Current assays overestimate 25-hydroxyvitamin D3 and underestimate 25-hydroxyvitamin D2 compared with HPLC: need for assay-specific decision limits and metabolite-specific assays

Ann Clin Biochem 2006;43:23-30
doi:10.1258/000456306775141650
© 2006 Association for Clinical Biochemistry

 

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Original Articles


P Glendenning,
M Taranto,
JM Noble,
AA Musk,
C Hammond,
PR Goldswain,
WD Fraser and
SD Vasikaran


Department of Core Clinical Pathology & Biochemistry, Royal Perth Hospital, Perth, Australia;
Department of Core Clinical Pathology & Biochemistry, Royal Perth Hospital, Perth, Australia;
Department of Generic Medicine, Royal Perth Hospital, Perth, Australia;
Department of Core Clinical Pathology & Biochemistry, Royal Perth Hospital, Perth, Australia;
Critical Care Division, Royal Perth Hospital, Perth, Australia;
Department of Generic Medicine, Royal Perth Hospital, Perth, Australia;
Department of Clinical Chemistry, University of Liverpool, Liverpool, UK;
Department of Core Clinical Pathology & Biochemistry, Royal Perth Hospital, Perth, Australia


Background: Clinical demand for quick, cheap, precise and accurate 25-hydroxyvitamin D (25(OH)D) results has led to the development of a variety of assay methods. Lack of standardization of these methods has resulted in intermethod disagreement and challenged whether current assays recognize 25(OH)D2 and 25(OH)D3 equally.

Methods: We studied 172 patient samples from hip fracture cases using DiaSorin (DS) and IDS radioimmunoassays and the Nichols Advantage-automated protein binding assay (NA-CLPBA) in comparison to high-performance liquid chromatography (HPLC). 52 patient samples were analysed before and after three months treatment with 1000 IU of daily ergocalciferol (vitamin D2).

Results: Linear regression analysis in pre-treatment samples demonstrated a positive Y-intercept for each immunoassay compared with HPLC, and a slope that varied from 0.64 (IDS) to 0.97 (DS, NA-CLPBA). Bland Altman analysis demonstrated that all the three assays had a proportional positive bias relative to HPLC at values from 20 to 50 nmol/L. Regression analysis of post-treatment samples demonstrated a slope that was not significantly different from zero for the IDS and NA-CLPBA and 0.2 for the DS method, with a positive intercept for all assays of between 8 and 22, indicating less than 50% of 25(OH)D2 measured by HPLC was detected.

Conclusions: These results demonstrate the need for assay-specific decision limits for 25(OH)D3 in order to define appropriate thresholds for treatment institution. Treatment with vitamin D2 may not be accurately monitored with any of the three commercialassays studied. Clinicians and biochemists who continue to use25(OH)D assays need to be urgently informed of these issues.

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