A-034 Multicenter Comparison of Analytical Interference of Vitamin D Immunoassay and Mass Spectrometry Methods by Endogenous Interferents and Cross-reactivity with 3-epi-25-OH-Vitamin D3
Authors: Lee J , Seo J , Lee K , Roh E , Yun Y , Lee Y , Cho S , Song J 
Published: September 2023

Abstract

Background
Vitamin D plays an important role in calcium homeostasis and bone metabolism and is known to increase calcium absorption from the intestinal tract and decrease calcium excretion from the kidneys. Vitamin D deficiency is prevalent in a substantial and continuously increasing proportion of the Korean population, thus highlighting the need for accurate vitamin D measurements. The current problems of vitamin D testing include significant inter-method and inter-laboratory differences in measurements, and interferences by both intrinsic and cross-reactive substances. In this study, the interfering effects by such intrinsic and cross-reactive substances were compared between several vitamin D immunoassays and mass spectrometry methods routinely used in Korea.

Methods
Two different mass spectrometry methods and 4 vitamin D immunoassays with different manufacturers (Abbott, Beckman Coulter, Roche, Siemens), from 5 different institutes were compared. Residual patient samples were utilized to collect either hemolyzed samples, icteric samples (total bilirubin &gt;20 mg/dL), lipemic samples (triglyceride &gt;500 mg/dL), of samples with rheumatoid factor &gt;200 IU/mL, samples from myeloma patients (paraprotein &gt;1.1 g/dL upon protein electrophoresis) or from patients undergoing hemodialysis. Four levels of National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 972 were prepared, with each level having different compositions and concentrations of vitamin D. Also, samples spiked with different levels of 3-epi-25(OH)D3 were prepared for interference analysis. The 10% bias limits for concentrations of intrinsic interferents and cross-reactivity percentages, provided by the respective immunoassay manufacturers, were referred to during data interpretation. The collection of intrinsic interferent samples, NIST material, and epimer spiked sample sets were delivered to each institute and measured on the same date.

Results
Consistently significant interference in vitamin D measurements was observed in hemolytic samples (Roche assay), icteric samples (Beckman and Siemens assays) and lipemic samples (all 4 immunoassays). General positive biases were also observed in myeloma samples (Abbott assay), hemodialysis samples (Siemens assay), and general negative biases were observed in high rheumatoid factor (Roche assay) and hemodialysis samples (Roche assay). Both the level 4 NIST material, which had a high concentration of 3-epi-25(OH)D3, and samples spiked with 3-epi-25(OH)D3, induced significant interference, yielding higher total vitamin D measurements in non-epimer separating MS methods, and the Beckman and Roche immunoassays.

Conclusion
In this study, we were able to observe interferences due to intrinsic substances and cross-reactive substances in common vitamin D tests used in Korea. Clinical awareness of possible causes of interference is important to increase the accuracy of vitamin D measurements. Moreover, due to the high cross-reactivity of 3-epi-25(OH)D3 with common vitamin D assays, particular care is due when interpreting vitamin D results of newborns, infants, and less commonly, pregnant women, who are known to have physiologically high levels of 3-epi-25(OH)D3.