Raeesa Gupte, PhD
Globally, approximately 18 million people die from cardiovascular diseases (CVD) each year,1 resulting in one-third of all deaths worldwide. High blood cholesterol is among the key risk factors of coronary heart disease (CHD) and stroke. Consequently, a lipid profile that measures levels of low-density lipoprotein (LDL or “bad” cholesterol), high-density lipoprotein (HDL or “good” cholesterol), triglycerides, and total cholesterol is the preferred test for screening and treatment of CVD.
Combined with genetic, demographic, and lifestyle factors, blood cholesterol measurements are used to stratify patients according to their risk of developing CVD. Risk stratification is the first step in identifying strategies for disease prevention and treatment. For instance, patients with fewer risk factors may be treated using lifestyle modifications, while those at higher risk of developing CVD may be treated using more aggressive pharmacological interventions. Therefore, accurate and precise measurement of blood cholesterol levels is imperative for appropriate patient classification and treatment strategies in accordance with clinical practice guidelines.
Need for standardization of biomarkers
Public health strategies are devised based on laboratory data obtained from several epidemiological studies. To develop evidence-based clinical practice guidelines, results are often compared across studies or over time. Variability in the measurement of biomarker levels across laboratories based on the clinical assay used may lead to inconsistent and inconclusive evidence. It is therefore crucial that the methods and materials used for biomarker detection be comparable and reliable across laboratories and over time.
Standardization: Process of ensuring accuracy and precision of clinical laboratory assays by comparing their performance against a reference measurement. In other words, analytical performance of a laboratory assay is assessed against the analytical performance goals based on a reference measurement.
Analytical accuracy: Indicates how closely a measurement aligns with the true (target or reference) value of a sample.
Analytical precision: Indicates how closely multiple measurements of the same quantity of a sample align with each other.
Bias: Systematic deviation of multiple measurements of a sample from its true or reference value.
Primary reference: Materials or procedures used to assign values to calibrators or control materials that are traceable to a globally recognized higher-order reference base or metrological system.
Secondary reference: High purity-grade materials quantified by comparison to a primary standard using a high precision comparator and making appropriate corrections for non-ideal conditions of measurement.
Standardization programs for cholesterol biomarkers
In 1988, the Laboratory Standardization Panel recommended that all cholesterol measurements from clinical laboratories be standardized and made traceable to the National Reference System for Cholesterol (NRS/ CHOL).3 The NRS/CHOL now forms the accuracy base for cholesterol measurements in the US. According to its hierarchy, National Institute of Standards and Technology (NIST)-certified materials and methods are the primary reference, while Centers for Disease Control and Prevention (CDC)-certified materials and methods are secondary references.3 However, since comparison to primary references is too cumbersome and expensive for routine use in clinical laboratories, CDC references have been used as the standard for cholesterol testing for over 30 years.
The CDC oversees the Reference Laboratory and Clinical Standardization Programs to ensure laboratory measurements of blood cholesterol levels are accurate and reliable. These programs include the following:
Cholesterol Reference Method Laboratory Network (CRMLN)
The CRMLN is a specialized network of laboratories across the world that provide reference measurement services to manufacturers and clinical laboratories for measuring total cholesterol, LDL, HDL, and triglycerides according to the CDC’s standardization protocols.4 CRMLN member laboratories determine the analytical accuracy and precision of laboratory-developed blood cholesterol tests and manufacturer-developed in vitro diagnostics and instruments. To obtain CDC certification, manufacturers and clinical laboratories must participate in a method comparison study with a CRMLN member laboratory. The CDC compares the results generated by the CRMLN laboratory with those of the manufacturer or clinical laboratory and issues a Certificate of Traceability if the analytical performance criteria for maximum allowable bias and imprecision are met, as outlined in Table 1. Validity of certificates for manufactures is two years and for clinical laboratories is six months.5
Lipids Standardization Program (LSP)
LSP evaluates the accuracy and precision of lipid measurements conducted by research and clinical laboratories over time.6 LSP provides laboratories with blinded standards of total cholesterol, glycerides, HDL cholesterol, apolipoprotein A-I (apo A-I), and apolipoprotein B (apo B). Standards are traceable to the CDC Reference Laboratory. Participating laboratories measure the blinded standard alongside their clinical or research samples each week for 12 weeks, and report measurement results to the CDC. The CDC provides participating laboratories with a statistical evaluation of the accuracy and precision of their results and issues certification if analytical performance criteria are met. Certification is a continuous process, with the LSP providing laboratories with new blinded standards each quarter. Laboratories are only certified for the quarter in which they participate.
Lipids Reference Laboratory (LRL)
LRL assigns target values to reference materials used by the CRMLN and LSP. It allows the same analyte (total cholesterol, LDL, HDL, or total glycerides) to be measured using different procedures that are standardized to the reference methods prescribed by the LRL.7 This ensures that thresholds for clinical decision-making remain consistent, irrespective of the procedure used to measure serum cholesterol levels. To avoid imprecision and bias, all LRL methods are calibrated to NIST standard reference materials.
Standardization of novel CVD biomarkers
Several biomarkers are emerging as promising candidates in the diagnosis of CVD. For example, elevated levels of cardiac troponin in peripheral blood indicates cardiac damage and is used as a predictor of myocardial infarction. However, results vary significantly between assays developed by different manufacturers.8 Clinical laboratories and manufacturers of in vitro diagnostics may harmonize assay results by recalibration with a common reference material. Nonetheless, standardization efforts have been hampered by the lack of a reference material with adequate commutability for all assays. Procedures for measuring other emerging inflammatory and natriuretic peptide biomarkers also share similar limitations on harmonization and standardization.
Standardization ensures accuracy and precision in biomarker measurements across procedures and over time. By introducing uniformity in clinical practice guidelines, biomarker standardization programs ensure reliable risk stratification and treatment of patients. Efforts to improve the testing and standardization of assays measuring lipids and lipoproteins continue. Institutional efforts to standardize emerging biomarkers of CVD are ongoing.
1. Roth, Gregory A., et al. "Global burden of cardiovascular diseases and risk factors, 1990–2019: update from the GBD 2019 study." Journal of the American College of Cardiology 76.25 (2020): 2982-3021.
2. Grundy, S. M., et al. "Guideline on the management of blood cholesterol: a report of the American College of Cardiology/American heart association task force on clinical practice guidelines." Journal of the American College of Cardiology 73.24 (2018): e285-350.
3. Vanderlinde, Raymond E., et al. "The national reference system for cholesterol." Clinics in Laboratory Medicine 9.1 (1989): 89-104.
4. "Cholesterol Reference Method Laboratory Network (CRMLN)." Centers for Disease Control and Prevention. https://www.cdc.gov/labstandards/crmln.html.
5. Myers, Gary L., et al. "A reference method laboratory network for cholesterol: a model for standardization and improvement of clinical laboratory measurements." Clinical Chemistry 46.11 (2000): 1762-1772.
6. "Lipids Standardization Program (LSP)." Centers for Disease Control and Prevention. https://www.cdc.gov/labstandards/lsp.html.
7. "Lipids Reference Laboratory." Centers for Disease Control and Prevention. https://www.cdc.gov/labstandards/lrl.html.
8. Tate, Jillian R., et al. "Standardisation of cardiac troponin I measurement: past and present." Pathology 42.5 (2010): 402-408.