Background: Performance of thyroid function assays can vary significantly. To address this issue, the Centers for Disease Control and Prevention (CDC) Clinical Standardization Programs conducted an interlaboratory comparison of free thyroxine (fT4) immunoassays (IAs) and laboratory-developed tests (LDTs). This assessment aimed to determine the current performance characteristics of these assays as a first step toward measurement standardization. Thyrotropin (TSH) IAs were also evaluated. Methods: Assays measured 41 blinded individual-donor sera, including a sample from a pregnant woman (for fT4 analysis only) and three serum pools, with 11.3-32.1 pmol/L (0.881-2.49 ng/dL) fT4 and 0.337-21.6 mIU/L TSH in duplicate over 2 days. Passing-Bablok regression analysis performed pre-recalibration compared assays performance to the CDC fT4 reference measurement procedure (RMP) or TSH all-lab mean (ALM). Additionally, the impact of linear regression-based recalibration of assays to the CDC fT4 RMP or TSH ALM was estimated. Inter-assay agreement of sample classification according to the assay-specific reference interval (RI) was assessed pre- and post-recalibration. Results: A total of 21 fT4 and 17 TSH assays participated. Pre-recalibration, median biases of TSH measurements to the ALM were -1.2% [confidence interval or CI -1.8% to -0.4%], and good classification agreement among TSH assays was observed. fT4 assays all showed a negative median bias to the RMP, with higher bias among IAs (median: -20.3%, CI [-21.5% to -19.4%]) than LDTs (median: -4.5%, [CI -6.1% to -3.2%]). Of the individual-donor sera, only 21 out of 40 samples were classified uniformly by all fT4 assays, indicating poor inter-assay agreement. Post-recalibration, agreement improved to 33 out of 40 individual-donor sera correctly classified by all tested IAs and LDTs. Similar improvement in post-recalibration median percent bias was observed for fT4 IAs (median: -0.2, [CI -1.2% to 0.6%]) and LDTs (median: -0.3%, [CI -2.5% to 1.4%]). Conclusions: The comparison among fT4 assays emphasizes the need for measurement standardization to improve accuracy and comparability. This and previous studies demonstrate the possibility to develop common fT4 RIs via standardization, enabling the use of evidence-based clinical guidelines universally in patient care. Recalibration can effectively address high variability in fT4 assays, ensuring consistent diagnostic classification.

BACKGROUND: Lipoprotein(a) [Lp(a)] is an independent risk factor for cardiovascular diseases (CVD). Recent clinical guidelines recommend measuring Lp(a); however, the lack of Lp(a) assay standardization presents challenges to using common clinical decision points. Assay standardization may minimize inter-assay variability. This improves consistency in CVD risk assessment and evaluations of Lp(a) therapeutic efficacy. Genetically determined size variations in the defining apolipoprotein(a) [apo(a)] protein contribute to inter-individual Lp(a) heterogeneity. Individuals who express 2 apo(a) isoforms have 2 sizes of apo(a) in circulation, further contributing to Lp(a) heterogeneity. OBJECTIVE: The Centers for Disease Control and Prevention's Clinical Standardization Programs (CDC CSP) recently launched an Lp(a) standardization program based on the International Federation of Clinical Chemistry endorsed liquid-chromatography mass spectrometry-based reference measurement procedure (RMP). As part of this program, CDC CSP conducted an interlaboratory comparison study to evaluate current Lp(a) inter-assay variability and to investigate potential factors contributing to measurement variability. METHODS: Eight clinical laboratories measured Lp(a) in 40 individual donor serum samples and 3 serum pools. Serum samples were immunophenotyped by Western blot analysis to determine Lp(a) isoform sizes. Sample concentrations were measured in duplicate over 2 independent runs. RESULTS: Assay-specific Lp(a) measurements demonstrated good linear correlation with the RMP. Lp(a) inter-assay measurement variations ranged from 3.3% to 69.1% across individual samples; however, Lp(a) inter-assay coefficients of variation did not increase in a concentration-dependent manner and were not correlated with Lp(a) isoform sizes. CONCLUSION: This study provides new insights into Lp(a) inter-assay variability and assay performance in clinical laboratories that will guide future standardization efforts.

Cardiovascular disease (CVD) is the leading cause of mortality in the United States and globally. This review describes changes in CVD lipid and lipoprotein biomarker measurements that occurred in line with the evolution of clinical practice guidelines for CVD risk assessment and treatment. It also discusses the level of comparability of these biomarker measurements in clinical practice. Comparable and reliable measurements are achieved through assay standardization, which not only depends on correct test calibration but also on factors such as analytical sensitivity, selectivity, susceptibility to factors that can affect the analytical measurement process, and the stability of the test system over time. The current status of standardization for traditional and newer CVD biomarkers is discussed, as are approaches to setting and achieving standardization goals for low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), triglycerides (TG), lipoprotein(a) (Lp(a)), apolipoproteins (apo) A-I and B, and non-HDL-C. Appropriate levels of standardization for blood lipids are maintained by the Centers for Disease Control and Prevention's (CDC) CVD Biomarkers Standardization Program (CDC CVD BSP) using the analytical performance goals recommended by the National Cholesterol Education Program. The level of measurement agreement that can be achieved is dependent on the characteristics of the analytes and differences in measurement principles between reference measurement procedures and clinical assays. The technical and analytical limitations observed with traditional blood lipids are not observed with apolipoproteins. Additionally, apoB and Lp(a) may more accurately capture CVD risk and residual CVD risk, respectively, than traditional lipids, thus prompting current guidelines to recommend apolipoprotein measurements. This review further discusses CDC's approach to standardization and describes the analytical performance of traditional blood lipids and apoA-I and B observed over the past 11 years. The reference systems for apoA-I and B, previously maintained by a single laboratory, no longer exist, thus requiring the creation of new systems, which is currently underway. This situation emphasizes the importance of a collaborative network of laboratories, such as CDC's Cholesterol Reference Methods Laboratory Network (CRMLN), to ensure standardization sustainability. CDC is supporting the International Federation of Clinical Chemistry and Laboratory Medicine's (IFCC) work to establish such a network for lipoproteins. Ensuring comparability and reliability of CVD biomarker measurements through standardization remains critical for the effective implementation of clinical practice guidelines and for improving patient care. Utilizing experience gained over three decades, CDC CVD BSP will continue to improve the standardization of traditional and emerging CVD biomarkers together with stakeholders.

BACKGROUND: Accurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. METHODS: Serum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. RESULTS: The described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. CONCLUSION: Our ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays.

Clinical laboratory measurements that are accurate and comparable across measurement systems and over time are critical for patient care and public health systems. Evidence-based clinical practice guidelines recommend specific decision points to guide clinical decisions. Electronic health records include patient laboratory data, transferrable across healthcare systems, that inform physicians about a patient’s health history. Laboratory data generated within and across healthcare systems are used to characterize patient populations, identify public health concerns, and track outcomes. Patients access their laboratory results, compare them with publicly available information, and discuss them with healthcare providers. These recent developments have advanced the roles of the clinical laboratory and introduced opportunities for laboratories to provide valuable new information to the patient and the healthcare team. However, these advancements and opportunities require accurate and reliable laboratory measurements. Reliability, in this context, comprises characteristics such as analytical sensitivity, specificity, precision, and consistency over time. Harmonization of laboratory measurement results and standardization, a more specific way of harmonizing results, can help to achieve this. Harmonization creates laboratory measurements that are applicable to practice guidelines, comparable, and interoperable across health systems and over time.