Enzygnost tat micro kit

Anesthetized male Wistar Han rats (150–200 g) (n = 6/group) were transplanted in sterile conditions with one of three different cell dosages, 5 × 10⁶ cells/kg, 1.25 × 10⁷ cells/kg or 5 × 10⁷ cells/kg, by intraportal injection, with or without intravenous administration of bivalirudin (0.75 mg/kg Angiox, Medicines Company, NJ, USA). The cell dose range originated from clinical cases of patients transplanted with HHALPCs [21 (link),33 (link)] or hepatocytes [34 (link),35 (link)]. For safety studies, the lowest cell dose, 5 × 10⁶ cells/kg, was infused by intraportal and peripheral infusion. Rats were sacrificed after 1 h, and their livers were harvested and fixed in 4% formaldehyde. Blood samples were taken from the portal vein, both before and after transplantation, with a syringe containing trisodium citrate 3.8% (dilution 1:9). Total blood counts were analyzed by an automatic hematology analyzer (Cell-DYN Emerald, Abbott Diagnostics, IL, USA). Plasma was obtained by centrifugation at 2700 g for 15 min and stored at −80 °C. Levels of fibrinogen and coagulation factors II, V, VIII and X were measured on an automated blood coagulation analysis system (ACL TOP 700 Werfen, Bedford, MA, USA). Thrombin–antithrombin (TAT) complex levels were measured by an ELISA assay (Enzygnost TAT micro kit, Siemens Healthineers, Germany).

Plasma D-dimer and TAT complexes were quantified using the Innovance™ D-dimer test on a BCS™ coagulation analyzer and the Enzygnost™ TAT micro kit, respectively (Siemens Healthcare).

Citrated plasma was thawed in a 37°C water bath for 15 min and tested for the following coagulation parameters using a Siemens BCS XP automated coagulation analyzer system: prothrombin time (PT; Dade Innovin), activated partial thromboplastin time (aPTT; Dade Actin FS APTT Reagent), vWF ristocetin cofactor activity (vWF:RCo; BC vWF Reagent), coagulation factor VIII (FVIII; Factor VIII Deficient Plasma), Protein C activity (Berichrom Protein C), D-dimer (Innovance D-Dimer Reagent kit). Additional analysis entailed immunoassays to measure vWF antigen (vWF:Ag; vWF ELISA, Imubind American Diagnostica, Inc.), thrombin-antithrombin III complex (TAT; Enzygnost TAT micro kit, Siemens), and t-PA (t-PA ELISA; Imubind, American Diagnostica, Inc.). Analyses for all samples were processed per manufacturer's directions.

TATc (Enzygnost® TAT micro kit, Siemens, Tarrytown, NY) and CXCL1 (DuoSet, R&D Systems, Minneapolis, MN) were quantified by ELISA according to manufacturers instructions.

Plasma was generated from patient blood samples by centrifugation (2500× g for 15 min) and was frozen at −80 °C immediately. Enzyme-linked immunosorbent assays (ELISAs) were performed according to respective instruction manuals to determine mediators of hemostasis and inflammation. Tissue plasminogen activator (t-PA) Combi Actibind, as well as plasminogen activator inhibitor-1 (PAI-1) and t-PA–PAI-1 complex, ELISA kits were purchased from Technoclone (Vienna, Austria). An ELISA kit from USCN (USCN Life Sciences Inc., Wuhan, China) was used to determine plasmin–antiplasmin (PAP) complex levels in plasma. An activated protein C–protein C inhibitor (APC–PCI) complex kit was obtained from BioPorto diagnostics (Hellerup, Denmark) and soluble thrombomodulin was measured with a kit from Asserachrom (Diagnostica Stago, Asnieres, France). Interleukin-6 levels were measured with an Immulite 1000 device (Siemens Healthcare, Erlangen, Germany). Thrombin–antithrombin (TAT) complex was determined with the Enzygnost© TAT Micro-kit from Siemens (Siemens Healthcare, Erlangen, Germany). Furthermore, ELISA measurements of histonylated, citrullinated DNA fragments (Cell Death Detection ELISA kit-plus, Roche, Basel, Switzerland) and PMN Elastase (alpha 1PI-complex, Milenia Biotec, Nauheim, Germany) were performed.

Plasma was prepared by centrifuging blood at 4,000 x g for 15 minutes at room temperature and aliquoted and frozen at -80°C until analysis. Thrombin-antithrombin (TAT) complex levels were measured at the end of the experiment by enzyme-linked immunosorbent assay (ELISA) (Siemens Enzygnost TAT Micro Kit). Plasma inflammatory mediators were measured using the Bio-Plex Pro Rat Cytokine 23-Plex Assay and HMGB-1 ELISA (IBL-International). Plasma creatinine was measured by ELISA (Invitrogen). All samples were performed in duplicate. Sample sizes vary due to differences in plasma sample collection volumes.