Standardized Serum Vitamin D Measurement

Blood withdrawal was performed according the protocol [33 (link)]. Measurement of serum vitamin D total was performed according to manufacturer’s protocol on an automated laboratory analyzer Cobas 8000 e602 (Roche Diagnostics, Mannheim Germany) using an electrochemiluminescence immunoassay (ECLIA) with competition principle (Roche Diagnostics Mannheim Germany). Traceability of the method was standardized against LC-MS/MS [34 (link)]and LC-MS/MS in turn was standardized against the NIST standard [35 (link)]. According to manufacturer information the limit of quantitation is 5 ng/ml. The primary measurement range is 5–70 ng/ml. Samples with concentration higher than 70 ng/ml were diluted manually 1:2 with Diluent Universal (Roche Diagnostics, Mannheim, Germany)
Between October 2011 and November 2014 VK of control level 1 varied between 6% and 17.5% (mean 10.13%), control level 2 varied between 3.4% and 12.2% (mean 6.89%). For the main regression analyses, we used the detected serum 25(OH)D concentrations as continuous variable. However, for additional analyses (see S2 Table) we subdivided the sample into groups based on recommendations from the literature [36 (link),37 (link),31 (link),12 (link)]: vitamin D deficiency (≤10 ng/ml), mild vitamin D deficiency (>10–20 ng/ml), vitamin D insufficiency (>20–30 ng/ml) and sufficient amount (>30 ng/ml).