The purpose of this study was to find out the cause of discrepancy between various automated immunoassays for 25-hydroxy-vitamin D (25-[OH]D).

National Institute of Standards & Technology Standard Reference Material (SRM) 972a is SRM for 25-(OH)D and consists of 4 vials of frozen serum with different concentrations of 25-(OH)D. Each concentration was measured 6 times in 3 different immunoassays: ADVIA Vitamin D Total assay (Siemens Healthcare, Erlangen, Germany), ARCHITECT 25-(OH)D (Abbott Laboratories, Abbott Park, IL, USA), and COBAS Vitamin D Total assay (Roche Diagnostics, Basel, Switzerland).

When using the certified reference values of SRM 972a as it is, discarding the cross-reactivity of each immunoassay, for ADVIA, the coefficient of determination (R²) as a score of regression analysis was 0.8995 and maximal difference between measured value and certified reference value was 3.6 ng/mL in level 3. The R² and maximal differences of ARCHITECT were 0.5377 and 6.9 ng/mL, respectively, in level 4. Those of COBAS were 0.3674 and 22.3 ng/mL, respectively, in level 4. When considering cross-reactivities of each immunoassays to various 25-(OH)D metabolites, the ADVIA had R² and maximal difference of 0.9254 and 3.3 ng/mL, respectively, in level 3. For ARCHITECT, the R² and maximal differences were 0.7602 and 5.1 ng/mL, respectively, in level 1. Those of COBAS were 0.9284 and 4.9 ng/mL, respectively, in level 1.

The cause of discrepancies between vitamin D immunoassays was mainly on the difference in cross-reactivities to various vitamin D metabolites. The discrepancies can be considerably decreased by considering cross-reactivities of each immunoassay.