Papers in Press. Published August 7, 2018 as doi: /clinchem

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1 Papers in Press. Published August 7, 2018 as doi: /clinchem The latest version is at Clinical Chemistry 64: (2018) Lipids, Lipoproteins, and Cardiovascular Risk Factors Comparability of Lipoprotein Particle Number Concentrations Across ES-DMA, NMR, LC-MS/MS, Immunonephelometry, and VAP: In Search of a Candidate Reference Measurement Procedure for apob and non-hdl-p Standardization Vincent Delatour, 1* Noemie Clouet-Foraison, 1 François Gaie-Levrel, 1 Santica M. Marcovina, 2 Andrew N. Hoofnagle, 3 Zsuzsanna Kuklenyik, 4 Michael P. Caulfield, 5 James D. Otvos, 6 Ronald M. Krauss, 7 Krishnaji R. Kulkarni, 8 John H. Contois, 9 Alan T. Remaley, 10 Hubert W. Vesper, 4 Christa M. Cobbaert, 11 and Philippe Gillery 12 BACKGROUND: Despite the usefulness of standard lipid parameters for cardiovascular disease risk assessment, undiagnosed residual risk remains high. Advanced lipoprotein testing (ALT) was developed to provide physicians with more predictive diagnostic tools. ALT methods separate and/or measure lipoproteins according to different parameters such as size, density, charge, or content, and equivalence of results across methods has not been demonstrated. METHODS: Through a split-sample study, 25 clinical specimens (CSs) were assayed in 10 laboratories before and after freezing using the major ALT methods for non- HDL particles (non-hdl-p) or apolipoprotein B-100 (apob-100) measurements with the intent to assess their comparability in the current state of the art. RESULTS: The overall relative standard deviation (CV) of non-hdl-p and apob-100 concentrations measured by electrospray differential mobility analysis, nuclear magnetic resonance, immunonephelometry, LC-MS/MS, and vertical autoprofile in the 25 frozen CSs was 14.1%. Within-method comparability was heterogeneous, and CV among 4 different LC-MS/MS methods was 11.4% for apob-100. No significant effect of freezing and thawing was observed. CONCLUSIONS: This study demonstrates that ALT methods do not yet provide equivalent results for the measurement of non-hdl-p and apob-100. The better agreement between methods harmonized to the WHO/IFCC reference material suggests that standardizing ALT methods by use of a common commutable calibrator will improve cross-platform comparability. This study provides further evidence that LC-MS/MS is the most suitable candidate reference measurement procedure to standardize apob-100 measurement, as it would provide results with SI traceability. The absence of freezing and thawing effect suggests that frozen serum pools could be used as secondary reference materials American Association for Clinical Chemistry Since the results of the Framingham epidemiological study on cardiovascular diseases (CVDs) 13 (1), correlations between lipid disorders and increased cardiovascular risk have been extensively studied (2 5), particularly evidencing the multifactorial origins of CVD. Therefore, to evaluate the patient risk of experiencing acute cardiovascular events and to establish 10-year risk profiles, current guidelines recommend the use of global approaches. These rely on identifying major risk factors such as age, 1 Laboratoire National de Métrologie et d Essais (LNE), Paris, France; 2 Northwest Lipid Metabolism and Diabetes Research Laboratories, University of Washington, Seattle, WA; 3 Department of Laboratory Medicine, University of Washington, Seattle, WA; 4 Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA; 5 Quest Diagnostics Nichols Institute, San Juan Capistrano, CA; 6 Laboratory Corporation of America Holdings, Morrisville, NC; 7 Children Hospital Oakland Research Institute, Oakland, CA; 8 VAP Diagnostics Laboratory Inc., Birmingham, AL; 9 Sun Diagnostics, LLC, New Gloucester, ME; 10 Lipoprotein Metabolism Section, National Heart, Lung, and Blood Institute, Bethesda, MD; 11 Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, the Netherlands; 12 University Hospital of Reims, Laboratory of Pediatric Biology and Research, Reims, France. * Address correspondence to this author at: Laboratoire National de Métrologie et d Essais, Chemistry and Biology Division, 1 rue Gaston Boissier, Paris Cedex 15, France. Fax ; vincent.delatour@lne.fr. V. Delatour and N. Clouet-Foraison contributed equally to the writing of this article. Received March 8, 2018; accepted July 10, Previously published online at DOI: /clinchem American Association for Clinical Chemistry 13 Nonstandard abbreviations: CVD, cardiovascular diseases; ALT, advanced lipoprotein testing; apob-100, apolipoprotein B-100; non-hdl-p, non-hdl particle number; LDL-P, LDL particle number; NMR, nuclear magnetic resonance; VAP, vertical autoprofile; IN, immunonephelometry; ES-DMA, electrospray differential mobility analysis; RMP, referencemeasurementprocedure; RM, referencematerial; DCM, designatedcomparison method; CS, clinical specimen; LNE, Laboratoire National de Métrologie et d Essais; LUMC, Leiden University Medical Center; FT, effect of freezing and thawing. 1 Copyright (C) 2018 by The American Association for Clinical Chemistry

2 sex, smoking, or hypertension, and on measuring the concentrations of circulating lipid markers such as total cholesterol, LDL cholesterol, and HDL cholesterol (6, 7). However, a considerable number of patients with low-risk profiles still experience cardiovascular events, and intervention studies on cholesterol-lowering therapies have shown that residual risk remains, even after achieving optimum cholesterol concentrations (2, 3, 8). In an era of precision medicine, this observation has stimulated further research on lipoprotein-related factors to provide physicians with more predictive and comprehensive biomarkers for CVD risk assessment (9). In this search, the development of new analytical methods, referred to as advanced lipoprotein testing (ALT), established the atherogenicity of lipoprotein particles themselves rather than their lipid content. In particular, a strong correlation was found between CVD risk and increased concentrations of apolipoprotein B-100 (apob- 100), an apolipoprotein present in VLDLs, intermediatedensity lipoproteins, LDLs, and lipoprotein(a) (10, 11). Current understanding of their biogenesis and immunolabeling studies indicate that these lipoproteins, also referred to as non-hdls, contain a unique molecule of apob-100 per particle. Therefore, the concentration of circulating apob-100 in blood can be converted to several circulating non-hdl particles (non-hdl-ps). LDL particle size and high concentrations of LDL particles (LDL- Ps) have been found to be good predictors of increased risk of premature CVD (12 15). With the growing interest in lipoprotein analysis for CVD risk prediction, ALT methods increasingly have been used in clinical trials and prospective studies to evaluate their added value in patient risk stratification (16 18). However, results are conflicting, and many believe there is insufficient evidence to support the widespread use of these biomarkers in routine clinical practice (19, 20). Because different ALT methods were involved in these studies, it has been suggested that the lack of agreement of clinical outcomes could be, in part, the result of a lack of agreement between analytical methods (21). Indeed, Grundy et al. demonstrated a poor comparability between apob-100 concentrations measured by nuclear magnetic resonance (NMR), vertical autoprofile (VAP), and immunonephelometry (IN) and stressed the need to improve between-method comparability (22). Similarly, Ensign et al., comparing VAP, NMR, tube gel electrophoresis, and gradient gel electrophoresis to determine LDL subclasses, demonstrated a strong disagreement in patient LDL phenotyping among these methods (23). For their part, Williams et al., although reporting comparable conclusions on the association of coronary artery stenosis and lipoprotein subclass concentrations when measured by NMR, VAP, electrospray differential mobility analysis (ES-DMA), and gradient gel electrophoresis, underlined the need to standardize ALT assays (15). Nevertheless, few studies have rigorously evaluated the comparability of non-hdl-p concentrations when measured with different ALT methods, and a thorough investigation of the current state of the art appears necessary. To improve measurement comparability, a preferred means consists of standardizing assay calibration through the establishment of a higher order reference measurement procedure (RMP) and the production of higher order reference materials (RMs). A candidate RMP needs to meet several requirements, the most important ones being that (a) the measurand, i.e., the entity intended to be measured, be well-defined, (b) results be traceable to the international system of units (SI), and (c) precision and accuracy of the method fit the clinical needs (24). With respect to ALT, no such method has yet been identified, and the only reference system available relies on an IN assay that was developed in the 1990s by Marcovina and colleagues (25). This assay was used to harmonize apob-100 immunoassays in clinical laboratories through the production of a secondary RM endorsed by the WHO and IFCC: the WHO/IFCC SP3 08. This initiative greatly improved comparability among apob-100 immunoassays, and IN has been considered as the designated comparison method (DCM) for ALT ever since. However, its suitability as higher order RMP to establish SI traceability of apob-100 and non-hdl-p measurements can be disputed. Therefore, a new candidate higher order RMP needs to be identified to standardize ALT assays. In this context, the aim of this study was to assess comparability of the major ALT methods available for the measurement of apob-100 and non-hdl-p concentrations, with the intent to evaluate the current state of the art. The possibility of establishing a higher order reference measurement system for apob-100 and non- HDL-P measurements consisting of a robust candidate RMP that would provide results with SI traceability is additionally discussed in this article. Materials and Methods SAMPLES Twenty-five clinical specimens (CS ) consisting of fresh human serum from individual donors (single donations), with apob-100 concentrations ranging from 0.55 g/l to 1.66 g/l (measured with the IN DCM), were collected and aliquoted according to the Clinical and Laboratory Standard Institute C37A guidelines (26) at the Solomon Park Research Laboratories (Kirkland, WA) using institutional review board approved protocol. Samples were aliquoted, and one-half of the aliquots were kept fresh and stored at 4 C until analysis. The other half was stored frozen at 80 C until analysis. The WHO/ 2 Clinical Chemistry 64:10 (2018)

3 In Search of a Candidate RMP for apob and non-hdl-p Table 1. Advanced lipoprotein testing methods involved in the cross-platform comparison. Methods Laboratory Covered measurands Calibrators ES-DMA-1 (27) Quest Diagnostics non-hdl-p, LDL-P, HDL-P, LDL size, full lipoprotein number concentration profile ES-DMA-2 (28) LNE non-hdl-p, LDL-P, LDL, and HDL size IN (25) NWLMDRL a (University apob-100 of Washington) LC-MS/MS-1A (offline digestion) (32) LC-MS/MS-1B (online digestion) (31) CDC apoa-i, apoa-ii, apoa-iv, apob-100, apoc-i, apoc-ii, apoc-iii, apoe LC-MS/MS-2A (33) LUMC apoa-i, apob-100, apoc-i, apoc-ii, apoc-iii, apoe None WHO/IFCC SP3-08 WHO/IFCC SP3-08 Peptide calibrators, value assigned by amino-acid analysis by isotope dilution LC-MS/MS Serum-based calibrators (CLSI b C37A), value assigned by the IN designated comparison method, calibrated with WHO/IFCC SP3-08 LC-MS/MS-2B (34) University of Washington apoa-i, apob-100 Native human serum samples, value-assigned by the IN designated comparison method, calibrated with WHO/IFCC SP3-08 NMR (LP3 software) (29) LabCorp - NIH non-hdl-p; 3 VLDL-P, 3 None LDL-P, 3 HDL-P subclasses; VLDL-P, LDL-P, HDL-P, sizes NMR (LP4 software) (30) LabCorp - NIH non-hdl-p; 5 TRL-P c, 3 LDL-P, 7 HDL-P subclasses; TRL-P, LDL-P, HDL-P, sizes None VAP (35) VAP Diagnostics d apob-100, LDL-P, cholesterol distribution profile a Northwest Lipid Metabolism and Diabetes Research Laboratories. b Clinical Laboratory Standard Institute. c Triglyceride-rich lipoprotein particle number. d Formerly Atherotech. e Canadian External Quality Assessment Laboratory (Vancouver, BC, Canada). Cholesterol profile: frozen serum from Solomon Park Research Laboratories, value assigned for cholesterol by the CEQAL e IFCC SP3 08 RM was obtained from the CDC (Atlanta, GA) and stored at 80 C until analysis. STUDY DESIGN The 25 fresh CSs were shipped in the presence of cooling elements to all study participants in the US, except the CDC laboratories and the 2 European laboratories: the French Metrology Institute [Laboratoire National de Métrologie et d Essais (LNE)] and the Leiden University Medical Center (LUMC). Fresh materials were delivered within 48 h after collection, and measurements were performed within 72 h after collection. The 25 frozen CSs and the WHO/IFCC SP3 08 RM were simultaneously shipped on dry ice to all study participants and delivered within 48 h after collection. Measurements were performed within 3 months after freezing. Methods involved, parameters measured, and calibrators used for each method are listed in Table 1. Most methods investigated were initially developed for routine measurements, and parameters measured included non- HDL-P, LDL-P, HDL-P, LDL, and HDL size, full lipoprotein number concentration profiles, full cholesterol distribution profiles, apoa-i, apoa-ii, apoa-iv, apob- 100, apoc-i, apoc-ii, apoc-iii, and apoe. ES-DMA and NMR measurements were each performed in 2 different laboratories (27 30). LC-MS/MS assays were performed in 4 laboratories, each using their own protocol and calibrators (31 34). IN and VAP assays were per- Clinical Chemistry 64:10 (2018) 3

4 formed in 1 laboratory each (25, 35). All samples were analyzed in triplicate with each method. DATA ANALYSIS Non-HDL-P and apob-100 concentrations measured in the 25 frozen CSs by each study participant were expressed in nanomoles per liter using a molecular weight of g/mol for apob-100 (36). Between-method agreement was assessed by calculating the relative standard deviation (CV) of the molar concentrations of apob-100 and non-hdl-p across the different methods and laboratories for the 25 frozen CSs. To identify a possible effect of apob concentration on the agreement between apob-100 and non-hdl-p methods, an F-test was performed and the corresponding P value was calculated. Reproducibility of apob-100 measurements across the 4 LC-MS/MS methods was estimated using the mean CV calculated on apob-100 concentrations measured in the 25 frozen CSs. The mean relative difference of each method against IN and the associated SD across the 25 frozen CSs were also calculated. The precision of each method was estimated using the mean CV of apob-100 and non-hdl-p concentrations measured in the 25 frozen CSs. Molar concentrations of apob-100 and non-hdl-p were additionally plotted against the consensus mean concentrations of the 25 frozen CSs and against apob-100 IN concentrations. The 95% CIs were calculated for the slope and the intercept of the linear regression curves obtained. ETHICS Clinical specimen collection was performed in agreement with local ethics rules. Single donations were obtained with donors informed consent and used accordingly. No personal identifiers were provided with the clinical specimens. EFFECT OF FREEZING AND THAWING The effect of freezing and thawing (FT) on apob-100 and non-hdl-p concentration measurements was evaluated by calculating the relative difference between the means of replicate measurements obtained on the fresh CSs and on the same CSs subjected to freezing and then thawing. The following equation was used for the calculation of the FT effect: FT % measurand Frozen measurand Fresh measurand Fresh 100 As each sample was measured before and after freezing, a paired sample t-test was performed to determine whether the effect of FT was statistically significant for each of the different considered methods. The null hypothesis assumes that there is no effect of freezing, i.e., that the mean difference of results obtained before and after freezing is equal to zero. Before the execution of this test, a Shapiro Wilk test was performed to ensure that the data were approximately gaussian and to verify the absence of outliers. A P value 0.05 was used for statistical significance. Results COMPARABILITY Results of apob-100 and non-hdl-p concentration measured by each method in the 25 frozen CSs are presented in Fig. 1 as a function of the consensus mean concentrations expressed in nanomoles per liter (see detailed results in the Data Supplement S1 that accompanies the online version of this article at clinchem.org/content/vol64/issue10). As Fig. 1 shows, modest agreement between methods can be observed. Between-method CV, calculated across the 25 frozen CSs, was 14.1%. No correlation between apob-100 concentration and between-method CV was observed (see the online Data Supplement S2). Supplemental data collected on HDL-P concentration measurements by ES- DMA and NMR highlight poor agreement between these 2 methods (see the online Data Supplement S3). Results obtained with the 4 different LC-MS/MS methods for apoa-i also demonstrated poor comparability to their respective consensus mean values (see the online Data Supplement S4 and S5). Methods precision and mean relative difference with the apob-100 IN DCM concentrations are shown in Fig. 2. For each method, the mean relative difference with the apob-100 IN DCM concentration is presented on the abscissa, and the mean CV of concentration measurements is presented on the ordinate. Method precision ranged from 0.9% for IN and VAP up to 8.7% for ES-DMA-1. The mean relative difference with the apob-100 IN DCM ranged from 27.5% for NMR LP3 and 20.6% for LC-MS/MS-1A up to 11.2% for ES-DMA-2. The mean relative differences of NMR methods against the IN DCM seemed to depend on the software used for data posttreatment, as comparability between NMR and IN was 27.5% for LP3 software vs 3.7% for LP4 software. Similarly, concentrations measured with the 2 ES-DMA methods were not in good agreement, with an 11.2% mean relative difference for ES-DMA-2 and a 0.9% mean relative difference for ES-DMA-1. The 4 different LC- MS/MS methods did not provide comparable results either (see detailed results in the online Data Supplement S6 and S7). LC-MS/MS methods 2A and 2B, the results of which are traceable to the IN DCM through calibration with the WHO/IFCC SP3 08 standard, compared well with the IN DCM with 1.7% and 2.7% mean relative differences, respectively. On the 4 Clinical Chemistry 64:10 (2018)

5 In Search of a Candidate RMP for apob and non-hdl-p Fig. 1. Comparisons between non-hdl-p and apob-100 concentrations measured by IN, LC-MS/MS, NMR, ES-DMA, and VAP in 25 frozen CSs against the consensus mean concentrations expressed in nanomoles per liter. Each point corresponds to the mean of 3 replicates performed on the same day for each frozen CS except for NMR, for which each point is a mean value of triplicates performed in 2 laboratories (NIH and LabCorp) using the same analyzers and software. The solid line corresponds to the identity line. contrary, LC-MS/MS methods 1A and 1B, calibrated with peptide standards of SI-traceable concentrations, resulted in large negative mean relative differences with the apob-100 IN DCM (Fig. 2). For some methods, comparability varied substantially from 1 sample to another. This was especially the case for the LC- MS/MS-1B method, for which a 13.4% CV across the 25 CSs could be calculated from the mean relative differences with apob-100 IN, and for ES-DMA-2, with a 10% CV of the mean relative differences with IN (Fig. 2). In complement to Fig. 2, Table 2 summarizes the correlations obtained between non-hdl-p, or apob- 100, measured with each method, and apob-100 concentrations measured by the IN DCM in the 25 frozen CSs (see also the online Data Supplement S6 and S7). The regression slopes between each method and IN ranged from (95% CI, ) to (95% CI, ). Slopes were not significantly different from unity, except for LC-MS/MS-2A and -1A, VAP, NMR LP3, and ES-DMA-1, and most intercepts were significantly different from zero (Table 2; see also the online Data Supplement S6 and S7). Correlation coefficients ranged from for LC-MS/MS-1B to for LC-MS/MS-2B. EFFECT OF FREEZING AND THAWING The relative mean difference between results obtained for the fresh and frozen CSs was 0.1% for IN, 4.2% for LC-MS/MS-2B, 1.4% for VAP, 2.3% for NMR LP3, 1.8% for NMR LP4, and 5.0% for ES-DMA-1 (Table 3). Results from paired t-tests indicated that the effect of FT was statistically significant for LC-MS/MS- 2B, VAP, NMR LP3, and NMR LP4 (P 0.001) but not for IN (P ) or ES-DMA-1 (P ). For ES-DMA-1, the effect of freezing varied widely from sample to sample and ranged from 14.7% to 31.7%. Methods comparability was also evaluated using the 25 fresh CSs, and results obtained were comparable with those obtained using frozen materials (see the online Data Supplement S8). Clinical Chemistry 64:10 (2018) 5

6 Fig. 2. Comparability of ALT methods as a function of the mean relative standard deviation and mean relative difference (and associated SD) to the IN DCM for apob-100 quantification in 25 frozen CSs. Dots represent methods that are traceable to the WHO/IFCC SP3 08 RM and to the IN DCM. Diamonds correspond to methods that are either traceable to another source or to the SI. Discussion The first aim of this study was to assess the current state of the art in comparability of non-hdl-p and apob-100 concentrations measured with the major ALT methods available. Results revealed that between-method comparability, evaluated as the between-method CV on a set of 25 frozen CSs, was 14.1%. This poor comparability could be the result of 3 major differences between these methods: (a) the different measurands targeted, (b) the fact that these Table 2. Linear regression comparisons between apob-100 concentrations measured by IN and non-hdl-p or apob-100 concentrations measured by LC-MS/MS, VAP, ES-DMA, and NMR. x Axis y Axis Measurand Method Measurand Method Slope (95% CI) Intercept (95% CI) r 2 apob-100 IN apob-100 LC-MS/MS 1A ( ) ( 6.10 to ) apob-100 IN apob-100 LC-MS/MS 1B ( ) ( to ) apob-100 IN apob-100 LC-MS/MS 2A ( ) ( ) apob-100 IN apob-100 LC-MS/MS 2B ( ) ( to ) apob-100 IN apob-100 VAP ( ) ( ) apob-100 IN non-hdl-p ES-DMA ( ) ( ) apob-100 IN non-hdl-p ES-DMA ( ) ( to ) apob-100 IN non-hdl-p NMR LP ( ) ( to 11.75) apob-100 IN non-hdl-p NMR LP ( ) ( to 27.42) Clinical Chemistry 64:10 (2018)

7 In Search of a Candidate RMP for apob and non-hdl-p Table 3. Study of the effect of FT CSs on apob-100 and non-hdl-p concentrations measured by IN, LC-MS/MS, VAP, NMR, and ES-DMA. a apob-100 non-hdl-p Samples IN, % LC-MS/MS-2B, % VAP, % NMR LP3, % NMR LP4, % ES-DMA-1, % CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS CS Mean difference SD P value FT effect E E E E a The difference between mean concentrations measured in the 25 fresh and frozen CSs is shown for each method and for each CS. Mean differences for each method and associated SDs were additionally calculated. A paired t-test was performed to evaluate if the FT effect was statistically significant, and the corresponding P value was calculated. A P value <0.05 implies that the FT effect is statistically significant. methods rely on highly different physical principles, and (c) the different calibration strategies chosen (12, 21). Indeed, ALT assays selectively detect and quantify lipoproteins depending on different characteristics of lipoprotein particles (21). NMR measures the intensity of the resonance signal of terminal methyl groups of lipids contained in lipoproteins and deconvolutes the obtained spectra to determine lipoprotein particle concentrations (29). ES-DMA selects lipoproteins according to their electrical mobility diameter and directly counts them by laser diffraction (27, 28). ApoB-100 quantification by LC-MS/MS relies on the quantification of characteristic peptides released after enzymatic proteolysis (31), whereas apolipoprotein quantification by immunoassays, such as IN, involves antibodies that target specific epitopes of apolipoproteins (25). Finally, VAP measures cholesterol concentrations after separating lipoproteins as a function of their sedimentation rate by ultracentrifugation in a density gradient (35). Thus, ALT methods target different components of lipoprotein particles with different specificities, which could explain, in part, the comparability issues observed. However, the poor comparability observed could also be the result of method-specific inaccuracies and Clinical Chemistry 64:10 (2018) 7

8 analytical errors. Indeed, ALT assays rely on vastly different analytical procedures, and preanalytical and postanalytical sources of error also are different. As discussed by Clouet-Foraison et al. in a recent review on the subject, each method suffers from specific limitations that could affect its accuracy and possibly the agreement with other methods. However, because it was the intent of this study to assess comparability in the current state of the art, the methods involved were run with protocols routinely used to measure patient samples in day-to-day clinical practice and, thus, were not fully optimized to fulfill the requirements of higher order RMPs. Therefore, results of this study need to be interpreted with an understanding that precision and accuracy could possibly be further improved. This last point finally raises the third potential explanation of the poor comparability observed, i.e., the lack of standardization of ALT methods. Indeed, different calibration strategies were used depending on the methods and laboratories, and their impact on comparability was confirmed. For instance, ES-DMA-1, which involved no calibration, provided significantly different results from ES-DMA-2, which was calibrated with the WHO/IFCC SP3 08. However, in this specific case, the high imprecision of ES-DMA methods (CV 8% for both procedures) makes it difficult to draw conclusions. The important role of the calibration strategy was also evidenced for the different LC-MS/MS methods. Indeed, LC-MS/MS methods 2A and 2B were both traceable to the WHO/IFCC SP3 08 and compared well with IN. On the contrary, LC-MS/MS methods 1A and 1B, calibrated with synthetic peptides according to isotope dilution mass spectrometry principles (37), resulted in large relative differences to IN. However, because peptide calibrators were value-assigned by amino acid analysis and isotope dilution mass spectrometry, results obtained with methods 1A and 1B are traceable to the SI, and not to IN, explaining the differences observed. As discussed in the introduction, the preferred means for improving comparability consists of standardizing measurements by establishing result traceability to the SI through an unbroken metrological chain of higher order calibrators and methods (24). In the context of ALT, no higher order reference measurement system exists, and IN has been considered as the DCM for apob- 100 quantification since the WHO/IFCC initiatives to harmonize apob-100 measurements with the production of the WHO/IFCC SP3 08 (25). However, although common calibration of routine apob-100 immunoassays with this RM did result in a great improvement of the agreement between the different assays, it was not originally designed to standardize ALT assays, and commutability of the WHO/IFCC SP3 08 material has never been evaluated to our knowledge. Most importantly, methods calibrated with this material cannot claim full traceability to the SI because the target value-assigned to the WHO/IFCC SP3 08 was obtained by calibrating IN with a purified LDL preparation that was itself valueassigned with the Lowry procedure (25). Therefore, IN does not provide results with SI traceability and, thus, cannot be considered as a potential higher order RMP for the standardization of ALT methods. Indeed, 1 of the first requirements of a higher order RMP for standardization is that results be traceable to the SI. However, this study reveals that most ALT methods do not yet meet this requirement. For NMR, SI traceability of non-hdl-p concentrations has not yet been achieved, and this study demonstrates that results seem to depend on proprietary postanalytical procedures. For VAP, although it presents the advantage of separating lipoproteins according to their sedimentation rate, i.e., the principle according to which lipoprotein classes were initially defined, result traceability to the SI has not been established either. As far as ES-DMA is concerned, it has been demonstrated that SI traceability could potentially be established. However, technical limitations remain, the first being insufficient precision for an RMP (28).In addition, the large variability of the relative differences with IN among the 25 CSs suggests that this method is sensitive to matrix effects, which also compromises its suitability as candidate RMP. Finally, LC-MS/MS appears to be the only method for which results SI traceability could be achieved through the choice of a calibration strategy involving high purity peptide standards, value-assigned by amino acid analysis and isotope dilution mass spectrometry, as demonstrated with LC- MS/MS methods 1A and 1B. However, traceability is not the only requirement of a higher order RMP, as such a method also must demonstrate a high degree of specificity for the defined measurand, robustness, and precision. In terms of specificity, it is well established that LC-MS/MS is a highly specific method. In terms of precision, Westgard et al. suggested a 3.5% goal for imprecision and an 11.6% total allowable error for apob-100 routine measurements based on between-patient variability and clinical needs (38). Given the role of the RMP in the metrological chain, it is well accepted that its analytical performance should be between 2 and 3 times better than that of routine methods. Results of this study reveal that LC-MS/MS methods providing SItraceable results do not yet meet these requirements, with 7.0% and 5.9% mean CVs, respectively. In addition to precision and robustness, an RMP also must demonstrate appropriate accuracy. However, accuracy can be evaluated only by comparison with the true value or through an interlaboratory comparison between higher order RMPs. For ALT methods, because no higher order RMP exists, accuracy cannot be evaluated, and the mean relative difference against the IN DCM cannot be considered as an estimate of the accuracy bias. Indeed, the relative differences to IN could be the results 8 Clinical Chemistry 64:10 (2018)

9 In Search of a Candidate RMP for apob and non-hdl-p of multiple independent parameters, for example, different specificities of the methods, inaccuracy of either IN or the method evaluated, or specific characteristics of the calibrator that would have resulted in its noncommutability for some methods but not others. However, although its value assignment may be disputable, the WHO/SP3 08 is still the only available anchor for apob-100 harmonization, and IN remains the DCM for apob-100 concentration measurements. Comparing ALT methods with the IN DCM will not provide information on accuracy; nevertheless, it will prove the amplitude of the potential drift in value that could occur if the traceability chain was modified from the IN DCM to the new candidate RMP. From that perspective, Fig. 2 shows that implementing a new traceability chain based on an LC-MS/MS method calibrated with high purity peptide standards will likely result in a large negative drift of apob-100 IN values in clinical practice, potentially implying defining new clinical thresholds and decision limits owing to recalibration of routine assays. However, this result needs to be carefully considered because accuracy of the LC-MS/MS methods 1A and 1B cannot be assessed. Although extensive work has been done over the past decade to establish reproducible, precise, and accurate LC-MS/MS assays with SI-traceable results, methods involving peptide standards still suffer from some limitations (37). Indeed, the accuracy of LC- MS/MS methods based on the bottom-up approach mainly relies on the complete digestion of the target protein and on an accurate value assignment of the peptide calibrators, which can be technically challenging in the case of long and/or modifiable peptides. The major limitation associated with the use of synthetic peptide calibrators instead of full-length protein calibrators or demonstrated equivalents is that they do not allow full control of the analytical work flow and particularly of the enzymatic digestion (37). Indeed, in such procedures, endogenous proteins undergo digestion whereas the peptide internal standards do not. This difference in preanalytical steps may result in variable balance between proteotypic peptides and peptide internal standards, thereby resulting in both increased imprecision and potential inaccuracy. Therefore, accuracy of LC-MS/MS methods should not be taken for granted. Although LC-MS/MS appears to be the most suitable candidate RMP to standardize and establish SI traceability of apob-100 and non- HDL-P measurements, results of this study highlight that the agreement between different LC-MS/MS methods still needs to be improved, as well as method robustness, before it can be implemented as the new RMP to standardize apob-100 immunoassays. Nevertheless, the good linearity and correlations observed between IN and most LC-MS/MS methods suggest that traceability could be successfully shifted from IN to LC-MS/MS (see the online Data Supplement S6). The fact that smaller relative differences to IN were observed for methods whose results were traceable to the WHO/IFCC SP3-08, whereas larger relative differences were observed for methods involving other calibrators (Fig. 2), indicates that standardization by use of a common calibration will likely improve methods comparability. A potential limitation of this study is that comparability was assessed on frozen CSs. Indeed, freezing may alter sample commutability and introduce matrix-related biases that could lead to erroneous conclusions on comparability (39). Therefore, the impact of freezing and thawing serum samples on apob-100 and non-hdl-p concentrations was investigated to confirm our observations. Although the effect of FT was found statistically significant for LC-MS/MS, VAP, and NMR methods, it is not meaningful from a clinical perspective because the relative mean differences between results obtained for the fresh and frozen CSs were only 4.2% for LC-MS/MS- 2B, 1.4% for VAP, 2.3% for NMR LP3, and 1.8% for NMR LP4. It should be noted that the effect of FT was not found statistically significant for ES-DMA-1, although the mean difference between frozen and fresh samples was the largest for that method (5.0%), most likely a result of the large imprecision of the method and important between-sample variability of the difference between fresh and frozen CSs. Although this study was performed over a short period, these results suggest that frozen human serum pools could be used as secondary RMs to disseminate SI traceability once it has been achieved. However, the mid- and long-term stability of these frozen pools needs to be carefully assessed, as well as their commutability for the different ALT methods. Conclusion This study demonstrates that, at present, ALT methods do not yet provide equivalent results, which prevents generalization of findings from clinical studies and stresses the urgent need to standardize these assays. The good agreement observed between methods whose results are traceable to the same calibrator is encouraging and indicates that calibration standardization could improve comparability. However, consensus needs to be reached concerning the choice of the most suitable candidate RMP. With its high specificity, multiplexing capability, and ability to provide results that are traceable to the SI under specified conditions, i.e., using peptides or proteins of well-characterized purity as primary calibrators, LC-MS/MS appears to be a suitable candidate primary RMP to establish an absolute accuracy-based reference system and standardize apolipoprotein measurements. However, further work remains to be done to improve robustness and comparability between different LC- MS/MS procedures and to reduce associated uncertainties. This work will be pursued as part of the IFCC work- Clinical Chemistry 64:10 (2018) 9

10 ing group on apolipoprotein standardization by mass spectrometry (40). It should be noted that, contrary to ES-DMA, VAP, and NMR, LC-MS/MS does not provide information on lipoprotein subclasses such as LDL-P or HDL-P, and that standardization of these advanced parameters, although needed, will be difficult to achieve in the current state of the art. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: F. Gaie-Levrel, LNE; A.N. Hoofnagle, Clinical Chemistry, AACC; M.P. Caulfield, Quest Diagnostics; J.D. References Otvos, LabCorp; K.R. Kulkarni, VAP Diagnostics Laboratory Inc., Birmingham, AL. Consultant or Advisory Role: R.M. Krauss, Quest Diagnostics. Stock Ownership: M.P. Caulfield, Quest Diagnostics. Honoraria: M.P. Caulfield, Quest Diagnostics. Research Funding: This work was carried out in the framework of the Joint Research Project SIB54 Bio-SITrace, co-funded by the European Metrology Research Program (EMRP). The EMRP is jointly funded by the EMRP-participating countries within the EURAMET and the European Union. A.N. Hoofnagle, funding to institution from NIDDK/NIH; M.P. Caulfield, Quest Diagnostics. Expert Testimony: None declared. Patents: M.P. Caulfield, ; K.R. Kulkarni, Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or final approval of manuscript. 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