Thrombin is routinely combined in surgical practice with a fibrinogen source to prepare fibrin sealant to promote hemostasis or with platelet concentrates to prepare platelet gels to enhance wound healing. The purpose of this study was to evaluate the robustness and reproducibility of a new sterile handheld disposable thrombin-processing device (TPD) to generate autologous human thrombin in the intraoperative setting, using whole blood as the starting source material. By using whole blood instead of plasma as the starting material, it is possible to eliminate the plasma separation step from whole blood and reduce the thrombin production time and increase its availability to the surgical team intraoperatively. Active thrombin was prepared by combining 4 mL of thrombin reagent (a mixture of calcium chloride and ethanol) to 11 mL of blood in a reaction chamber containing negatively charged particles. The whole blood, reagent and particle mixture was incubated for 25 minutes at either 18 degrees C or 24 degrees C (n = 25/group) to assess stability of the thrombin activity. The mean activity of the thrombin produced at 18 degrees C and 24 degrees C was 52 +/- 14 (n = 25) and 61 +/- 12.2 IU/mL (n = 25), respectively. The average volume of thrombin harvested from each aliquot of blood at 18 degrees C and 24 degrees C was 10 +/- 0.4 and 9 +/- 0.6 mL, respectively. The thrombin concentration generated was shown to rapidly (<5 seconds) coagulate fibrinogen concentrate and retained clotting activity for 1 hour at room temperature (18-26 degrees C) and up to 4 hours when stored on ice. The results show that the TPD is able to consistently generate high thrombin activity from human whole blood. The device offers a robust and rapid approach for preparing active thrombin from whole blood.