Cobas e801

Blood and urine samples were collected from all participants. Serum free T4, TSH, and thyroid peroxidase antibodies (TPOAb) were analyzed with electrochemiluminescence immunoassay. Free T4, TSH, and TPOAb levels were measured using an E- free T4 kit (Cobas e 411, Roche Diagnositics GmbH Mannheim, Germany), E-TSH kit (Cobas e 801, Roche Diagnostics GmbH, Mannheim, Germany), and E-Anti-TPO kit (Cobas e 801, Roche Diagnostics GmbH, Mannheim, Germany). The reference ranges for free T4 and TPOAb were 0.89 to 1.76 ng/mL and 0–34 IU/mL, respectively. As TSH is affected by iodine intake status, and excess iodine is prevalent in Korea [23 (link)], serum TSH levels between 0.62 and 6.68 mIU/L, based on population data [24 (link)], were considered as reference ranges. Urine iodine concentration was measured by inductively coupled plasma mass spectroscopy (ICP-MS: PerkinElmer ICP-MS, Waltham, USA) and was adjusted for creatinine concentration.
Serum total cholesterol, TG, and HDL-cholesterol were measured by enzymatic methods using a commercial kit (Sekisui, Osaka, Japan) with a Hitachi Automatic Analyzer 7600 (Hitachi, Tokyo, Japan). LDL-cholesterol was calculated using the Friedewald formula: LDL-cholesterol = total cholesterol − HDL-cholesterol − (TG/5) [25 (link)]. Non-HDL-cholesterol was calculated by subtracting the quantity of HDL-cholesterol from total cholesterol [26 (link)].

We collected specimen from the antecubital vein after an overnight fast. Biomarkers including fasting insulin, 25(OH)VD, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), prealbumin (PA), albumin (ALB), free triiodothyronine (FT3), free tetraiodothyronine (FT4), triglycerides (TG), high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), plasm total cortisol (PTC) and hemoglobin (Hb) were measured. Except Hb was used with whole blood, the remaining blood was centrifuged at 3000rpm for 10 minutes to obtain serum. TP, ALB, FT3, FT4, TG, VLDL, HDL, PTC were measured by electrochemiluminescence (cobas e801, Roche). ALT and AST were measured by enzyme method (cobas e801, Roche). PA was measured by Turbidimetric inhibition immuno assay (IMMAGE 800, Beckman). Some clinic signs such as weight, height, calf circumference (CC), waistline, mid-arm circumference (MAC), thickness of triceps skinfold (TST), hip circumference (HIPL) were also measured. Trained interviewers collected questionnaire data through face-to-face, one-on-one personal interviews. Trained technicians performed the anthropometric and bioimpedance measurements.

Anti-SARS-CoV-2 nucleocapsid IgG antibodies were measured using electrochemiluminescent immunoassay (ECLIA) Elecsys^® anti-SARS-CoV-2 assay on Roche Cobas e801 (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions. Results were expressed as a cutoff index (COI; signal sample/cutoff). The analyzer automatically calculates the cutoff based on the measurement of negative and positive calibrators. A cutoff index equal to or higher than 1.0 was interpreted as positive. Clinical sensitivity and specificity of this test were 99,5% and 99.8% respectively.

We will measure bone turnover markers at three time points (T0, 0 weeks; T2, 12 weeks; T4, 24 weeks) during and immediately after the intervention to evaluate the effect of the intervention on bone metabolism. CTX is a known bone resorption marker, and higher levels are adversely associated with bone loss [39 (link)]. Fasting blood samples will be collected from the antecubital veins of participants, and CTX will be assayed via the Roche Cobas E801 electrochemiluminescence immunoassay (Roche Diagnostics, Basel, Switzerland) using the Elecsys beta-CrossLaps serum assay [40 (link)].

Venous whole blood samples were collected after an overnight fasting into Vacutainer® tubes either containing EDTA or lithium/heparin as anticoagulants, to measure biochemical variables and for extracting DNA from peripheral blood mononuclear cells.
After centrifugation at 1500 g for 10 min at room temperature, lithium heparin plasma was separated, stored in aliquots and kept frozen at −70°C until measurement. Fe was determined by the routine method used in the local laboratory (Roche Diagnostics). Ferritin concentration was measured with an automated chemiluminescence method, on Roche Cobas e801 (Roche Diagnostics). DNA was extracted from peripheral blood mononuclear cells by Wizard Genomic DNA Purification Kit (Promega Corporation). Genotyping for one-carbon-related polymorphisms (MTHFR 677C > T, cSHMT 1420C > T, DHFR 19bp ins/del, RFC1 80G > A) was analysed by different methods as previously described^{(14 (link))}.
A plasma Fe concentration > 10·74 μmol/l and ferritin range values 20–200 µg/l were considered as adequate concentrations^{(15 (link))}.

Procalcitonin (PCT) and ferritin were measured with electrochemiluminescent immunoassays (ECLIA) on a Roche Cobas e801 instrument (Roche Diagnostics, Monza, Italy). C-reactive protein (CRP) was measured with an immunoturbidimetric method and lactate dehydrogenase (LDH), alanine and aspartate aminotransferase (ALT, AST) with the International Federation of Clinical Chemistry (IFCC) optimized methods on a Roche Cobas c702 instrument (Roche Diagnostics, Monza, Italy). Fibrinogen and D-dimer were measured with ACL Top (Werfen, Milano, Italy). Hemocytometry analysis was performed with Sysmex XN 9000 (Dasit, Cornaredo, Italy).

Samples were collected in fasting state from 7.00 to 10.00 am by venepuncture in test tubes with clot activator (4 or 7 mL Vacuette, Greiner Bio-One GmbH, Kremsmünster, Austria). After clotting, blood samples were centrifuged at 2200xg for 10 minutes and fresh serum samples were used for analysis. All measurements, except androstenedione, were performed on Roche Cobas analysers e601 until March 2020 and e801 from April 2020 (Roche Diagnostics GmbH, Mannheim, Germany) by ECLIA method. Two different generations of Roche analysers (e601 and e801) were compared in our laboratory using at least 20 patient samples with values covering all measuring range. The comparison results showed no clinically significant difference (Supplement 1). Androstenedione was measured only with Roche Cobas e801 ECLIA method and implemented in routine practice in October 2020. All calibrations, internal and external quality control assessment were carried out according to the laboratory-defined standard operating procedures throughout all study duration.