Price of High-Throughput 25-Hydroxyvitamin D Immunoassays: Frequency of Inaccurate Results

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1 Price of High-Throughput 25-Hydroxyvitamin D Immunoassays: Frequency of Inaccurate Results Nicole V. Tolan, 1, * Edward J. Yoon, 1, Ashley R. Brady, 1 and Gary L. Horowitz 1, Background: With a 10-year sustained increase in 25-hydroxyvitamin D [25(OH)D] testing, laboratories have swapped their LC-MS/MS methods for high-throughput automated immunoassays. Although it is generally wellknown that immunoassays have poor recoveries for 25-hydroxyvitamin D2 [25(OH)D2], the frequency and extent to which this impacts total 25(OH)D have not been previously demonstrated. We evaluated 3 automated immunoassays against the first FDA-cleared CDC/NIST-traceable LC-MS/MS method. Methods: Method comparison was performed for the Siemens ADVIA Centaur, Roche Elecsys Cobas, and Abbott Architect 25(OH)D immunoassay methods in real patient samples (n = 105). We calculated the mean bias in samples containing >20 ng/ml 25(OH)D2 and estimated the percent 25(OH)D2 cross-reactivities. We determined the prevalence of appreciable concentrations of 25(OH)D2 in our patient population through random sampling (n = 120) and projected the frequency of inaccurate 25(OH)D immunoassay results. Results: Linear regression for 25(OH)D was y = 1.09x 4.44 (Centaur), y = (Cobas), and y = 0.83x 0.48 (Architect). The mean biases of 25(OH)D concentrations were 5.6 (11.0) ng/ml (Centaur), 17.5 (7.2) ng/ml (Cobas), and 20.3 (9.8) ng/ml (Architect) in samples containing >20 ng/ml 25(OH)D2. The observed percent cross-reactivities for 25(OH)D2 were 115% (Centaur), 52% (Cobas), and 44% (Architect). We estimate that 8% of our population has >20 ng/ml 25(OH)D2, thereby compromising the accuracy of 25(OH)D results in >3000 samples annually. Conclusions: We demonstrate that immunoassay manufacturer package inserts indicate much better recoveries of 25(OH)D2 than what is observed in unadulterated real patient samples. We estimate the frequency of inaccurate total 25(OH)D determination by these immunoassay methods to be largely dependent on the concentration of 25(OH)D2 in each sample. IMPACT STATEMENT Considering variable immunoassay performance over time, clinical laboratorians, endocrinologists, and primary care physicians will benefit from reviewing our study, focusing on the current limitations of commercial 25-hydroxyvitamin D [25(OH)D] immunoassay methods. We demonstrate the prevalence of inaccurate 25(OH)D determination, as a result of poor recoveries of 25-hydroxyvitamin D2 [25(OH)D2], and present a straightforward method for estimating the percent cross-reactivity of immunoassays. Given the relatively few standard reference materials and proficiency testing materials containing sufficient concentrations of 25(OH)D2, our study highlights the requirement to thoroughly evaluate the performance of commercial immunoassay methods and the need to communicate their limitations to practicing clinicians. May : JALM 1 Copyright 2017 by American Association for Clinical Chemistry.

2 Price of High-Throughput 25(OH)D Immunoassays Vitamin D was first demonstrated in the 1980s to be related to colorectal cancer (1). Since then, studies have demonstrated the connection between decreased 25-hydroxyvitamin D [25(OH)D] 2 and increased risk of cardiovascular disease, stroke, cancer, fractures, and mortality (see supplemental reading list available at clinchem.org/content/vol61/issue3) (2). A correlation between increased 25(OH)D concentrations and decreased risk for autoimmune diseases, hypertension, heart disease and stroke, upper respiratory tract infections, wheezing disorders, asthma, and falling has been observed (3). As a result, the 2011 Endocrine Society Guidelines recommended that 25(OH)D concentrations from 20 ng/ml (50 nmol/l) to <30 ng/ml (75 nmol/l) be considered insufficient and levels <20 ng/ml represent deficiency (4). Considering only the effect on bone health, the Institute of Medicine maintains a 20 ng/ml threshold for vitamin D sufficiency (5). Very few epidemiologic studies have related deficiency with a risk for nonskeletal diseases. Several metaanalyses have also failed to show that low 25(OH)D concentrations are associated with risk for any of the aforementioned nonskeletal chronic conditions, with the possible exception of fractures. Despite these findings, and that no professional societies have recommended generalized screening (3), testing has remained high. Much of this is attributed to enthusiastic reporting of the potential health benefits of vitamin D in the lay literature (6). Like many laboratories, we have seen the volume of 25(OH)D testing increase during the past decade and has since remained high, at nearly 800 tests/week. Introduction of more rigorous and traceable LC- MS/MS methods into routine clinical laboratories has provided increased accuracy for the determination of 25(OH)D (7). These methods incorporate standard reference materials (SRMs), protein precipitation and extraction from the vitamin D- binding protein (VDBP), chromatographic separation, and correction for procedural losses. However, because of such high testing volumes, more complicated laboratory-developed LC-MS/MS methods have been increasingly replaced with high-throughput automated immunoassays in recent years. For individuals with clinical indications, it is necessary to accurately determine total 25(OH)D concentrations to assess overall vitamin D stores and ensure patients are not at risk of hypervitaminosis D toxicity (8). Both vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol) are available as overthe-counter supplements, but until recently, the only form of high-dose (e.g., IU) prescription vitamin D in the US has been vitamin D2. Current automated immunoassays do not distinguish the 2 metabolites, 25-hydroxyvitamin D3 [25(OH)D3] and 25-hydroxyvitamin D2 [25(OH)D2]. Nevertheless, as long as these assays accurately detect both forms, the total 25(OH)D concentrations they provide are adequate for clinical needs. Yet, it is generally well-known that immunoassays have poor recoveries of the vitamin D2 metabolite. Previous studies have evaluated the accuracy of 25(OH)D2 determination by various immunoassays (9 11). However, we perform an evaluation 1 Department of Pathology and Laboratory Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA. Current affiliation: SCIEX, Clinical Diagnostics and Department of Pathology and Laboratory Medicine, Tufts Medical Center, Boston, MA. Current affiliation: New York Blood Center, New York, NY. Current affiliation: Department of Pathology and Laboratory Medicine, Tufts Medical Center, Boston, MA. *Address correspondence to this author at: SCIEX, Clinical Diagnostics, 500 Old Connecticut Path, Framingham, MA Fax: ; nicole.tolan@sciex.com. DOI: /jalm American Association for Clinical Chemistry 2 Nonstandard abbreviations: 25(OH)D, 25-hydroxyvitamin D; SRM, standard reference material; VDBP, vitamin D-binding protein; 25(OH)D3, 25-hydroxyvitamin D3; 25(OH)D2, 25-hydroxyvitamin D2; FDA, Food and Drug Administration; 3-epi-25(OH)D3, 3-epi-25-hydroxyvitamin D3; 3-epi-25(OH)D2, 3-epi-25-hydroxyvitamin D2; PT, proficiency testing; BIDMC, Beth Israel Deaconess Medical Center; IS, internal standard; CLSI, Clinical and Laboratory Standards Institute; IQR, interquartile range. 2 JALM :06 May 2018

3 Price of High-Throughput 25(OH)D Immunoassays ARTICLE against the first FDA-cleared LC-MS/MS 25(OH)D method. This method demonstrates ±5% mean bias to the CDC and University of Ghent Reference Method and an overall imprecision of <10% over the concentration range of 4.1 to 325 ng/ml ( nmol/l); it also received certification in the CDC Vitamin D Standardization Program, effective April 29, 2015 (12). In addition, 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3] and 3-epi-25-hydroxyvitamin D2 [3-epi-25(OH)D2] are chromatographically separated, eliminating their interference in 25(OH)D determinations. The disagreement among immunoassays has been attributed, in part, to the ineffective liberation of 25(OH)D from the VDBP (13, 14). It may also be necessary to consider the various VDBP phenotypes in the patient population, because of their varying binding affinities. However, their accurate quantification is challenging (15). Regardless, without complete irreversible disassociation from the binding protein, immunoassays are vulnerable to inaccuracies. In contrast, LC-MS/MS methods incorporating protein precipitation and normalization for extraction efficiency are more likely to recover accurate concentrations. However, their accuracy can be affected by the presence of the 3-epimers (16), the biologically inactive forms of vitamin D found in pediatric and adult populations (17). More recently, rapid LC-MS/MS methods have been developed to separate out these interferences (18). Since the availability of the 2009 Ghent University and CDC reference measurement procedures incorporating SRMs, performance of 25(OH)D assays has improved on accuracy-based proficiency testing (PT) surveys, such as the Accuracy-Based Vitamin D and Vitamin D External Quality Assessment Scheme (14). However, many of these SRMs and PT materials do not contain sufficient concentrations of 25(OH)D2 to fully test the accuracy of measuring this form of supplementation, resulting in a void in the challenge for total 25(OH)D. That is, the accuracy of methods for determining total 25(OH)D will be falsely reassuring if the PT material is largely composed of 25(OH)D3. Here, we assess the accuracy of 3 automated immunoassays for 25(OH)D in real patient samples using the first FDA-cleared CDC/NISTtraceable LC-MS/MS method for 25(OH)D. We calculated the observed percent cross-reactivities for 25(OH)D2, as compared with that indicated in the manufacturers' package inserts. Through random sampling, we estimated the prevalence of appreciable 25(OH)D2 concentrations in our patient population and determined the resulting frequency of inaccurate 25(OH)D concentrations. To the best of our knowledge, the prevalence of inaccurate total 25(OH)D determination because of poor recoveries of 25(OH)D2 by immunoassays has not previously been reported. METHODS These investigations were performed under quality assurance activities, which do not constitute research by the Institutional Review Board at Beth Israel Deaconess Medical Center (BIDMC), Boston, MA. The main hospital consists of a major urban academic institution with robust endocrinology and general primary care services. The laboratory receives 150 to 200 samples/weekday for 25(OH)D testing, the majority of which come from outpatients seen at hospital clinics and affiliated physicians' offices. In addition to our participation as 1 of the 3 clinical trial sites in the FDA application, we evaluated the accuracy of 3 automated commercial immunoassay methods for total 25(OH)D: ADVIA Centaur (Siemens), Elecsys Cobas (Roche), and Architect (Abbott Diagnostics). Precision of each immunoassay method was evaluated using quality control material (UTAK, UTAK Laboratories, Valencia, CA) in which 2 levels were run each day to total n=20 measurements. The %CV was 3.8% to 5.3% ( ng/ml) for Centaur, 3.8% to 7.0% ( May : JALM 3

4 Price of High-Throughput 25(OH)D Immunoassays ng/ml) for Cobas, and 4.0% to 4.8% ( ng/ml) for Architect. The FDA-cleared CDC/NIST-traceable LC-MS/MS method for 25(OH)D on the Topaz LC-MS/MS System (SCIEX, Framingham, MA) was used as the reference method. This system comprises a triple quadrupole mass spectrometer and a heated atmospheric pressure ionization source with integrated autosampler and liquid chromatography system using online sample cleanup. In this method, patient serum samples undergo automated protein precipitation using the MICROLAB STARlet IVD automated liquid handler (Hamilton, Reno, NV). Solutions of 25(OH)D3 and 25(OH)D2 calibrators, internal standards (IS), PBS, precipitation reagent, mobile phases, and trap and analytical columns used in this method are commercially available in the Vitamin D 200M assay (SCIEX). Briefly, 200 μl of a proprietary mixture of IS solution [25(OH)D3-d6], 25 μl of ZnSO 4, and 100 μlof each patient sample were pipetted into a 96-well filter plate. After 10-min agitation to liberate 25(OH)D from VDBP, the plate is centrifuged at 1500g for 5 min into a 96-well collection plate. This method incorporates duplicate injections of a zero calibrator, 6 standards for 25(OH)D3 ( ng/ml) and 25(OH)D2 ( ng/ml), and 3 levels of quality control for 25(OH)D3 (15.4, 35.4, 96.9 ng/ml) and 25(OH)D2 (15.9, 35.7, 88.8 ng/ml) in each run. For LC-MS/MS analysis, 40 μl is injected into a 50-μL sample loop before gradient liquid chromatography separation for 6.2 min/injection incorporating a Vitamin D 200 Series trap and analytical columns (SCIEX) in positive ion mode, providing chromatographic resolution of 25(OH)D2, 25(OH)D3, 3-epi-25(OH)D3, and 3-epi-25(OH)D2. The concentrations of 25(OH)D3 and 25(OH)D2 in patient samples were determined by monitoring 2 precursor/product pairs by multiple reaction monitoring using ClearCore MD 1.1 software (SCIEX). Precision was determined following the multisite evaluation study (including BIDMC) outlined in the Clinical and Laboratory Standards Institute (CLSI) guideline EP05-A3 (19). At each of 3 sites, 7 samples (approximately 4 90 ng/ml) were assayed as 5 replicate extractions each day, over 5 days. The intraassay and interassay precision %CV ranged from 5.6% to 7.3% ( ng/ml) for 25(OH)D3, 5.4% to 6.3% ( ng/ml) for 25(OH)D2, and 3.8% to 5.4% ( ng/ml) for total 25(OH)D. Method comparison was performed, following CLSI guideline EP09-A3 (19) against the CDC and Ghent University Reference Method (20). Passing Bablok linear regression for 120 real patient samples is described by the line y = 1.011x 0.503, R 2 = Additionally, at BIDMC, 4 NIST and 10 CDC SRMs (n = 14) were analyzed, and the median bias (±SD) was 0.34 (0.58) ng/ml for 25(OH)D2, 2.23 (1.66) ng/ml for 25(OH)D3, and 2.57 (1.69) ng/ml for total 25(OH)D. The ability to confirm the accuracy of the LC-MS/MS reference method for 25(OH)D2 to that of the gold standard CDC/Ghent University is of particular importance for our study. Given the limited concentration of 25(OH)D2 in SRMs, we also evaluated the method accuracy by 2 NIST, 1 CDC, and 11 Vitamin D Standardization Program survey materials [target 25(OH)D2 concentrations ranging from 0.81 to ng/ml]. The best-fit line was determined to be y = 0.986x , R 2 = (n = 14), an absolute mean bias of 0.19 (±0.79) ng/ml, and a corresponding bias of 2% (±0.17) for 25(OH)D2. Linearity studies were performed following CLSI guideline EP06-A (21) using 3 different reagent lots. Two pools (high and low) were used to prepare 9 linearity samples, with equally spaced concentrations, according to the CLSI guidelines. Studies were also performed to establish the lower limit of the measuring interval following the CLSI guidelines EP17-A2 (22) and C62-A (23), in which 6 SRMs were run in replicates of 5 over 3 days and across 2 different lots of reagents. The reportable range was determined from the linearity studies as 2.2 to 160 ng/ml for 25(OH)D3, 1.9 to 165 ng/ml for 25(OH)D2, and 4.1 to 325 ng/ml for total 25(OH)D. 4 JALM :06 May 2018

5 Price of High-Throughput 25(OH)D Immunoassays ARTICLE Carryover testing was performed following the EP Evaluator method (Data Innovations, Burlington, VT) in which the carryover was defined as the amount of increased analyte concentration in low samples (1.6 ng/ml) following replicate injection of a high-concentration sample (160 ng/ml). The acceptability criteria for carryover was defined as <3 SD of the low sample run in replicate without carryover. Studies were performed in replicates of 5 over 3 different reagent lots, and carryover did not exceed the acceptability criteria. Method comparison and observed cross-reactivity Fig. 1. Chromatogram of LC-MS/MS assay demonstrating separation of the 3-epimer metabolites of 25(OH)D3 and 25(OH)D2, 3-epi-25(OH)D3 and 3-epi-25(OH)D2, respectively. Analytical specificity testing was performed in which an interference was defined as any compound demonstrating >10% change in the concentration or suppression of signal intensity (cps) by >30%. In addition, following CLSI guideline C62-A (23), interferences at the upper limit of the measuring interval were required to be <5% of the IS, and interference from the IS could not contribute >20% of the test analyte lower limit of the measuring interval response. Following the study design outlined in CLSI guideline EP07-A2 (24), 79 potential interferents, including vitamin D analogs and metabolites, endogenous substances, and potential isobaric compounds or fragments, were tested, and no interferents were identified. Most notably, interference from 3-epi-25(OH)D3 and 3-epi- 25(OH)D2 is prevented through chromatographic separation (Fig. 1). Further, cross-talk between analytes and IS met the acceptability criteria and was demonstrated to be 3.4% and 0% at the lower limit of the measuring interval and 0.8% and 0.6% at the upper limit of the measuring interval for 25(OH)D3 and 25(OH)D2, respectively. Real patient samples (n = 105) received for clinical testing by the Centaur method in our laboratory were also tested by the Cobas, Architect, and LC-MS/MS methods. Method comparison was performed, in which the concentration of total 25(OH)D determined by immunoassay was plotted as a function of total 25(OH)D determined by LC- MS/MS. The accuracy of each immunoassay was evaluated using the robust Theil Sen linear regression model (RStudio, Median-Based Linear Models version 0.12), which minimizes the impact of outliers. In addition, Bland Altman difference plots were constructed, and mean bias ± SD was determined. Following the grading criteria used for accuracybased PT surveys, CAP Accuracy-Based Vitamin D and Vitamin D External Quality Assessment Scheme, we determined the number of samples in which the concentration of total 25(OH)D fell >25% outside of the LC-MS/MS reference method concentration. We also used a second, slightly more stringent but clinically more reasonable, set of accuracy limits: ±5 ng/ml for 25(OH)D <20 ng/ml or ±20% for samples with 25(OH)D concentrations >20 ng/ml. We evaluated the accuracy of the immunoassays in specifically detecting 25(OH)D2 by plotting the difference of the immunoassay method for total 25(OH)D as a function of LC-MS/MS 25(OH)D2 concentration. The mean bias of the immunoassay May : JALM 5

6 Price of High-Throughput 25(OH)D Immunoassays Fig. 2. Distribution of total 25(OH)D concentrations obtained in the patient sample set (n = 105) by the LC-MS/MS reference method and 3 immunoassay methods. Tukey box and whisker plot demonstrates the IQR, median, and range of concentrations without exclusion of outliers. The total 25(OH)D concentrations determined by Cobas and Architect are statistically different from the LC-MS/MS reference method (P < 0.01 and P < 0.04, respectively). methods was calculated for samples containing appreciable concentrations of 25(OH)D2 (>20 ng/ ml). The observed percent cross-reactivity of each immunoassay method was estimated by subtracting the concentration of 25(OH)D3 determined by LC-MS/MS in each sample, assuming complete (100%) cross-reactivity with 25(OH)D3, and evaluating the accuracy of the residual 25(OH)D2 concentration against the LC-MS/MS 25(OH)D2. Estimating prevalence in our patient population Because it is not possible to predict which samples contain significant concentrations of 25(OH)D2 by the total 25(OH)D immunoassay result alone, we estimated the prevalence of inaccurate total 25(OH)D immunoassay results through random sampling. Using a random number generator, we selected 20 specimens across 5 days (n = 120) during a 1-month period to test by LC-MS/MS. Of these, any with 25(OH)D2 concentrations >5 ng/ml were also tested for total 25(OH)D concentrations using the Centaur, Cobas, and Architect methods. RESULTS The distribution of total 25(OH)D concentrations determined by each method in the patient sample set (n = 105) are illustrated in Fig. 2. As determined through unpaired t-tests, the results were determined to be significantly lower for the Cobas (P < 0.01) and Architect (P < 0.04) immunoassay methods but not for those measured by Centaur (P > 0.5) as compared with LC-MS/MS. The 6 JALM :06 May 2018

7 Price of High-Throughput 25(OH)D Immunoassays ARTICLE Fig. 3. Method comparisons of 25(OH)D determination by immunoassay vs LC-MS/MS. Theil Sen linear regression is shown as black solid line and ±5% (<20 ng/ml) or ±20% (>20 ng/ml) acceptability limits are shown as dashed gray lines against a solid gray line of identity. median (±SD), range, and 25th to 75th percentile interquartile ranges (IQRs) were calculated to be: LC-MS/MS median: 40.6 (15.9); range, 9.1 to 80.4; IQR, 29.0 to 51.2 Centaur median: 39.0 (19.5); range, 4.0 to 98.0; IQR, 26.0 to 51.0 Cobas median: 34.6 (15.0); range, 7.9 to 76.0; IQR, 21.6 to 46.2 Architect median: 30.4 (19.0); range, 7.6 to 97.0; IQR, 20.0 to 44.4 Plotting the total 25(OH)D concentration determined by each immunoassay method as a function of that determined by LC-MS/MS, Fig. 3 illustrates the accuracy of each method. The Theil Sen robust linear regression model resulted in the following best-fit lines: Centaur: y = 1.09x 4.44, R 2 = Cobas: y = 0.84x , R 2 = Architect: y = 0.83x 0.48, R 2 = Setting the accuracy threshold to ±25% of the LC- MS/MS total 25(OH)D, we determined that the number of unacceptable recoveries was 24% (25 of 105) for Centaur, 33% (35 of 105) for Cobas, and 50% (52 of 105) for Architect. Using a more stringent accuracy requirement of ±5 ng/ml [25(OH)D <20 ng/ ml] or 20% [25(OH)D >20 ng/ml], the number of unacceptable results increases to 29% (30 of 105) for Centaur, 38% (40 of 105) for Cobas, and 59% (62 of 105) for Architect. Overall, the mean bias (±SD) for total 25(OH)D determined by Centaur, Cobas, and Architect immunoassays was 0.7 (8.1) ng/ml, 5.6 (9.2) ng/ ml, and 5.0 (14.5) ng/ml, respectively (Fig. 4A). However, a proportional bias is observed when these differences in total 25(OH)D are plotted as a function of 25(OH)D2 concentration, as illustrated in Fig. 4B. Further, for samples containing >20 ng/ml 25(OH)D2, the absolute mean biases increase to 5.6 (11.0) ng/ml, 17.5 (7.2) ng/ml, and 20.3 (9.8) ng/ml, respectively. Assuming complete cross-reactivity of the immunoassay with 25(OH)D3, the residual concentration remaining after subtracting the concentration of 25(OH)D3 (as determined by LC-MS/MS) represents an estimate of the 25(OH)D2 recovered by each immunoassay. As illustrated in Fig. 5, these calculated concentrations of 25(OH)D2 are plotted against the 25(OH)D2 concentrations measured by LC-MS/MS for each assay, for specimens containing >20 ng/ml 25(OH)D2 (n = 24). The Theil Sen linear regression correlations are described by the best-fit lines (with 95% CIs): Centaur: y = 1.51x 14.00, R 2 = (m, ; y intercept, to 8.25) May : JALM 7

8 Price of High-Throughput 25(OH)D Immunoassays Fig. 4. Bland Altman difference plots shown as a function of (A) total 25(OH)D and (B) 25(OH)D2 as determined by LC-MS/MS. The mean bias is shown by dashed gray lines for (A) of all differences calculated against LC-MS/MS for total 25(OH)D and (B) 25(OH)D differences calculated in only those samples containing >20 ng/ml 25(OH)D2. Cobas: y = 0.55x 1.22, R 2 = (m, ; y intercept, 3.53 to 0.53) Architect: y = 0.42x 0.99, R 2 = (m, ; y intercept, 5.07 to 5.65) The percent cross-reactivities were calculated by dividing the estimated 25(OH)D2 concentration in each sample by the concentration of 25(OH)D2 determined by LC-MS/MS. We estimated the median percent cross-reactivities (25 75th IQR) to be 115% (85% 133%), 52% (49% 60%), and 44% (34% 59%) for each Centaur, Cobas, and Architect, respectively, as compared with the manufacturers' Fig. 5. Method comparison of the estimated 25(OH)D2 for each immunoassay method plotted against the concentration of 25(OH)D2 measured by the LC-MS/MS reference method. The immunoassay 25(OH)D2 concentrations are calculated by subtracting the concentration of 25(OH)D3 determined by LC-MS/MS in each specimen. Here, the Theil-Sen linear regression is shown as a black solid line. 8 JALM :06 May 2018

9 Price of High-Throughput 25(OH)D Immunoassays ARTICLE Fig. 6. Method comparison of the prevalence study samples (n = 120) in which only those with LC-MS/MS 25(OH)D2 concentrations >5 ng/ml are plotted (n = 22). Discrepant samples with sufficient total 25(OH)D ( 30 ng/ml) by the mass spectrometry method but are discordantly classified as insufficient/deficient (<30 mg/dl) by immunoassay are shown as solid points. package inserts stating the following cross-reactivities for 25(OH)D2: 104.5% (Centaur), 92% (Cobas), and 82% (Architect). Finally, through random sampling, we estimated the frequency of inaccurate total 25(OH)D determination by the immunoassay methods, given the bias resulting from poor recoveries of 25(OH)D2. Of the 120 patient samples collected at random over 5 days, 22 (18%) contained >5 ng/ml 25(OH)D2. As shown in Fig. 6, we performed a second method comparison plotting total 25(OH)D in these samples against the LC-MS/MS reference method. We determined that 2.5% (3 of 120) of Centaur and 6.7% (8 of 120) of Cobas and Architect randomly selected samples were clinically discordant, whereby specimens representing vitamin D sufficiency ( 30 ng/ml) by LC-MS/MS were classified as insufficiency/deficiency (<30 ng/ml) by immunoassay. We estimate that 8% (9 of 120) of our population have 25(OH)D2 levels >20 ng/ml and that approximately 3000 samples (8% of 150 samples/weekday, 50 weeks/year) could be inaccurate annually, and negatively impact patient treatment. DISCUSSION As assessed by the Vitamin D External Quality Assessment Scheme PT surveys, interlaboratory imprecision has decreased by >15% since the implementation of the 2009 reference measurement procedures incorporating SRMs (14). However, of the SRMs available through NIST and CDC, only 10 contain detectable 25(OH)D2, and of these, only a single SRM contains sufficient levels (NIST SRM 972a, level 3 = 13.3 ng/ml) to test the accuracy of 25(OH)D methods. The remaining SRMs contain 25(OH)D2 at concentrations ranging from 0 to 2.6 ng/ml and offer little to support the standardization of 25(OH)D2 quantification. Unless accuracy-based surveys include an appropriate number of samples from patients supplemented with 25(OH)D2, the discrepancies noted in this study will be missed, and participating laboratories may gain a false sense of security regarding the accuracy of their methods. We believe that the 25(OH)D2 cross-reactivities provided in the manufacturers' package inserts are incorrect and misleading for the immunoassays evaluated here. This could be a result of the way the cross-reactivity is calculated or the materials used in the determination of the recovery. In real patient samples, our method demonstrates that the mean bias (Fig. 4B) and estimated percent cross-reactivities are different from what is indicated in the manufacturers' package inserts. Similarly, results from previous studies may differ from ours because of the equation used to determine cross-reactivity (10 12). We attempt to provide a May : JALM 9

10 Price of High-Throughput 25(OH)D Immunoassays relatively straightforward method for estimating 25(OH)D2 and percent D2 cross-reactivity, and our study differs from previous reports in 2 important ways: 1. It does not rely on the regression analysis of immunoassay vs LC-MS/MS in 25(OH)D3-only samples, in which imprecision can confound the best-fit line. The error in estimated D3 concentration propagates into the estimate of D2 concentration, and ultimately the percent cross-reactivity. 2. Our approach includes only samples with appreciable concentrations of 25(OH)D2, as inclusion of low concentrations of 25(OH)D2 may generate large percent cross-reactivity errors and wide IQRs. It is not apparent that the discordances in total 25(OH)D are a function of 25(OH)D2 (Fig. 3) until the differences are plotted against the LC-MS/MS 25(OH)D2 concentrations (Fig. 4B). Because the inaccuracies are not dependent on the concentration of total 25(OH)D measured by immunoassay, laboratories using immunoassay methods cannot identify which samples require LC-MS/MS confirmation for accurate assessment of vitamin D. As such, it is important to determine the prescribing practices within the local and affiliated sites and the prevalence of high-dose vitamin D2 supplementation. If laboratories choose to use one of these immunoassays, we believe that ordering physicians must be made aware of the method limitations at the time they place the order, be given the opportunity to request a more accurate method, and/or a comment be appended, indicating that caution is necessary when interpreting results in patients receiving vitamin D2 supplementation. Our study demonstrated that within our patient population, 18% of samples had detectable 25(OH)D2 (>5 ng/ml) and 8% had levels >20 ng/ ml. Through random sampling, we demonstrate that >3000 samples could be impacted on an annual basis. Significant concentrations of 25(OH)D2 compromise the accuracy of 25(OH)D immunoassay determination to the extent of potential negative impact on patient treatment. For example, the 25(OH)D would appear to remain low in patients supplemented with IU of vitamin D2 and could raise questions about compliance and lead to unnecessary medical evaluation for potential malabsorption issues, as well as continuation of, or perhaps increased dose or duration of, vitamin D supplementation. CONCLUSIONS Our study demonstrates that although there has been great improvement in 25(OH)D assay standardization under the Vitamin D Standardization Program (8), issues of unequal cross-reactivity with 25(OH)D2 remain for some immunoassays. Failure to include an appropriate number of patients supplemented with 25(OH)D2 in evaluations will not effectively demonstrate method accuracy. Although we do not consider the differences between immunoassay and LC-MS/MS pose a severe patient safety risk, the immunoassay methods evaluated in this study appear to be performing differently from what one might expect based on the manufacturers' package inserts. We believe that manufacturers would benefit from reviewing their methods for calculating the degree of crossreactivity with 25(OH)D2. Further, the incorporation of appreciable concentrations of 25(OH)D2 in accuracy-based surveys on a regular basis would best test the accuracy of all methods JALM :06 May 2018

11 Price of High-Throughput 25(OH)D Immunoassays ARTICLE Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 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; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: SCIEX provided materials and reagents for this study. Expert Testimony: None declared. Patents: None declared. Role of Sponsor: The sponsor, SCIEX, (a) contributed final FDA-studies data to the manuscript and (b) reviewed and approved the final manuscript. Acknowledgments: The authors thank SCIEX for supporting this study by providing materials and reagents for the LC-MS/MS reference method. SCIEX has provided financial support to BIDMC for participating in data submissions to the FDA. REFERENCES 1. Garland CF, Garland FC. Do sunlight and vitamin D reduce the likelihood of colon cancer? International journal of epidemiology. 1980;9: Scott MG, Gronowski AM, Reid IR, Holick MF, Thadhani R, Phinney K. Vitamin D: the more we know, the less we know. Clin Chem 2015;61: Holick MF. The D-lemma: to screen or not to screen for 25-hydroxyvitamin D concentrations. Clin Chem 2010;56: Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2011; 96: Institute of Medicine Committee to Review Dietary Reference Intakes for Vitamin D, Calcium. The National Academies Collection: reports funded by National Institutes of Health. In: Ross AC, Taylor CL, Yaktine AL, Del Valle HB, editors. Dietary reference intakes for calcium and vitamin D. Washington (DC): National Academies Press (US) National Academy of Sciences; Kolata G. D is for Dilemma. Why are so many people popping vitamin D? The New York Times April National Institutes of Health, Office of Dietary Supplements. Vitamin D initiative. Research/VitaminD.aspx (Accessed February 2016). 8. Carter GD, Phinney KW. Assessing vitamin D status: time for a rethink? Clin Chem 2014;60: Shu I, Pina-Oviedo S, Quiroga-Garza G, Meng QH, Wang P. Influence of vitamin D2 percentage on accuracy of 4 commercial total 25-hydroxyvitamin D assays. Clin Chem 2013;59: Le Goff C, Peeters S, Crine Y, Lukas P, Souberbielle JC, Cavalier E. Evaluation of the cross-reactivity of 25- hydroxyvitamin D2 on seven commercial immunoassays on native samples. Clin Chem Lab Med 2012;50: Li L, Zeng Q, Yuan J, Xie Z. Performance evaluation of two immunoassays for 25-hydroxyvitamin D. J Clin Biochem Nutr 2016;58: Centers for Disease Control and Prevention (CDC) Vitamin D Standardization-Certification Program (VDSCP): Total 25-hydroxyvitamin D certified procedures. Hormone and vitamin D standardization programs. Vitamin_D_Procedures.pdf (Accessed May 2017). 13. Heijboer AC, Blankenstein MA, Kema IP, Buijs MM. Accuracy of 6 routine 25-hydroxyvitamin D assays: influence of vitamin D binding protein concentration. Clin Chem 2012;58: Carter GD. 25-Hydroxyvitamin D: a difficult analyte. Clin Chem 2012;58: Denburg MR, Hoofnagle AN, Sayed S, Gupta J, de Boer IH, Appel LJ, et al. Comparison of two ELISA methods and mass spectrometry for measurement of vitamin D- binding protein: implications for the assessment of bioavailable vitamin D concentrations across genotypes. J Bone Miner Res 2016;31: van den Ouweland JM, Beijers AM, van Daal H. Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25- hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum. J Chromatogr Analyt Technol Biomed Life Sci 2014;967: Lensmeyer G, Poquette M, Wiebe D, Binkley N. The C-3 epimer of 25-hydroxyvitamin D(3) is present in adult serum. J Clin Endocrinol Metab 2012;97: Yang Y, Rogers K, Wardle R, El-Khoury JM. Highthroughput measurement of 25-hydroxyvitamin D by LC-MS/MS with separation of the C3-epimer interference for pediatric populations. Clin Chim Acta 2016;454: Evaluation of precision of quantitative measurement procedures; approved guideline. 3rd Ed. CLSI document.. May : JALM 11

12 Price of High-Throughput 25(OH)D Immunoassays EP05-A3. Wayne (PA): Clinical Laboratory Standards Institute (CLSI); Stepman HC, Vanderroost A, Van Uytfanghe K, Thienpont LM. Candidate reference measurement procedures for serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 by using isotope-dilution liquid chromatography-tandem mass spectrometry. Clin Chem 2011;57: Evaluation of the linearity of quantitative measurement procedures: a statistical approach; approved guideline. CLSI document EP06-A. Wayne (PA): Clinical Laboratory Standards Institute (CLSI); Evaluation of detection capability for clinical laboratory measurement procedures; approved guideline. 2nd Ed. CLSI document EP17-A2. Wayne (PA): Clinical Laboratory Standards Institute (CLSI); Liquid chromatography-mass spectrometry methods; approved guidelines. CLSI document C62-A. Wayne (PA): Clinical Laboratory Standards Institute; Interference testing in clinical chemistry; approved guideline. 2nd Ed. CLSI document EP07-A2. Wayne (PA): Clinical Laboratory Standards Institute (CLSI); JALM :06 May 2018