Serum samples from each individual were obtained at the time of diagnosis, before any therapeutic measures were started. Samples were centrifuged at 1500 × g for 10 min at −4°C. The supernatant was stored at −80°C. The periostin levels were determined by ELISA with the commercial periostin ELISA Ready-SET-Go kit (eBioscience, San Diego, CA). The levels of CEA were measured by electrochemiluminescence immunoassays on Roche Elecsys 1010 analyzer (Roche Diagnostics; Mannheim, Germany). The upper normal limit for the CEA is 5 ng/ml. All samples were blinded to the technologists running the assays, and the code was broken to the statisticians after the database was constructed.
Elecsys 1010 analyzer
Manufactured by Roche
Sourced in United States
The Elecsys 1010 analyzer is a compact and fully automated immunoassay analyzer designed for routine in vitro diagnostic testing. It utilizes electrochemiluminescence technology to perform a wide range of immunoassay tests. The Elecsys 1010 is capable of performing multiple sample processing and analytical functions in a single, self-contained instrument.
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9 protocols using elecsys 1010 analyzer
1
Serum Periostin and CEA Levels
2
Venous Blood Sampling and Biomarker Analysis
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Fasting venous blood samples were collected into silicone test tubes. They were centrifuged upon cooling at 6,000 rpm 430 g for 3 minutes. Then, plasma was refrigerated immediately and stored at a temperature no higher than -35 ºС until they were analysed. Samples were processed according to the recommendations of the manufacturer of the analytical technique. SUA level was measured by enzymatic methods using a chemical analyzer Beckman (Synchron LX20, city, state). Analytical range average for SUA was 0.5-82 mmol/L. N-terminal pro-brain natriuretic peptide (NT-pro-BNP) was measured by immune-electro-chemiluminescence method using sets by R&D Systems (city, state, USA) on Elecsys 1010 analyzer (Roche, Mannheim, Germany). Calibration of the assay was performed according to the manufacturer’s recommendations and values were normalized to a standard curve. Concentrations of total cholesterol (TC) and high-density lipoprotein (HDL) cholesterol were determined with Dimension Clinical Chemistry System® (Dade Behring Inc, Newark, NJ). Low-density lipoprotein (LDL) cholesterol was calculated using Friedewald formula.20 (link)
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Fasting venous blood samples were collected into silicone test tubes. They were centrifuged upon cooling at 6,000 rpm 430 g for 3 minutes. Then, plasma was refrigerated immediately and stored at a temperature no higher than -35 ºС until they were analysed. Samples were processed according to the recommendations of the manufacturer of the analytical technique. SUA level was measured by enzymatic methods using a chemical analyzer Beckman (Synchron LX20, city, state). Analytical range average for SUA was 0.5-82 mmol/L. N-terminal pro-brain natriuretic peptide (NT-pro-BNP) was measured by immune-electro-chemiluminescence method using sets by R&D Systems (city, state, USA) on Elecsys 1010 analyzer (Roche, Mannheim, Germany). Calibration of the assay was performed according to the manufacturer’s recommendations and values were normalized to a standard curve. Concentrations of total cholesterol (TC) and high-density lipoprotein (HDL) cholesterol were determined with Dimension Clinical Chemistry System® (Dade Behring Inc, Newark, NJ). Low-density lipoprotein (LDL) cholesterol was calculated using Friedewald formula.20 (link)
3
Investigating Metabolic Biomarkers in Blood
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Venous blood samples were collected after an overnight fast. Plasma glucose and insulin were measured as well as other parameters including cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein (HDL-C), creatinine, high-sensitivity C-reactive protein, troponin-I, and B-type natriuretic peptide. Fasting insulin (FINS) levels were measured using a double-antibody sandwich immunoassay (Elecsys 1010 analyzer, Roche Diagnostics, Mannheim, Germany). IR was assessed using the homeostasis model assessment for insulin resistance (HOMA-IR) and the following formula: HOMA-IR (mmol/L×µU/mL) = fasting glucose (mmol/L)×fasting insulin (µU/mL)/22.5.16 (link)
4
Biomarker Determination in Blood Samples
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Blood samples were taken in the morning (at 7 - 8 A.M.) and poured into cooled silicone test tubes. The samples were processed according to the recommendations of the manufacturer of the utilized analytical technique.
Circulating NT-pro-BNP level was measured by immunoelectro chemoluminescent assay using sets by R&D Systems (USA) on Elecsys 1010 analyzer (Roche, Mannheim, Germany). Moreover, the serum concentrations of TNF-alpha, sFas, and sFas ligand were determined in duplicate using commercially available enzyme-linked immunosorbent assay kits (Bender MedSystems GmbH, Vienna, Austria). Overall, 100 μL of the serum samples was assayed in parallel to determine the standard concentrations for each biological marker. The mean intra-assay coefficients of variation were < 10% of all the cases. The concentrations of Total Cholesterol (TC) and High-Density Lipoproteins (HDLP) were measured by fermentation method. On the other hand, Low-Density Lipoproteins (LDL-C) concentration was computed according to the Friedewald formula (1972). All the biomarkers were determined at baseline.
Circulating NT-pro-BNP level was measured by immunoelectro chemoluminescent assay using sets by R&D Systems (USA) on Elecsys 1010 analyzer (Roche, Mannheim, Germany). Moreover, the serum concentrations of TNF-alpha, sFas, and sFas ligand were determined in duplicate using commercially available enzyme-linked immunosorbent assay kits (Bender MedSystems GmbH, Vienna, Austria). Overall, 100 μL of the serum samples was assayed in parallel to determine the standard concentrations for each biological marker. The mean intra-assay coefficients of variation were < 10% of all the cases. The concentrations of Total Cholesterol (TC) and High-Density Lipoproteins (HDLP) were measured by fermentation method. On the other hand, Low-Density Lipoproteins (LDL-C) concentration was computed according to the Friedewald formula (1972). All the biomarkers were determined at baseline.
5
Biomarker Measurement Protocol for Cardiovascular Assessment
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After an overnight fast blood samples were drawn in the morning (at 7–8 a.m.) into cooled silicone test tubes wherein 2 mL of 5% Trilon B solution were added; then they were immediately centrifuged upon permanent cooling at 6000 rpm for 10 min. Then, plasma was refrigerated immediately to be stored at a temperature − 70 °С. All laboratory tests were performed using standard methods to measure the serum fasting plasma glucose, fasting lipid profiles and other biomarkers.
N-terminal pro-brain natriuretic peptide (NT-pro-BNP) level was measured by immunoelectrochemoluminescent assay using sets by R&D Systems (USA) on Elecsys 1010 analyzer (Roche, Mannheim, Germany). High-sensitive C-reactive protein (hs-CRP) was measured by commercially available standard kit (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany). Galectin-3 was measured using an ELISA kit (BG Medicine, Germany). Concentrations of total cholesterol (TC), cholesterol of high-density lipoproteins (LDL-C), and cholesterol of high-density lipoproteins (HDL-C) were measured by enzymatic colorimetric method according standardized methodology on Beckman Synchron LX20 chemistry analyzer.
N-terminal pro-brain natriuretic peptide (NT-pro-BNP) level was measured by immunoelectrochemoluminescent assay using sets by R&D Systems (USA) on Elecsys 1010 analyzer (Roche, Mannheim, Germany). High-sensitive C-reactive protein (hs-CRP) was measured by commercially available standard kit (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany). Galectin-3 was measured using an ELISA kit (BG Medicine, Germany). Concentrations of total cholesterol (TC), cholesterol of high-density lipoproteins (LDL-C), and cholesterol of high-density lipoproteins (HDL-C) were measured by enzymatic colorimetric method according standardized methodology on Beckman Synchron LX20 chemistry analyzer.
6
Biomarker Measurement Protocol for Research
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All biomarkers were determined at baseline. To measurement of biological marker concentrations, blood samples were drawn in the morning (at 7–8 a.m.) into cooled silicone test tubes. Samples were processed according to the recommendations of the manufacturer of the analytical technique used. They were centrifuged upon permanent cooling at 6000 rpm for 3 min. Then, plasma was refrigerated immediately to be stored at a temperature − 70 °С until measurement.
Circulating NT-pro-BNP level was measured by immunoelectrochemoluminescent assay using sets produced by R&D Systems (USA) on Elecsys 1010 analyzer (Roche, Mannheim, Germany). The high-sensitivity C-reactive protein (hs-CRP) levels were measured by using nephelometric technique on AU640 analyzer manufactured by Diagnostic Systems Group (Japan).
Concentrations of total cholesterol (TC) and cholesterol of high-density lipoproteins (HDLP) were measured by the fermentation method. Concentration of cholesterol of low-density lipoproteins (LDL-C) was calculated according to the Friedewald formula (1972).
A total of 100 μL of serum samples was assayed in parallel to known standard concentrations for each biological marker. The mean intra-assay coefficients of variation were < 10% of all cases.
Circulating NT-pro-BNP level was measured by immunoelectrochemoluminescent assay using sets produced by R&D Systems (USA) on Elecsys 1010 analyzer (Roche, Mannheim, Germany). The high-sensitivity C-reactive protein (hs-CRP) levels were measured by using nephelometric technique on AU640 analyzer manufactured by Diagnostic Systems Group (Japan).
Concentrations of total cholesterol (TC) and cholesterol of high-density lipoproteins (HDLP) were measured by the fermentation method. Concentration of cholesterol of low-density lipoproteins (LDL-C) was calculated according to the Friedewald formula (1972).
A total of 100 μL of serum samples was assayed in parallel to known standard concentrations for each biological marker. The mean intra-assay coefficients of variation were < 10% of all cases.
7
Biomarker Measurement in Stable Patients
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Blood samples were collected at baseline in the morning (at 7–8 am) into cooled silicone test tubes, when patients were discharged from the hospital with stable clinical status. Samples were processed according to the recommendations of the manufacturer of the analytical technique used. They were centrifuged upon permanent cooling at 6000 rpm for 3 min. Then, plasma samples were stored at a temperature ≤−200 °C.
Circulating OSN level was determined by ELISA method (Bender MedSystems GmbH, Vienna, Austria). N-terminal pro-brain natriuretic peptide (NT-pro-BNP) concentration was measured by immunoelectrochemoluminescent assay using commercial kits produced by Roche (Mannheim, Germany) on Elecsys 1010 analyzer (Roche, Mannheim, Germany).
Concentrations of total cholesterol (TC) and cholesterol of high-density lipoproteins were measured by fermentation method.
Circulating OSN level was determined by ELISA method (Bender MedSystems GmbH, Vienna, Austria). N-terminal pro-brain natriuretic peptide (NT-pro-BNP) concentration was measured by immunoelectrochemoluminescent assay using commercial kits produced by Roche (Mannheim, Germany) on Elecsys 1010 analyzer (Roche, Mannheim, Germany).
Concentrations of total cholesterol (TC) and cholesterol of high-density lipoproteins were measured by fermentation method.
8
Plasma Biomarker Analysis for Cardiovascular Risk
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To determine TSP-2, N-terminal pro-brain natriuretic peptide (NT-pro-BNP), total cholesterol (TC) and cholesterol fractions, blood samples were drawn in the morning (at 7-8 a.m.) into pre-chilled silicone test tubes. Samples were processed according to the recommendations of the manufacturer of the analytical instruments used. They were centrifuged at 6,000 rpm for 3 minutes. Then, plasma was refrigerated immediately and stored at a temperature not higher than -35°C. Circulating galectin-3 (Gal-3) and TSP-2 levels were determined by ELISA (Bender MedSystems GmbH, Vienna, Austria). NT-pro-BNP content was measured by immunoelectrochemoluminescent assay using sets by R&D Systems (USA) on Elecsys 1010 analyzer (Roche, Mannheim, Germany). Concentrations of TC and high-density lipoprotein cholesterol (HDL-C) were measured by fermentation method. Concentration of low-density lipoprotein cholesterol (LDL-C) was calculated according to the Friedewald formula (1972).
9
Multisite Glycemic Biomarker Measurement
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Serum insulin concentrations were measured with standardized assays at each institution (reported coefficient variations ≤10%); NIH: Roche Cobas 6000 analyzer (Roche Diagnostics, Indianapolis, IN), CHOP: ELISA, ALPCO Diagnostics (Salem, NH), and Baylor: Elecsys 1010 Analyzer (Roche Diagnostics, Indianapolis, IN). HbA1c was measured with high-performance liquid chromatography. Data for HbA1c (n = 7), glucose (n = 1), and insulin (n = 2) in some participants were not available due to technical difficulties.
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