Accuracy of three automated 25-hydroxyvitamin D assays in hemodialysis patients
Highlights
► Not all automated 25(OH)D assays are equally accurate in hemodialysis patients. ► 25(OH)D measurements on Architect (Abbott) deviate most from ID-XLC-MS/MS. ► Architect falsely assigns 48.5% of hemodialysis patients a low vitamin D status. ► Method validation data in normal sera cannot be considered universally relevant. ► Matrix interferences may affect 25(OH)D testing in hemodialysis patients.
Introduction
The increased scientific and public attention to the prevalence and clinical consequences of vitamin D deficiency has enormously elevated laboratory test frequency and stimulated manufacturers' development of automated immunoassays for the past decade. Patients with chronic kidney disease (CKD) stages 3–5 (NKF KDOQI guidelines) and those who follow dialysis therapy form an important subgroup for vitamin D measurements with high needs for accurate results and monitoring. Renal dysfunction and diminished active vitamin D production combined with nephrotic damage increases the risk for vitamin D deficiency. Proteinuria and proximal tubular cell injury, associated with the clinical course of kidney disease, are responsible for significant urinary losses of 25(OH)D (25-hydroxyvitamin D) and its binding protein DBP [1]. Vitamin D deficiency is a major direct stimulus for PTH secretion, thereby increasing the risk for secondary hyperparathyroidism, a hallmark of CKD with several deleterious consequences [2]. Therefore, KDOQI guidelines for bone metabolism and disease in CKD stipulate measurement of serum 25(OH)D if PTH is above target range for the stage of CKD. Also the KDIGO guidelines recommend measurement of 25(OH)D and treatment of vitamin D deficiency or insufficiency [3]. Supplementation of hemodialysis patients with vitamin D is recommended by the KDOQI guidelines if 25(OH)D < 30 ng/ml [4]. Consequently, 25(OH)D measurement is a necessary practice for management of CKD patients.
Current assays for 25(OH)D measurement are radio- and chemiluminescence immunoassays, DBP assays, high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry (LC–MS/MS). Advantages of chromatographic analyses are the high accuracy and possibility to distinguish cross-reactants like 25(OH)D2 and 3-epi-25(OH)D. High throughput automated assays without manual sample preparation try to fulfill the increasing clinical demand and laboratory workload of 25(OH)D analyses. These assays are issued by three challenges for development: Firstly, the Food and Drug Administration (FDA) stipulates concomitant measurement of both 25(OH)D3 (cholecalciferol, the predominant endogenous circulating form) and 25(OH)D2 (ergocalciferol) besides the already difficult measurement of the single molecule with small immunogenic character. In the absence of vitamin D2 supplementation, the ratio of 25(OH)D3/25(OH)D2 lies around 95/5 [5], [6], [7], [8]. Secondly, the strong binding between highly hydrophobic 25(OH)D and DBP creates competition with the capturing antibodies or DBP. Thirdly, the low 25(OH)D circulating serum concentration (10% of total body presence) requires highly sensitive assays [9], [10], [11]. For clinical decisions, the limit of quantification (LoQ) for any 25(OH)D assay should be < 10 ng/ml [12].
The National Institute of Standards and Technology (NIST) released in 2008 Standard Reference Material (SRM) to improve the analytical performance and standardization of 25(OH)D assays [13]. This material has recently (2012) been modified to obtain human-based serum instead of spiked equine serum [14]. SRM material is certified and assigned reference values for 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3 by three isotope-dilution mass spectrometry approaches performed at NIST and at the Centers for Disease Control and Prevention (CDC). Recently, two candidate reference methods have been developed by Tai et al. [15] and Stepman et al. [16] that can be used by manufacturers and laboratories to calibrate and validate their methods.
Despite standardization efforts, unresolved discrepancies and unacceptable bias persist among immunoassays compared to the reference methods, particularly in samples from diseased patients with atypical plasma or serum matrix composition such as hemodialysis patients. As CKD patients represent an important target population for vitamin D testing, often forgotten in method validation studies, we specifically evaluated the performance of three automated assays in hemodialysis patients. To exclude sample type variability as a cause for inter-method differences by not using the same matrix for each method, as occurred in a previous study [17], we wanted to confirm these data by using only serum.
The aim of this study was to evaluate the accuracy of three different assays on Architect i2000sr (Abbott), Modular E170 (Roche), and iSYS (IDS) analyzers for 25(OH)D measurement in comparison to a higher reference isotope dilution/online solid-phase extraction liquid chromatography tandem mass spectrometry (ID-XLC-MS/MS) method [17], in serum from hemodialysis patients.
Section snippets
Samples
We studied 99 hemodialysis patients (47 women, 52 men, age 24–94 years) and a healthy control group of 50 blood donors (34 women, 16 men, age 20–65 years). During the periodic control of hemodialysis patients (2 days) in our hospital, blood samples were collected just before start of the regular dialysis run, allowed to clot, and centrifuged (10 min at 4000 rpm), and serum was divided automatically (RSA Pro, Roche Diagnostics) into 4 aliquots (minimum 350 μl) and frozen (− 20 °C). Serum samples of
Vitamin D concentrations
ID-XLC-MS/MS measured 25(OH)D concentrations between 11.6–86.0 ng/ml in the hemodialysis patients and 7.2–47.2 ng/ml in the healthy subjects. Measuring ranges of Architect, Modular E170 and iSYS were 10.0–52.0 ng/ml, 4.0–70.0 ng/ml, and 10.1–64.1 ng/ml for the hemodialysis patients and 10.6–39.3 ng/ml, 10.4–51.4 ng/ml, and 7.1–47.4 ng/ml for the healthy subjects, respectively. Box and Whisker plots illustrate the differences between 25(OH)D immunoassays and ID-XLC-MS/MS for hemodialysis patients (Fig. 1
Discussion
We report that distribution of measured 25(OH)D concentrations showed differences between automated assays and ID-XLC-MS/MS, with greater deviations in sera from hemodialysis patients than from healthy subjects. Compared to ID-XLC-MS/MS, Modular E170 measurements showed the best fit that crossed the line of identity for dialysis patients (slope 0.9552, intercept − 5.4077) and healthy subjects (slope 0.9200, intercept 2.2602) and the highest Weighted correlation coefficient for the dialysis
Conclusion
We conclude that not all automated 25(OH)D assays may be considered equally accurate in samples from hemodialysis patients compared to samples from healthy subjects. We found most deviating results with Abbott (Architect) measurements compared to ID-XLC-MS/MS in hemodialysis patients as well as for healthy subjects. In the hemodialysis groups, we did not observe the previously reported [17] concentration-dependent difference with ID-XLC-MS/MS (possibly due to the use of EDTA-plasma instead of
Acknowledgments
The authors are grateful to Roche Diagnostics and IDS for logistic support and for providing the reagent kits for 25(OH)D measurement. We thank Linda Thienpont and Katleen Van Uytfanghe from the Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences of Ghent University for statistical advice and guidance.
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