1. Accuracy of 6 Routine 25-Hydroxyvitamin D assays; Influence of Vitamin D Binding Protein Concentration A.C. Heijboer, M.A. Blankenstein, I.P. Kema, and M.M. Buijs March 2012 www.clinchem.org/content/article/58/3/543.full © Copyright 2012 by the American Association for Clinical Chemistry

2. © Copyright 2009 by the American Association for Clinical Chemistry Introduction I  Vitamin D High percentage of the population is vitamin D deficient Vitamin D has skeletal and extraskeletal effects Laboratory vitamin D requests on the increase Therefore automation is desirable

3. © Copyright 2009 by the American Association for Clinical Chemistry Introduction II  Beware: vitamin D is a difficult analyte!  Editorial of Carter, Clin Chem March 2012  Analysis of vitamin D  Prerequisite: release vitamin D from binding protein (DBP)  Traditional methods: RIA and HPLC preceded by extraction  Automated methods: unclear whether displacement of vitamin D from DBP is sufficient

4. © Copyright 2009 by the American Association for Clinical Chemistry Question I  What problem can arise if the displacement of vitamin D from its binding protein (DBP) is not sufficient?

5. © Copyright 2009 by the American Association for Clinical Chemistry Aims  To test the accuracy of the currently available 25(OH)D assays  To assess the sensitivity of the various assays to differences in circulating DBP concentrations

6. © Copyright 2009 by the American Association for Clinical Chemistry Material & Methods  Plasma and serum samples 51 healthy individuals, 52 pregnant women, 50 hemodialysis patients, 50 intensive care (IC) patients  Methods vitamin D  ID-XLC-MS/MS (accuracy established by measuring standard & control with reference method)  6 routine assays Architect i2000SR (Abbott Diagnostics) Centaur XP (Siemens Diagnostics) iSYS (IDS) Liaison (Diasorin) Elecsys (Roche Diagnostics) RIA preceded by extraction (Diasorin)  Method DBP ELISA (R&D systems)

7. © Copyright 2009 by the American Association for Clinical Chemistry Question II  As compared with healthy controls, do you expect DBP to be increased or decreased?  In pregnant women  In hemodialysis patients  In intensive care patients

8. © Copyright 2009 by the American Association for Clinical Chemistry Figure. 1. 25(OH)D concentrations [mean (SD)] measured by different methods in healthy individuals (n=51), pregnant women (n=52), hemodialysis patients (n=50), and IC patients (n=50). 25(OH)D was not measured in plasma from IC patients using the iSYS. To convert 25(OH)D concentrations to ng/mL, multiply by 0.4. Main Results

9. © Copyright 2009 by the American Association for Clinical Chemistry Figure 2. DBP concentrations in the circulation of healthy individuals, pregnant women, dialysis patients, and IC patients. *P <0.001. Main Results

10. © Copyright 2009 by the American Association for Clinical Chemistry Main Results Table 1. Correlation coefficient, slope, and intercept according to Passing–Bablok regression for each 25(OH)D assay compared with ID-XLC-MS/MS in each clinical subject group. a To convert 25(OH)D concentrations to ng/mL, multiply by 0.4 b Significantly different from 1.00 (slope) and 0.00 (intercept) (P< 0.05) c ND, not determined

11. © Copyright 2009 by the American Association for Clinical Chemistry Figure 3. Correlation (R) between the difference of 25(OH)D results between 6 routine 25(OH)D assays and the ID-XLC-MS/MS, and DBP concentration. For the Architect (P< 0.005), Centaur, iSYS, and Liaison (P< 0.0001), the correlation was statistically significant. To convert 25(OH)D concentrations to ng/mL, multiply by 0.4. Main Results

12. © Copyright 2009 by the American Association for Clinical Chemistry Question III  Did you anticipate an inverse relationship between DBP concentrations and deviations of measured 25(OH)D concentrations from ID-XLC-MS/MS results?  Based on the answer to Question II, what is your explanation for this finding?

13. © Copyright 2009 by the American Association for Clinical Chemistry Conclusions  Some of the assays used to measure 25(OH)D are not well standardized and report significantly different results from measurements performed with ID-XLC-MS/MS  The deviations which are sometimes serious are different in various patient groups, and are dependent on the concentration of DBP and other still-unknown interfering factors  Laboratory specialists, physicians, researchers, reviewers and authorities who provide advice on cutoffs for sufficiency and supplementation of 25(OH)D should carefully consider the 25(OH)D assay used in studies, before any conclusions are drawn or decisions made

14. © Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at www.clinchem.org Follow us