and reporting
- The need to standardize Lp(a) measurements
- Current state of the art of similar lipid and lipoprotein measurement and standardization
- Developing strategies for concerted efforts towards standardization of Lp(a) measurement globally from stakeholders who have already embarked on Lp(a) standardization
- Potential for adapting the current infrastructure offered by the Centers for Disease Control and Prevention (CDC) for other lipid and lipoprotein tests towards Lp(a) standardization
- Strategies for developing optimal reference standards for Lp(a)
- Leveraging knowledge and experience from other clinical laboratory tests and standardization efforts like those for measuring hemoglobin A1c
CDC and FDA resources
CDC provides reference measurements for total cholesterol (TC), high density lipoprotein cholesterol (HDLc), low density lipoprotein cholesterol (LDLc), and triglycerides (TG) through its Cardiovascular Disease Biomarker Standardization program, which assesses the accuracy of TC, TG, LDLc, and HDLc tests via the Cholesterol Reference Method Laboratory Network. The Lipids Standardization Program is a broader program that also includes apolipoprotein A1 and apolipoprotein B in addition to the lipid profile. The CDC is considering adding Lp(a) to its standardization programs. The Food and Drug Administration (FDA) provides the regulatory requirements for reviewing and approving the clinical use of the developed assays/biomarkers.
Current methods of standardizing Lp(a)
A working group at the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) in Milano, Italy has developed a proteomics approach with isotopically labelled internal peptide standards to establish a mass spectrometry (MS) based reference measurement system for apo(a). This system will have the highest order traceability, i.e., traceability of apo(a) to international system of units (SI). To ensure accurate results, molar units of apo(a) should be used rather than mass units of the entire Lp(a) particle.
Northwest Lipid Research Laboratories at the University of Washington in Seattle is also developing an MS reference method for apo(a) using recombinant apo(a) as primary reference material. It has SI-traceability and is validated by several ways such as linearity, limit of quantification, and parallelism to ensure the reference material and plasma sample behave similarly.
Reference materials
Reference materials are commonly divided into two groups: primary and secondary. Primary reference material is at the top of the traceability chain and could be based on a protein/peptide MS standard that is traceable to apo(a). The source of the primary reference material may be a full recombinant apo(a) protein or synthetic peptides. Secondary reference material will likely be serum-based material that could be used to calibrate commercial assays and should contain different Lp(a) levels and different isoforms e.g., small isoforms with low Lp(a) levels, large isoforms with high Lp(a) levels.
A limitation of current commercial Lp(a) assays is that the choice of secondary calibrators is often not well described or known to the end user. However, the source of calibrators may not be as important as the assessment that the assay is certified by the appropriate reference method.
Antibody for commercial assays
An accurate assay would be needed to establish reliable Lp(a) thresholds for predicting disease risk. There was a general consensus that an isoform independent monoclonal antibody would be the preferable way to measure Lp(a), but more analytical work is needed to establish such an assay.
Lp(a) assay units and use of percentile cut points for Lp(a) assays
Discontinuing the use of mass measures (mg/dL) and universal use of molar measures (nmol/L) would create an opportunity to standardize and harmonize Lp(a) assays. Because there are significant limitations of using mass measurements, several clinical laboratories have already switched to molar units.
On the other hand, clinicians are accustomed to assay results reporting with mass units, and switching to molar units can cause confusion. Measurements in mass units and molar units can be highly correlated by using the same antibody. However, the conversion factor between mass units and molar units varies as a function of Lp(a) levels if the conversion is performed between assays that use different antibodies.
A suggestion of using a percentile as cutpoint (e.g., 75-80%) to improve existing assays was proposed. However, different populations (e.g., statin use, renal disease, familial hypercholesterolemia, different ethnicities) may have different optimum cutoffs for risk and more data is needed to carefully establish percentile for risk in various populations. Thus, absolute Lp(a) levels may be more relevant than percentile levels.
Collaborations for standardization and role of metrological institutes
Current assays can be used for clinical measurements. However, they are not well standardized and lack optimal accuracy. Thus, a more accurate assay is needed. A network of laboratories that can perform the reference method or a definitive comparison method will be important to ensure accuracy over time.
CDC is well positioned to facilitate bringing together stakeholders including the Reference Laboratory Network. Metrological institutes such as the National Institute of Standards and Technology (NIST) can ensure purity and stability of the reference material. However, there will be a need for reference materials and single donor materials in sufficient amounts. The European Commission Joint Research Centre provides resources for making reference materials worldwide.
Issues/barriers
MS measurement of Lp(a) is relatively expensive and requires rigor/consistency in making the reference material. It may be suitable for developing a reference method but cannot be used at this time for routine laboratory testing for diagnostic purposes.
Workshop participants identified lack of insurance coverage for Lp(a) testing as a barrier to develop better tests. Manufacturers report difficulties navigating regulatory requirements and want the scientific community to lead standardization. The recent European Society of Cardiology/European Atherosclerosis Society guidelines recommend Lp(a) be measured in all adults at least once in a lifetime to assess the risk of cardiovascular disease. This provides a strong rationale to standardize Lp(a) assays globally.
Research Opportunities and Further Development
The workshop identified the following opportunities:
- Encourage strong and constructive collaboration among all stakeholders to ensure standardization and harmonization of Lp(a) assays
- Develop assays that are isoform independent using a monoclonal antibody that is specific to apo(a)
- Support studies for the validation of mass spectrometry methods and their potential role as primary reference methods
- Educate clinicians, laboratory service providers, and manufacturers on the need for standardization of assay methods and uniformity in reporting the results
- Work closely with FDA for educating stakeholders on regulatory needs for approval of Lp(a) molar concentration assays
Further Development:
- Primary and secondary reference methods that will be sustainable and scalable, especially for the primary reference method
- Comparing and validating whether a full recombinant apo(a) or synthetic peptides should be the preferred choice as primary reference material
- Strategic collaborations among different agencies including NIH, CDC, NIST, and IFCC to ensure sustained long-term availability of reference standards and methods.
Publication Plans
The workshop members will work to prepare a manuscript for publication in a peer-reviewed journal.
Workshop Participants
Chairs:
Sotirios Tsimikas, MD, University of California San Diego, CA
Alan Remaley, MD, PhD, NIH/NHLBI, Bethesda, MD
Members:
- Paula Caposino, PhD, FDA, Silver Spring, MD
- Christa Cobbaert, PhD, Leiden University Medical Center (LUMC), the Netherlands
- Christopher Conn, PhD, Amgen, Thousand Oaks, CA
- Leslie Donato, PhD, DABCC, Mayo Clinic, Rochester, MN
- Chris Farina, MS, RAC, Abcentra LLC, Los Angeles, CA
- Julia Hoover, Novartis, United Kingdom
- Kazuhiko Inoue, PhD, Kaneka Pharma America LLC, Newark, CA
- Helina Kassahun, MD, Amgen, Thousand Oaks, CA
- Marlys Koschinsky, PhD, Robarts Research Institute, Canada
- Susan Kuklenyik, PhD, CDC, Atlanta, GA
- Randie Little, PhD, University of Missouri, Columbia, MO
- Santica Marcovina, PhD, ScD, University of Washington, Seattle, WA
- Keiko Matsumoto, Denka Seiken, Japan
- Manuel Mayr, MD, PhD, Kings College London, United Kingdom
- Joe McConnell, PhD, Salveo Diagnostics, Richmond, VA
- Patrick Moriarty, MD, University of Kansas Medical Center, Kansas City, KS
- Borge Nordestgaard, MD, DMSc, Copenhagen University Hospital, Denmark
- Akiko Ono, Denka Seiken, Japan
- Marc Penn, MD, PhD, Quest, Cleveland, OH
- Urban Prinzing, PhD, Roche, Germany
- Nathan Provo, MS, Mercodia Inc., Winston-Salem, NC
- Giacomo Ruotolo, MD, PhD, Eli Lilly and Company, Indianapolis, IN
- Banu Sankaran, Novartis, Boston, MA
- Ahmed Al Sattari, Kaneka Pharma America LLC, Newark, CA
- Sandra Revill Tremulis, MBA, Lipoprotein(a) Foundation
- Hubert Vesper, PhD, CDC, Atlanta, GA
- Calvin Yeang, MD, PhD, University of California San Diego, CA
NHLBI Contacts:
Lijuan Liu, Ph.D.
Simhan Danthi, Ph.D.
Michelle Olive, Ph.D.
Division of Cardiovascular Sciences
National Heart, Lung, and Blood Institute
National Institutes of Health
Phone: 301-435-0582
Email: lliu@mail.nih.gov