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15 protocols using n high sensitivity crp

1

Cardiovascular Risk Factors Assessment

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A number of evaluations were performed as part of MESA Visit 1. HsCRP was measured using a BNII nephelometer (N-High Sensitivity CRP; Dade Behring Inc, Deerfield, IL). Sociodemographic characteristics, family history of CHD, tobacco use, and medication use were self-reported using a standardized questionnaire. Resting blood pressure and waist circumference were measured following standardized protocols [18 (link)]. Fasting levels of total and high density lipoprotein (HDL) cholesterol, and blood glucose were measured at the MESA central laboratory at the University of Minnesota.
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2

Inflammatory Biomarker Measurement Protocol

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To obtain the inflammatory profile, 15 mL of blood was taken via vein puncture, always in the morning. It was stored in a tube containing protease inhibitor and EDTA, then centrifuged at 3000 rpm for 15 min at 4 °C. The resulting plasma was transferred to micro centrifuge tubes (1.5 mL) and stored at −80 °C. This sample was later sent for analysis of Interleukin-6 (IL-6), Interleukin-10 (IL-10), Cortisol, and C-reactive Protein (CRP).
IL-6, IL-10, and cortisol were measured by the Enzyme-Linked Immuno-Sobent Assay (ELISA) method and Chemiluminescent Competitive Immunoassay following the manufacturer’s recommendations. The quantitative determination of CRP was performed by nephelometry (Dade-Behring N High Sensitivity CRP).
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3

Biomarker Analysis in Coagulation Disorders

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Baseline and follow‐up blood samples were analyzed in cases and controls using the following methods: fibrinogen (clot‐rate assay, STA‐R instrument, Diagnostica Stago, Parsippany, NJ, USA), factor VIII and IX activity (clotting time on mixing with factor VIII or IX deficient plasma using STA‐Deficient VIII or IX; STA‐R instrument, Diagnostica Stago), von Willebrand factor, antithrombin and D‐dimer (immunoturbidometric or colorimetric assays, Liatest von Willebrand factor, Liatest D‐Di, Stachrom ATIII; STA‐R instrument, Diagnostica Stago), PAI‐1, PAP and prothrombin antigen (in‐house immunoassays),12, 13 prothrombin fragment 1.2 (ELISA, Dade Behring, Marburg, Germany), protein C antigen, free and total protein S antigen (Asserachrom ELISA, Diagnostica Stago), CRP (nephelometry, N High Sensitivity CRP, Dade‐Behring, Deerfield, IL, USA). Distributions of each biomarker were examined blind to case control status. Analytical outliers were defined based on knowledge of the biology and excluded from analysis as follows: factor VIIIc or prothrombin antigen <10%, fragment 1.2 > 7.2 nmol/L (>3 SD above the mean), PAI‐1 > 70 ng/mL.14
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4

Comprehensive Lipid and Inflammatory Biomarker Profiling

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Fasting blood samples were collected in the early morning with patients in the supine position. Routine laboratory parameters were measured on a daily basis as previously described[18 (link)]. The lipoproteins were separated using a combined ultracentrifugation–precipitation method (β-quantification). Cholesterol and triglycerides were measured with enzymatic reagents from WAKO (Neuss, Germany) on a WAKO 30 R analyser. Apolipoproteins A-I, and B were measured by turbidimetry (Rolf-Greiner Biochemica, Flacht, Germany). Creatinine was measured with the Jaffé method on a Hitachi 717 analyzer (Roche Diagnostics, Mannheim, Germany). The CRP concentrations were determined using immunonephelometry (N High Sensitivity CRP, Dade Behring, Marburg, Germany). Total PCSK9 was measured using the Quantikine Human PCSK9 sandwich immunoassay (R&D, Minneapolis, MN, USA).
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5

Metabolic Profile Characterization

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Total and high-density lipoprotein (HDL) cholesterol, triglycerides, and glucose levels were measured from blood samples obtained after a 12-h fast. Low-density lipoprotein cholesterol was calculated by the Friedewald equation. Diabetes was defined as fasting glucose >125 mg/dl or use of hypoglycemic medication. C-reactive protein (CRP) was quantified by a high-sensitivity assay (N-High-Sensitivity CRP; Dade Behring, Deerfield, IL; inter-assay coefficient of variation: 2.1–5.7%).
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6

Measurement of High-Sensitivity C-Reactive Protein

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hsCRP was measured using the BNII nephelometer (N High Sensitivity CRP; Dade Behring Inc., Deerfield, Illinois) at the Laboratory for Clinical Biochemistry Research (University of Vermont, Burlington, Vermont). Analytical intra-assay coefficient of variations ranged from 2.3% to 4.4%, and inter-assay coefficient of variation ranged from 2.1% to 5.7% with a detection level of 0.18 mg/L.
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7

Ranolazine Effects on Systemic Inflammation

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Human samples were analyzed for CRP in patients undergoing ranolazine treatment within a previous study (44 (link)). Briefly, 51 patients aged between 32 and 65 y were included in a chronic phase of CAD at least 12 mo after acute myocardial infarction. All patients were treated with β-blockers, antiplatelet therapy, angiotensin-converting inhibitors or sartans, and statins. The therapy was not changed at least 6 wk prior to inclusion and during the study. In this double-blind study, patients were randomized to ranolazine 375 mg twice daily for the first 4 wk and 500 mg twice daily for 8 wk or to control treatment. Baseline patient characteristics including age, body mass index, cholesterol, high-density lipoprotein, LDL, and triglyceride levels can be found in SI Appendix, Table 1. Blood was drawn before and after 12 wk of treatment. High-sensitivity CRP was measured with a fully automated, latex-enhanced nephelometric immunoassay (N High Sensitivity CRP; Dade Behring).
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8

Citrate-Anticoagulated Blood Sampling

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Blood samples were obtained using antecubital venipuncture with minimal compression and a 21 G needle. At each sampling time (baseline and 21 days after treatment withdrawal), the first 3 mL of blood were discarded and then 21 plastic tubes with 0.109 M trisodium citrate (Vacutainer®, BD Biosciences, Erembodegem, Belgium) were filled. The samples were immediately sent to the laboratory, which performed testing for hs-CRP (N High Sensitivity CRP, Dade Behring®; normal range: <5 mg/L) on a Behring Nephelometer II System and for DD (Acute Care™ D-dimer test pack, Siemens Healthcare Diagnostics, Newark, DE, USA; normal range: <500 µg/L).
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9

Biomarker Measurement in Clinical Study

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Blood samples were collected, centrifuged, and serum and plasma was separated and shipped overnight on ice packs to the Laboratory for Clinical Biochemistry Research at the University of Vermont. High sensitivity CRP was analyzed by particle-enhanced immunonephelometry using the BNII nephelometer according to the manufacturer’s instructions (N High Sensitivity CRP; Dade Behring Inc., Deerfield, IL); the interassay coefficients of variation (CV) were 2.1%–5.7%. Albumin and glucose were measured in serum by colorimetric reflectance spectrophotometry using the Ortho Vitros Clinical Chemistry System 950IRC instrument (Johnson & Johnson Clinical Diagnostics; Rochester, NY); the CV was < 5% for albumin and 1% for glucose.
Complete blood counts were performed the day after sample collection using a Beckman Coulter LH 755 Hematology Workcell (Beckman Coulter, Incorporated; Fullerton, California); CVs of 5% and 10% for WBC and PLTC, respectively, were considered acceptable. Albumin, WBC, and PLTC were measured among consecutive individuals as part of an ancillary study that began after enrollment had started, resulting in 19,393 participants with serum albumin measured, 18,334 with WBC information, and 17,850 with PLTC measured.
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10

Inflammatory Biomarker Measurement in Cardiovascular Research

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Serum hsCRP was measured using a high‐sensitivity assay (N‐High‐Sensitivity CRP; Dade Behring, Deerfield, IL) (intra‐assay coefficient of variation [CV] ranged from 2.3% to 4.4% and the interassay CV ranged from 2.1% to 5.7%). IL‐6 was measured by ultrasensitive enzyme‐linked immunosorbent assay (Quantikine HS Human IL‐6 Immunoassay; R&D Systems, Minneapolis, MN) (analytical CV 6.3%).39 Serum fibrinogen was measured by immunoprecipitation using the BNII nephelometer (N‐Antiserum to Human Fibrinogen; Dade Behring) (intra‐assay and interassay CV as 2.7% and 2.6%, respectively). We evaluated inflammation markers as continuous. We also categorized hsCRP ≥2 mg/L as suggested in a previous study.40
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