Cobas 8000 e602

Free thyroxine (free T4), thyroid-stimulating hormone (TSH), and TPO Ab levels were assessed via blood sampling. Free T4, TSH, and TPO Ab were measured using Roche COBAS 8000 E-602 (Roche, Mannheim, German) with immunoassays. An internal validation was performed monthly. The monthly coefficients of variation were within an acceptable range (for TSH, free T4, and TPO Ab: ≤7%, 7%, and 10%, respectively). External quality assessment was performed by both the College of American Pathologists and the Korean Association of External Quality Assessment Service. The detailed quality control reports were described previously^{13 ,14} .

Serum levels of IGF-I were determined by an in-house radioimmunoassay (RIA) after separation from IGFBPs by acid ethanol extraction and cryoprecipitation. To minimize interference of remaining IGFBPs, des (1-3) IGF-I was used as radio-ligand (33 (link)). The intra-and inter-assay CV were 4% and 11%, respectively and detection limit 6 µg/L. The concentrations of fasting IGFBP-1 in serum were determined by an in-house RIA according to Póvoa et al. (34 (link)). The sensitivity of the RIA was 3 µg/l and the intra- and inter-assay CV were 3% and 10%, respectively. In healthy adults a linear inverse correlation, with no gender differences, exists between logarithmic transformed IGF-I levels and age. To correctly compare IGF-I levels between subjects of different age, IGF-I values were also expressed as SD scores (IGFSD) calculated from the regression of the IGF-I values of healthy adult subjects aged 20-95 years (14 (link), 35 (link), 36 (link)). Fasting levels of insulin were measured using the clinical standard method electrochemiluminescence immunoassay (ECLIA) [Roche Cobas 8000 (e602)] at the Karolinska University Laboratory, with a reference range (according to the manufacturer Roche) of 2.0-25. mIE/L. Fasting serum insulin between 14-25 mIE/L indicates insulin resistance.

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).

FSH, LH, Prolactin, TSH: Electrochemiluminescence immunoassay ECLIA, cobas kit insert Elecsys and cobas e analyzers (2013–10, V 19), Roche Diagnostics GmbH/Roche Diagnostics GmbH, Sandhofer Strasse 116, D-68305 Mannheim, Germany. Equipment: cobas 8000 e 602, Roche Diagnostics. Estradiol: radioimmunological assay, Spectra kits ¹²⁵I Coated Tube Radioimmunoassay kit insert, Orion Diagnostica, Espoo, Finland.. Equipment: Gammamaster 1270, Wallac Oy, Turku, Finland. AMH: AMH Gen II ELISA. Equipment: Beckman Coulter, Inc. 250 s. Kraemer Blvd. Brea, CA 92821 U.S.A.