Teg 5000 thrombelastography hemostasis analyzer

Whole blood was collected in 3.2 % sodium citrate tubes at a 1:10 ratio, based on a standardized model of performing TEG in rodents (16 (link)). Individual microcentrifuge tubes were prefilled with citrate and marked to an appropriate fill level to ensure reproducible ratios of whole blood to citrate. Citrated native thrombelastography assays were re-calcified and run according to the manufacturer's instructions on a TEG 5000 Thrombelastography Hemostasis Analyzer (Haemonetics, Niles IL). The CL30 parameter was used to quantify fibrinolysis. This variable corresponds to the percent of clot strength remaining 30 minutes after reaching maximum amplitude. Blood not used for TEG was spun to yield plasma for protein analysis. Whole blood was centrifuged at 6,000 g for 10 minutes at 4 °C. Plasma was removed and then spun at 12,500 g for 10 minutes at the same temperature to remove contaminating platelets and acellular debris. The remaining plasma was flash frozen in liquid nitrogen and stored at -80 °C until it was analyzed.

Viscoelastic assays were completed by a team of trained PRA with extensive experience in multiple types of TEG assays. Citrated blood samples were analyzed using the TEG 5000 Thrombelastography Hemostasis Analyzer (Haemonetics Corp., Niles, IL, USA). The following indices were obtained from the tracings of the TEG: reaction time (R-time min.), angle (°), maximum amplitude (MA [mm]), and lysis 30 min after MA (LY30 [%]). Fibrinolysis phenotypes were based on LY30 and previously reported ranges of phenotypes (1 (link)); shutdown LY30 <0.9%, physiologic 0.9–2.9%, and hyperfibrinolysis >2.9%.

Viscoelastic assays were completed by a team of trained PRA with extensive experience in multiple types of TEG assays. Citrated blood samples were analyzed using the TEG 5000 Thrombelastography Hemostasis Analyzer (Haemonetics, Niles, IL, USA). The following indices were obtained from the tracings of the TEG: reaction time (R-time min.), angle (°), maximum amplitude (MA [mm]), and lysis 30 min after MA (LY30 [%]). Modified assays to quantify sensitivity to fibrinolysis were run in parallel with rapid TEG assay (r-TEG, activated by tissue factor and kaolin). The same TEG analyzer was used for this assay without an activator (native TEG), but prior to re-calcification exogenous t-PA (t-TEG) was added. This t-PA challenge of whole blood has been previously validated to quantify t-PA sensitivity and resistance in vitro to assess the effects of different proteins (14 , 15 (link)), as well as clinically in trauma patients (9 (link)). In brief, 500 microliters of whole blood were pipetted into a customized vial containing lyophilized t-PA (Molecular Innovation, Novi, MI, USA) to a final concentration of 75 ng/mL of t-PA, and mixed by gentle inversion. A 340-μL aliquot of this mixture was then transferred to a 37 °C TEG cup, preloaded with 20 μL of 0.2 mol/L CaCl2.