Enzygnost tat micro

After collection, BALFs were immediately put on ice and then centrifuged for 10 min at 900 ×g at 4 °C. The supernatant was aliquoted and stored at − 80 °C until assays were performed. The levels of prothrombin fragment 1 + 2 (F_{1 + 2}), thrombin–antithrombin complex (TATc), soluble TF (sTF) antigen, plasminogen activator inhibitor 1 (PAI‐1) and tissue‐type plasminogen activator (t‐PA) were measured with specific enzyme immunoassays, according to the manufacturers’ instructions (F_{1 + 2}, Enzygnost F_{1 + 2} [Siemens, Marburg, Germany]; TATc, Enzygnost TAT micro [Siemens]; sTF, Imubind Tissue Factor ELISA [Sekisui Diagnostics, Stamford, CT, USA]; PAI‐1 activity, Technozym PAI‐1 Actibind ELISA [Technoclone, Vienna, Austria]; PAI‐1 antigen, Quantikine Human Total Serpin E1/PAI‐1 Immunoassay [R&D Systems, Minneapolis, MN, USA]; and t‐PA antigen and activity, Technozym t‐PA Combi Actibind ELISA [Technoclone]). The F_{1 + 2}, TATc and sTF assays are sensitive to detect even low values in plasma and BALF of healthy volunteers as previously described 22, 23, 24. The lower limits of quantification were 20 pmol L⁻¹ for F_{1 + 2}, 2 μg L⁻¹ for TATc, 0.05 ng mL⁻¹ for sTF, 0.49 IU mL⁻¹ for PAI‐1 activity, 0.313 ng mL⁻¹ for PAI‐1 antigen, 0.05 IU mL⁻¹ for t‐PA activity and 0.1 ng mL⁻¹ for t‐PA antigen). Fibrinogen was measured with the Clauss method in an accredited routine laboratory.

Leucocyte, haemoglobin and platelet counts were obtained on EDTA anticoagulated blood. For coagulation assays, blood (four parts) was collected in tubes containing 3.8% sodium citrate (one part). Fibrinogen levels were rapidly measured by standard procedures. Immunoassay methods were used to determine quantitative thrombin–antithrombin complexes (TAT) (Enzygnost^® TAT micro, Siemens, Munich, Germany). Fibrin monomer (Liatest FM^® Stago, Asnières, France) was performed by immunoturbidimetric assay. The quantitative determination of vWF antigen (Ag) was measured by turbidimetric assay (vWF Ag^® Reagent, Siemens, Nederland) (n = 70–100%).
TNFα and interleukin-6 (Il-6) were measured in serum. Plasma levels were detected by ELISA method with porcine anti-TNFα antibodies (Quantikine^® Porcine TNFα, R&D Systems, USA) and anti-Il-6 antibodies (Quantikine^® Porcine Il-6, R&D Systems, USA).
Blood and urinary creatinine levels (Cobas^® 8000 modular analyser, Roche Diagnostics, Switzerland) were measured at each time except T30. We computed creatinine clearance, a surrogate marker of GFR, with standard formula [creatinine clearance (CrCl) = (creatinine urinary concentration × rate of urine formation)/Creatinine plasma concentration]. Diuresis, a marker of both GFR and tubular function, was measured at each time except T30.

Citrated plasma samples were thawed for 15 minutes at 37°C and gently mixed before the concentration of TAT complexes was determined with a sandwich enzyme immunoassay (Enzygnost®TAT micro, Siemens Healthcare Diagnostics) previously validated for canine plasma [27 (link)], according to the manufacturer’s protocol. Plasma samples had been thawed once previously. The standards included in the Enzygnost®TAT micro kit ranged from 2.0 to 60 µg/L. In samples with values above the highest standard, the software extrapolated the concentration of TAT, and the absolute values above 60 µg/L should therefore be interpreted with caution.

The influence of the two different abdominal swab materials on the activation of the complement system, blood clotting, activation of platelets, and activation of neutrophils was evaluated by the detection of different activation markers using ELISAs, which were performed according to the manufacturer’s instructions. For this purpose, the shock frozen plasmas were used. The following activation markers were measured: terminal complement complex (sC5b-9) (MicroVue™ Complement, Quidel, Osteomedical GmbH, Sissach, Switzerland) in EDTA plasma, thrombin–antithrombin III complex (TAT) (Enzygnost^® TAT micro, Siemens Healthcare, Erlangen, Germany) and the polymorphonuclear (PMN)-elastase (PMN-elastase ELISA, demeditec, Kiel, Germany) in citrated plasma, β-thromboglobulin (β-TG) (Asserachrom^® β-TG, Diagnostica Stago, Düsseldorf, Germany) in CTAD plasma.

Plasma concentrations of D-dimer were established using a Sysmex^® CA-7000 System Automated Coagulation Analyzer with reagents obtained from Siemens Healthcare Diagnostics (Marburg, Germany). D-dimer measurements were performed using the INNOVANCE^® D-dimer assay [Reference range: < 550 ng/mL]. TAT complexes were quantified by a commercially available immunoassay (Enzygnost^® TAT Micro, Siemens Healthcare Diagnostics, Marburg, Germany) [Reference range: 2.0–4.2 ng/mL].