Method evaluation study of a new generation of vitamin D assays

Introduction Recently several diagnostic manufacturers have launched new 25-hydroxy-vitamin D (25[OH]D) assays, which are aligned to the National Institute of Standards and Technology (NIST) Standard Reference Materials (SRM) (NIST, Gaithersburg, Maryland). The aim of this study was to compare the performance of one liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, one enzyme linked immunosorbent assay (ELISA), and one recalibrated and previous version of a chemiluminescence immunoassay (CLIA). Material and methods Serum-aliquots of 198 patient samples from routine 25(OH)D analysis were measured by the ClinMass® LC-MS/MS Complete Kit (RECIPE Chemicals + Instruments GmbH, Munich, Germany), the ORGENTEC 25(OH)D3/D2 ELISA (ORGENTEC Diagnostika GmbH, Mainz, Germany), the recalibrated Immunodiagnostic Systems (IDS)-iSYS 25(OH)DS and the previous used IDS-iSYS 25(OH)D CLIA (Immunodiagnostic Systems Ltd, Boldon, United Kingdom). Bland-Altman and Deming regression analyses were calculated for methods comparison of all tested 25(OH)D assays. The LC-MS/MS method was defined as the reference method. Within-run and between-run precision measurements were performed for all methods with three different concentration levels. Results Compared to the LC-MS/MS method, the new IDS-iSYS 25(OH)DS and ORGENTEC 25(OH)D3/D2 assay demonstrated mean relative biases of 16.3% and 17.8%. The IDS-iSYS 25(OH)D assay showed the lowest mean bias of 1.5%. Deming regression analyses of the recalibrated IDS-iSYS 25(OH)DS and the ORGENTEC 25(OH)D3/D2 assay showed proportional differences, when compared to the reference method. All assays showed a within-run and between-run imprecision of ≤ 20% at each of the evaluated concentration levels. Conclusions The evaluated standardized immunoassays and LC-MS/MS are useful methods for measuring 25(OH)D serum-levels in clinical laboratories.


Introduction
The clinical interest in the physiological importance of the steroid hormone vitamin D and its possible roles in the pathophysiological processes of many diseases have increased the demand for the measurement of vitamin D and its metabolites (1). On the one hand, vitamin D deficiency results in abnormalities in bone metabolism known as rickets, osteomalacia, and osteoporosis (2,3). On the other hand, it is associated with non-skeletal diseases such as type one diabetes mellitus (2,4,5), multiple sclerosis (2), cancer (2,5), hypertension (5) or cardiovascular disease (5,6).
In the liver vitamin D is converted to the 25-hydroxy-vitamin D (25[OH]D) and transported in the circulation by the vitamin-D binding protein (DBP). In the kidneys the biologically active form 1,25-dihydroxy-vitamin D (1,25[OH]2D) is created from 25(OH)D (7). This active form has a circulating halflife of only 4-6 hours and serum-levels of about 1000 fold less than 25(OH)D (1,8,9). The major circulating form of vitamin D is 25(OH)D, which has a half-life of approximately 2-3 weeks (8). Therefore, the total 25(OH)D is principally used as the biomarker indicating the vitamin D status (9,10). Enko D. et al. A

new generation of vitamin D assays
In the past, 25(OH)D measurements have proven to be a major challenge with a wide spread variation in the results (1). The sometimes huge between-method discrepancies have been known for many years from data obtained from the International Vitamin D External Quality Assessment Scheme (DEQAS) (11)(12)(13). The DEQAS was already founded in 1989 and meanwhile has become the largest vitamin D quality assessment program worldwide (14).
To overcome the problem of inter-laboratory as well as inter-assay discrepancies, the Vitamin D Standardization Program (VDSP) was established in 2011. This program is conducted as a collaboration between the US Office of Dietary Supplements (ODS) of the National Institute of Health (NIH), the Center for Disease Control and Prevention (CDC), the National Centre for Environmental Health (NCEH), the National Institute of Standards and Technology (NIST), and the Belgian Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University (10,13). The NIST, in collaboration with the ODS, has developed and certificated Standard Reference Materials (SRM 2972 and 972a) for vitamin D metabolites in human serum (10,15).
The 3-epi-25(OH)D 3 (C 3 -epimer) is a vitamin D metabolite, which is considered as a confounder in 25(OH)D measurements. The biological role and its clinical significance are still unknown. The presence of the C 3 -epimer is considered to affect the quantification of 25(OH)D 3 measurements in routine LC-MS/MS methods, especially in infant populations (16). For that reason, the measurement of this metabolite is one important objective of the VDSP (10).
The recent release of a new generation of 25(OH)D assays, which are aligned to the NIST SRM, is anticipated to show an improved analytical performance. Considering the difficulties of 25(OH)D measurements in the past, the hypothesis of this study was, that the standardization of this new generation of 25(OH)D assays represents an improvement of inter-assay accordance in daily clinical 25(OH)D determination.

Subjects
For the method evaluation of the above mentioned 25(OH)D assays, leftover blood samples from routine 25

IDS-iSYS 25(OH)D S assay (Immunodiagnostic Systems Ltd, Boldon, United Kingdom)
In December 2013, this new recalibrated version replaced the IDS-iSYS 25(OH)D assay on the market. This immunoassay also was performed on the IDS-iSYS Multi-Discipline Automated Analyzer. According to the previous version of this assay, the denaturation of the DBP was also done with NaOH (part of the reagent used for CLIA and ELISA methods) inside the analyzer. In contrast, this assay is aligned to the NIST SRM 2972 (NIST, Gaithersburg, Maryland). The measurement range of this assay is 7-125 ng/mL (information of the manufacturer). The IDS-iSYS 25(OH)D S Control Set (IS-2730S) (Immunodiagnostic Systems Ltd, Boldon, United Kingdom) was used for QC.

ORGENTEC 25(OH)D 3 /D 2 assay (ORGENTEC Diagnostika GmbH, Mainz, Germany)
This recently launched assay is based on a competitive ELISA. A single (not duplicate) serum sample was pipetted manually into well number one of an eight-well-micro strip. The extraction of 25(OH)D 2 /D 3 was done automatically inside the Alegria® Random Access Analyzer. Serum samples were mixed with tracer reagent (ORGENTEC Diagnostika GmbH, Mainz, Germany) and the 25(OH) D 2 /D 3 was delivered from the DBP. The extraction procedure was followed by analysis. This assay is also aligned to the NIST SRM 2972 (NIST, Gaithers-burg, Maryland). The measurement range of this assay is 5-170 ng/mL (information of the manufacturer   (17), equal parts of the high-and low-level QC material were mixed to create the mid-level. At least one run per day at five consecutive workdays (from monday to friday) with a specific sequence (mid-, high-, low-, mid-, mid-, low-, low-, high-, high-, and mid-level) without change, interruption or intervening samples was analyzed for each immunoassay to determine the between-run precision (N = 5) for each level. In addition to the required measurements in the EP10-A2 protocol, one tenfold measurement (N = 10) of the low-, mid-and high-level of each immunoassay was performed at one day to determine the within-run precision.

Statistical analysis
Bland-Altman and Deming regression plots were calculated for methods comparison of all tested 25(OH)D assays. The LC-MS/MS method was defined as the reference method. All other methods were compared to the reference method. Withinrun and between-run precision at each concentration level were assessed by calculating the mean, the standard deviation (SD) and the coefficient of variation (CV) of the above mentioned replicates and sequences. The CV was calculated based on the formula: CV (%) = 100 x standard deviation (SD)/mean (ng/mL). According to the literature (18), the precision goal for each concentration level for the within-run and between-run was not to exceed 20% of the CV. Analyse-it® software version 2.30 (Analyse-it Software, Ltd, Leeds, United Kingdom) was used for statistical analysis. A P-value < 0.05 was considered statistically significant.

Characteristics of the study population
The study population mainly consisted of adults and a total of five adolescents between 15

Immunoassays vs. LC-MS/MS method
Bland-Altman plots are illustrated in Figure 1 A-C.

Precision studies of the immunoassays
The results of the precision studies of the immunoassays are demonstrated in Table 1. All immunoassays showed a within-run and between-run imprecision of ≤ 20% at each concentration level (low, mid, high). The highest within-run CV (19.0%) was observed at the low-level precision measurements with the IDS-iSYS 25(OH)D S assay. The highest between-run CV (19.1%) was shown at the lowlevel precision measurements with the IDS-iSYS 25(OH)D assay.

Precision studies of the LC-MS/MS method
The results of the precision studies of the LC-MS/ MS method are shown in Table 2. All within-run and between-run precision measurements of each 25(OH)D 2 and 25(OH)D 3 concentration level had a CV of ≤ 10%. Enko D. et al. A new generation of vitamin D assays

25(OH)D 2 and C 3 -epimer detection by the LC-MS/MS method
In all blood samples (N = 198), no 25(OH)D 2 and no C 3 -epimer were detected with the LC-MS/MS method.

Discussion
The     However, recent studies with commercially available 25(OH)D assays before standardization have reported significant bias between 25(OH)D assays (19,20). For example, one immunochemical method performed on the Architect i2000 (Abbott GmbH, Vienna, Austria) showed a mean bias of 27.0% compared to the LC-MS/MS method (19). Furthermore own published data of a previous work presented a significant negative absolute mean bias of -22.8 nmol/L between the Cobas® Vitamin D 3 assay (Roche Diagnostics GmbH, Mannheim, Germany) and the LC-MS/MS method, leading to a misjudgment of the actual 25(OH)D status of a patient (21). Meanwhile the manufacturer has withdrawn this assay from the market. High inter-assay disagreement in the 25(OH)D measurements (20,22) can lead to an underestimation (23) or overestimation (24) of the 25(OH)D serum-levels. The lack of standardization, matrix effects, poor antibody specificity, and cross-reactivity with other 25(OH)D metabolites could be possible reasons for reported high inter-assay disagreement before standardization (21).
The C 3 -epimer is one of the vitamin D metabolites, which is considered to be a potential confounder in 25(OH)D measurements, especially in infants (16). In the present study, no newborns or young children were included. Although the C 3 -epimer is also described in adults (25,26), no C 3 -epimer was detected with the LC-MS/MS method.
Not only the C 3 -epimer but also other vitamin D metabolites are considered as possible reasons for significant inter-assay differences of previous published 25(OH)D comparative studies. Many chemiluminescence assays use antibodies to measure 25(OH)D in unextracted serum. These antibodies also recognize other vitamin D metabolites, such as 24, 25(OH) 2 D. Such assays not only provide the total 25(OH)D but also include the metabolites (27,28). Furthermore, some of these assays do not always recognize 25(OH)D 2 and 25(OH)D 3 (27). An increased antibody specificity could be one potential reason for the improvement of the reproducibility and comparability of the new NIST SRM aligned 25(OH)D assay generation.
According to the literature (18) In comparative studies, the LC-MS/MS is widely used as the reference method (12). It is a reliable diagnostic tool and able to distinguish 25(OH)D 2 and 25(OH)D 3 (9,29). A low batch-to-batch variation and a low limit of detection are further advantages of this method (30). In the present study, we used an LC-MS/MS method, which has met the performance target set by the international DEQAS Advisory Panel in 2013 and 2014 (data not shown). The method was aligned to the NIST SRM 972a and enabled the separation and qualitative detection of the C 3 -epimers in a single analytical run. Nevertheless the biggest problem in 25(OH)D measurements in the last few years was the lack of a common standard. Furthermore not all LC-MS/ MS methods used in previous comparative studies (20) could separate C 3 -epimers. The LC-MS/MS method in particular was recommended to be aligned to the NIST SRM and to be able to discriminate the C 3 -epimer (10,20). The strength of this study is that these recommendations of the VSDP have been completely fulfilled. The reported new generation of 25(OH)D assays (except the previous used IDS-iSYS 25[OH]D assay) tested are aligned to the NIST SRM 2972 or 972a.
The limitation of this study is that the precision studies of the immunoassays and the precision studies of the LC-MS/MS method were not performed with the same protocol. Therefore, the within-run and between-run precision measurements of the immunoassays are not comparable with the LC-MS/MS method.
In conclusion, the new generation of the NIST SRM aligned immunoassays and LC-MS/MS evaluated in this study are useful methods for measuring 25(OH)D serum-levels in clinical laboratories. The performance characteristics are suitable for routine diagnostic purposes.