Architect c8000

Blood samples were collected in plain plastic tubes during the emergency service admission. After standing for 30 minutes, the blood samples were centrifuged for 5 minutes at 3,000 rpm. The sera were separated and allocated to tubes (Eppendorf, Hamburg, Germany) and stored at −80°C until assay. The Lp-PLA₂ activity was determined with a commercial kit (DiaDexus, South San Francisco, CA, USA) and analyzer (Architect C8000; Abbott Laboratories, Abbott Park, IL, USA). Calibration and control procedures were performed, samples were dissolved, and enzyme activity (nmol/min/mL) was determined from absorbance at 412 and 524 nm. Serum Lp-PLA₂ activity was defined as low (<100 nmol/min/mL), medium (100 to 150 nmol/min/mL), or high (>150 nmol/min/mL).
The hs-CRP level was determined with a commercial kit (Abbott Laboratories) and analyzer (Architect C8000; Abbott Laboratories). After the samples were dissolved, a commercial test was performed (Denka Seiken, Tokyo, Japan) and validated by a commercial method (Dade Behring; Siemens Healthcare, Erlangen, Germany) and analyzed with a spectrophotometric method. The hs-CRP level was reported in mg/dL (reference range <0.5 mg/dL). Plasma hs-CRP level was defined as low (<3 mg/dL), medium (3 to 15 mg/dL) or high (>15 mg/dL). The assays were performed at the Selçuk University School of Medicine Biochemistry Laboratory.

We recorded the following general information for each subject: Age, sex, time of disease progression. Counts of white blood cells (WBC), red blood cells (RBC), neutrophils, monocytes, lymphocytes and platelets were performed in samples of peripheral blood obtained by standard venipuncture in K²EDTA BD vacutainers using an automatic flow cytometry analyzer (CELL-DYN Ruby System; Abbott Diagnostics). Using the hematological data, we calculated the monocyte/lymphocyte ratio (MLR), neutrophil/lymphocyte ratio (NLR) and platelet/lymphocyte ratio (PLR) by dividing the number of respective subtypes of blood cells (monocytes, neutrophils and platelets) by lymphocyte number (21 (link)) and also a systemic immune-inflammation index (SII) according to the formula: SII=platelet count x neutrophil count/lymphocyte count (22 (link)). The erythrocyte sedimentation ratio (ESR) was assessed according to the Westergren method. C-reactive protein (CRP) analysis was performed using an automated immunoassay analyzer (Cobase411; Roche Diagnostics GmbH). Fibrinogen was measured using an ACL Top 500 coagulometer (Instrumentation Laboratory, USA). Total proteins, albumin, alanine-aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (Palk) and γ-glutamyl transpeptidase (GGT) were assessed using an automated analyzer Architect c8000 (Abbott Diagnostics).

AMH and insulin measurements were carried out on Cobas e 411 analyzer (Roche Diagnostics). The intra- and interassay variability at AMH concentration of 2.44 and 12.3 ng/mL was 1.2%, 3.3% and 1.1%, 3.7%, respectively. For insulin, the intra- and interassay variability at concentration of 21.9 and 74.3 μU/mL was 3.2%, 4.2% and 3.7%, 4.6%, respectively. Glucose, TG, and HDL-C were measured on an ARCHITECT C8000 chemistry analyzer (Abbott Diagnostics). The interassay variability of TG at 1.00 and 2.65 mmol/L was 0.89% and 1.54%, respectively. For HDL-C, at concentration of 0.54 and 2.04 mmol/L, the intra- and interassay variability was 1.7%, 1.0% and 1.1%, 0.5%, respectively. Adiponectin measurement was based on enzyme-linked immunosorbent assay (ELISA) method using RayBio^® Human Acrp30 ELISA Kit (RayBiotech). The intra- and interassay variability was <10% and <12%, respectively. HOMA-IR index was calculated based on fasting plasma glucose concentration and serum insulin with standard formula: HOMA-IR = fasting concentration of insulin (μU/mL) × fasting concentration of glucose (mmol/L)/22.5.

Blood was sampled at fasting using an intravenous stationary catheter. Glucose, triacylglycerides (TG) and high-density lipoproteins (HDL) were analyzed according to local protocols. In Uppsala, glucose was analyzed using an Architect c8000 instrument (Abbott Diagnostics, Solna, Sweden) and by a Gluco-quant Glucose-Kit (Roche Diagnostics, Mannheim, Germany) in Salzburg. Uppsala quantified TG and HDL using an Architect c800 instrument (Abbott Diagnostics) and in Salzburg an enzymatic photometric test (Modular Analytics System, P-Modul, 917; Roche Diagnostics) was used. Validation of analyses was performed between the laboratories in Uppsala and Salzburg using reference blood samples.
Selected samples underwent immediate centrifugation at 2500g for 10 minutes at 4°C, subsequently aliquoted, and frozen at −80°C. Plasma was later used for central analyses of insulin, proinsulin, and C-peptide in Uppsala. Single-plex enzyme-linked immunosorbent assay kits for each analyte were used (Mercodia AB, Uppsala, Sweden). Standardized control samples (Mercodia AB) were used to control for interplate variability.