All biomarkers were analysed through commercially available kits. PTX3 was quantified in plasma samples through an enzyme-linked immunosorbent assay (ELISA) kit (Human Pentraxin 3/TSG-14 Quantikine ELISA Kit, R&D Systems, Minnesota, USA). Three classical inflammatory biomarkers were evaluated in serum samples: high-sensitivity (hs) CRP by immunoturbidimetry (Cardiac C-Reactive Protein (Latex) High Sensitive assay, Roche Diagnostics, Basel, Switzerland), IL-6, and TNF-α (Human IL-6 Quantikine HS and Human TNF-alpha Quantikine HS, R&D Systems). Lipid profile, including total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), was performed using laboratorial routine procedures (Cobas Integra 400 Plus autoanalyser; Roche Diagnostics, Basel, Switzerland); plasma oxidized LDL (oxLDL) and serum adiponectin, and leptin levels were determined through ELISA kits (Oxidized LDL ELISA Kit, Mercodia AB, Uppsala, Sweden; Human Total Adiponectin/Acrp30 Quantikine ELISA Kit and Human Leptin Quantikine ELISA Kit, R&D Systems). Levels of plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) and serum tissue inhibitor of metalloproteinase-1 (TIMP-1) (Human proBNP and Human TIMP1 ELISA kits, Abcam, Cambridge, UK) were assessed as cardiac and renal fibrosis markers, respectively.
Human il 6 quantikine hs
Manufactured by R&D Systems
Sourced in United States, Switzerland
The Human IL-6 Quantikine HS is a quantitative sandwich enzyme immunoassay designed to measure human interleukin-6 (IL-6) levels in cell culture supernates, serum, and plasma. It has a high sensitivity detection range.
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Lab products found in correlation
Human pentraxin 3 tsg 14 quantikine elisa kit, by R&D Systems (3 mentions)
Cardiac c reactive protein latex high sensitive assay, by Roche (3 mentions)
Human tnf alpha quantikine hs, by R&D Systems (2 mentions)
Human gdf15 elisa kit, by Abcam (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Oxidized ldl elisa kit, by Mercodia (1 mentions)
Human leptin quantikine elisa kit, by R&D Systems (1 mentions)
Human total adiponectin acrp30 quantikine elisa kit, by R&D Systems (1 mentions)
Human leptin, by Merck Group (1 mentions)
Free style, by Nipro (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
K 1000, by Sysmex (1 mentions)
Vacutainer, by BD (1 mentions)
12 protocols using human il 6 quantikine hs
1
Comprehensive Biomarker Assessment Protocol
2
Cytokine Biomarker Sampling Protocol
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Blood samples were collected at 8 am, centrifuged, and stored at −80°C until thawed for assay. Participants were instructed to avoid exercise and alcohol 24 hours prior to blood withdrawal. Also, participants were instructed to avoid rushing and taking public transportation to the laboratory to avoid physical overexertion before blood sampling. Serum concentrations of IL-6 and TNF-alpha were determined using highly sensitive immunoassays according to the manufacturer’s instructions (Human IL-6 Quantikine HS and Human TNF-alpha Quantikine HS; R & D Systems, Minneapolis, MN, USA).
3
Cytokine Profiling in Serum Samples
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The serum samples were analyzed for the cytokines IL-1 RA, IL-6, and TNF α. All assays were carried out with quantitative enzyme-linked immunosorbent assay (ELISA) kits in accordance with the manufacturers’ instructions (Human IL-6 Quantikine HS, Human TNF α Quantikine HS; Human IL-1 RA Quantikine, all from R&D Systems, Minneapolis, MN, USA). A highly sensitive ELISA was used for IL-6 and TNF α. According to the manufacturer, the intra-assay coefficient of variation was between 6.9% and 7.8% for IL-6, between 3.1% and 8.5% for TNF α, and between 3.7% and 7.3% for IL-1 RA, with a minimum detectable amount of 0.039 pg/ml for IL-6, 0.106 pg/ml for TNF α, and 6.26 pg/ml for IL-1 RA.
4
Plasma Biomarkers in Longitudinal Study
5
Exercise-Induced Changes in Iron Metabolism
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Blood samples were collected from an antecubital vein before exercise (before the start of the exercise period), immediately after exercise, and 3 h after exercise. The samples were centrifuged (3,000 rpm, 4°C), and serum and plasma samples were stored at -80°C until subsequent analyses. Serum ferritin, iron, and hepcidin levels were measured. Ferritin and iron levels were measured at a clinical laboratory (SRL Inc., Tokyo, Japan), and hepcidin levels were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). Plasma IL-6 levels were measured using an ELISA kit (Human IL-6 Quantikine HS; R&D Systems). Glucose and lactate levels were measured immediately after the collection of blood using a glucose analyzer (Freestyle, Nipro Co., Osaka, Japan) and a lactate analyzer (Lactate Pro; Arkray Co., Kyoto, Japan), respectively.
6
Quantification of Inflammatory Biomarkers and Genetic Profiling
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Total leukocyte cell count was assessed in whole-blood using an automatic blood cell counter (Sysmex K1000; Sysmex, Hamburg, Germany).
All inflammatory biomarkers were analyzed through commercially available kits, according to the manufacturer’s instructions. Plasma samples were used to quantify PTX3 (Human Pentraxin 3/TSG-14 Quantikine ELISA Kit, R&D Systems, Minnesota, USA; sensitivity 0.026 ng/mL) and GDF15 (Human GDF15 ELISA Kit, Abcam, Cambridge, UK; sensitivity 2 pg/mL) through enzyme-linked immunosorbent assays (ELISA). In serum we measured IL6 by ELISA (Human IL6 Quantikine HS, R&D Systems; sensitivity 0.09 pg/mL) and hsCRP by immunoturbidimetry [Cardiac C-Reactive Protein (Latex) High Sensitive assay, Roche Diagnostics, Basel, Switzerland; sensitivity 0.15 mg/L].
Genomic DNA was extracted from buffy-coat samples, using genomic DNA extraction kit (GRiSP, Research Solutions, Porto, Portugal), quantified by NanoDrop-1000 (ThermoFisher Scientific, Wilmington, DE, USA) and analyzed by agarose gel electrophoresis. Trademark TaqMan SNP genotyping assays (Human; ThermoFisher Scientific) were performed to assess the allelic frequencies of IL6 (rs1800795) and PTX3 (rs2305619) polymorphisms, using a real-Time PCR system (StepOnePlus, ThermoFisher Scientific).
All inflammatory biomarkers were analyzed through commercially available kits, according to the manufacturer’s instructions. Plasma samples were used to quantify PTX3 (Human Pentraxin 3/TSG-14 Quantikine ELISA Kit, R&D Systems, Minnesota, USA; sensitivity 0.026 ng/mL) and GDF15 (Human GDF15 ELISA Kit, Abcam, Cambridge, UK; sensitivity 2 pg/mL) through enzyme-linked immunosorbent assays (ELISA). In serum we measured IL6 by ELISA (Human IL6 Quantikine HS, R&D Systems; sensitivity 0.09 pg/mL) and hsCRP by immunoturbidimetry [Cardiac C-Reactive Protein (Latex) High Sensitive assay, Roche Diagnostics, Basel, Switzerland; sensitivity 0.15 mg/L].
Genomic DNA was extracted from buffy-coat samples, using genomic DNA extraction kit (GRiSP, Research Solutions, Porto, Portugal), quantified by NanoDrop-1000 (ThermoFisher Scientific, Wilmington, DE, USA) and analyzed by agarose gel electrophoresis. Trademark TaqMan SNP genotyping assays (Human; ThermoFisher Scientific) were performed to assess the allelic frequencies of IL6 (rs1800795) and PTX3 (rs2305619) polymorphisms, using a real-Time PCR system (StepOnePlus, ThermoFisher Scientific).
7
Biomarker Analysis and Genetic Profiling
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All biomarkers were analyzed through commercially available kits, according to the manufacturer's instructions. Plasma samples were used to quantify PTX3 (Human Pentraxin 3/TSG-14 Quantikine ELISA Kit, R&D Systems, Minnesota, USA) and GDF15 (Human GDF15 ELISA Kit, Abcam, Cambridge, UK) through enzyme-linked immunosorbent assay (ELISA). In serum we measured IL6 by ELISA (Human IL6 Quantikine HS, R&D Systems, by ELISA) and high-sensitivity (hs)CRP by immunoturbidimetry [Cardiac C-Reactive Protein (Latex) High Sensitive assay, Roche Diagnostics, Basel, Switzerland].
Genomic DNA was extracted from buffy-coat, using genomic DNA extraction kit (GRiSP, Research Solutions, Porto, Portugal), quanti ed by NanoDrop™-1000 (ThermoFisher Scienti c, Wilmington, DE, USA) and analyzed by agarose gel electrophoresis. Trademark TaqMan® SNP genotyping assays (Human; ThermoFisher Scienti c) were performed to assess the allelic frequencies of IL6 (rs1800795) and PTX3 (rs2305619) polymorphisms, using a real-Time PCR system (StepOnePlus, ThermoFisher Scienti c).
Genomic DNA was extracted from buffy-coat, using genomic DNA extraction kit (GRiSP, Research Solutions, Porto, Portugal), quanti ed by NanoDrop™-1000 (ThermoFisher Scienti c, Wilmington, DE, USA) and analyzed by agarose gel electrophoresis. Trademark TaqMan® SNP genotyping assays (Human; ThermoFisher Scienti c) were performed to assess the allelic frequencies of IL6 (rs1800795) and PTX3 (rs2305619) polymorphisms, using a real-Time PCR system (StepOnePlus, ThermoFisher Scienti c).
8
Inflammatory Marker Analysis in Plasma Samples
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Plasma sampled at baseline was frozen at -70 0C and stored in a central core lab (Cedars-Sinai, LA, CA) for subsequent measurement of inflammatory markers. Levels of IL-6 was measured using a commercially available enzyme-linked immunosorbent assay kit (Quantikine hs human IL-6, R&D Systems, Minneapolis, Minn.). Levels of high-sensitivity CRP (hsCRP) and SAA were measured by a high-sensitivity method on the Hitachi 911 analyzer using reagents from Denka Seiken (Niigata, Japan). All samples were assayed within 5 years of collection at a core laboratory (Ridker P, Brigham and Women’s Hospital, Boston, Mass) using previously validated techniques [8 (link)].
9
Plasma IL-6 Quantification Protocol
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The fasting whole blood samples (10 mL) were collected in the morning (9–11 am) and placed into EDTA tubes. The tubes were immediately centrifuged (at 3000 r/min for 15 min) at 4°C; then, the plasma was divided into multiple aliquots and frozen in a − 80°C freezer until assay. The plasma level of IL-6 was examined by a high sensitivity enzyme-linked immunosorbent assay (ELISA, Quantikine HS Human IL-6, R&D Systems, Minneapolis, Minnesota) according to the manufacturer’s protocol. The assay has a lower limit of quantitation of 0.2 pg./mL, a range of 0.2–10 pg./mL, and a sensitivity of 0.09 pg./mL. All samples were tested in duplicate. The experimental operators were blinded to all the clinical data.
10
Cytokine Profiles in Transfusion Patients
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In a subgroup of patients (10 patients randomly selected from each transfusion policy allocation group) IL-6, IL-10, and TNFα were measured. Peripheral venous blood was drawn at the following time points: preoperatively, 6 hours, one day, and three days postoperatively. All samples were collected in sterile tubes (Vacutainer, Becton-Dickinson, Heidelberg, Germany) and were immediately centrifuged and the supernatant was stored at −60°C until assay. Quantitative determination of cytokine levels was performed using commercially available sensitive immunoassay kits (Quantikine HS human IL-6, Quantikine HS IL-10, and Quantikine HS human TNFα for IL-6, IL-10, and TNFα, resp.) (R&D Systems Inc. 614 McKinley Place NE, MN, USA), according to the recommendations of the manufacturer. Detection sensitivity was 0.039 pg mL−1 for IL-6, 3.9 pg mL−1 for IL-10, and 0.106 pg mL−1 for TNFα. The coefficient of variability of the method was 6.5–9.6% for IL-6, 4.3–7.5% for IL-10, and 5.3–16.7% for TNFα. All assays were performed in duplicate and averaged data were used in the subsequent analysis.
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