Competitive Protein‑binding assay‑based Enzyme‑immunoassay Method, Compared to High‑pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children

Maliheh Zahedi Rad, Tirang Reza Neyestani, Bahareh Nikooyeh, Nastaran Shariatzadeh, Ali Kalayi, Niloufar Khalaji, Azam Gharavi


Background: The most reliable indicator of Vitamin D status is circulating concentration of 25‑hydroxycalciferol (25(OH) D) routinely determined by enzyme‑immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein‑binding assays (CPBA)‑based EIA with the gold standard, high‑pressure liquid chromatography (HPLC). Methods: Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9–11 years were determined by two methods of CPBA and HPLC.

Results: Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut‑offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%.

Conclusions: Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes. Keywords: Competitive protein-binding assays, high-pressure liquid chromatography, Vitamin D deficiency, Vitamin D measurement

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