25-Hydroxyvitamin D assays: Potential interference from other circulating vitamin D metabolites

Area of Science:

• Clinical Chemistry
• Endocrinology
• Laboratory Medicine

Background:

• The Vitamin D External Quality Assessment Scheme (DEQAS) is a global program assessing 25-hydroxyvitamin D (25-OHD) measurement accuracy.
• DEQAS expanded in 2015 to include 24,25-dihydroxyvitamin D (24,25(OH)2D) assessment.
• Since 2013, DEQAS 25-OHD assessments are accuracy-based, utilizing the NIST reference measurement procedure (RMP).

Purpose of the Study:

• To evaluate the accuracy and specificity of routine 25-OHD laboratory methods.
• To investigate the interference of other vitamin D metabolites in 25-OHD assays.
• To assess the performance of various analytical methods in resolving different vitamin D metabolites.

Main Methods:

• DEQAS distributes proficiency testing samples to approximately 1100 participants worldwide.
• Samples are spiked with known concentrations of 3-epi-25-OHD3, 24R,25(OH)2D3, and 24S,25(OH)2D3 to assess metabolite interference.
• Analytical methods evaluated include competitive protein binding assays, antibody-based assays, HPLC/UV, and LC-MS/MS.

Main Results:

• The 3-epimer showed 56% cross-reactivity in competitive protein binding assays but was undetectable in antibody-based methods.
• HPLC/UV and LC-MS/MS methods could not always resolve 3-epi-25-OHD3, leading to overestimated total 25-OHD.
• Cross-reactivity of 24,25-dihydroxyvitamin D metabolites varied widely ( <5% to 643%) in ligand binding assays, with HPLC/UV and LC-MS/MS resolving these metabolites effectively.

Conclusions:

• Significant interference from vitamin D metabolites, particularly 3-epi-25-OHD3 and 24,25-dihydroxyvitamin D, impacts the accuracy of routine 25-OHD assays.
• Method-specific interference varies, with antibody-based and ligand-binding assays showing greater susceptibility compared to HPLC/UV and LC-MS/MS.
• Accurate measurement of 25-OHD requires methods capable of resolving interfering metabolites, highlighting the importance of external quality assessment schemes like DEQAS.