Standardization of setting

General anesthesia was managed by a group of cardiac anesthesiologists using a standardised care protocol. The study was conducted in a dedicated cardiac theatre with standardization of ambient air temperature set to 21 °C (69.8 F). All patients were fasted 6-h for solids and 2-h for clear fluids. Prior to induction of anesthesia there was no additional intravenous fluid administered. Induction of anesthesia consisted of a standard technique using propofol (0.5–1 mg/kg), fentanyl (5–10 μg/kg) and a non-depolarizing neuromuscular blocker. Maintenance of anesthesia was achieved using sevoflurane or isoflurane in 50 % oxygen: 50 % air ratio titrated to a bispectral index (BIS) of 40–60. No patient received any antifibrinolytic therapy (e.g. tranexamic acid). Intra-operative autologous hemodilution was not utilised as part of the anesthesia technique for any patient. Prior to initiation of CPB fluid intervention was restricted but if clinically indicated, a balanced crystalloid solution was used. No colloid solutions were used in the pre CPB period. Prior to commencement of CPB heparin (300 U/kg) was administered to achieve a therapeutic activated clotting time (ACT) of greater than 480 s. If ACT was sub-therapeutic repeated boluses of heparin (5000 U) were administered. The CPB circuit was primed with 10,000 U heparin. For the pump prime crystalloid solution, all patients received 2000 ml of Plasma-Lyte 148^® (Baxter, Sydney, NSW). Cardiopulmonary bypass was performed using a membrane oxygenator (Quadrox-i, Maquet Cardiopulmonary, Hirrlingen, Germany). The pump rate was set at 2.4 l m⁻² min and body temperature was maintained between 32 and 34 °C (89.6–93.2 F). A standard induction cardioplegia solution (Baxter Viaflex^®, AHK 5560, Toongabie, NSW) followed by a maintenance cardioplegia solution (Baxter Viaflex^® AHK6029, Toongabie, NSW) was administered to all patients.

During CPB the ACT was maintained above 480 s with additional boluses of heparin if required. If volume supplementation was required during CPB, fluid boluses of Plasma-Lyte 148 were used. The use of additional blood products was at the discretion of the attending clinical perfusionist and anesthesiologist. Normothermia was maintained in all patients after separation from CPB by using forced air warming devices and a fluid warmer ensuring delivery of fluid at 42 °C (107.6 F). At the termination of CPB, 750 ml of unprocessed residual blood remaining within the reservoir and tubing of the CPB circuit was collected and transferred to a standardised non-citrated, 1000 ml sterile clear bag (Baxter Healthcare, Toongabbie, NSW) over a 5-min period. Systemic heparin was reversed with a standardised dose of protamine (1.5 mg/kg for very 100 U of heparin) to achieve an ACT of less than 150 s. If ACT was above 150 s, additional protamine (50 mg) was titrated in small aliquots. When the ACT was less than 150 s, the unprocessed residual blood was then reinfused into the patient over a 15-min period.

Blood was sampled from the patient at two key time points to evaluate changes in coagulation before and after the re-infusion of the unprocessed residual blood.

5 min prior to the reinfusion of the unprocessed residual blood.

15 min after the reinfusion of the unprocessed residual blood.

Sampling was performed by aspirating blood from the indwelling arterial catheter using a standard 10 ml syringe (Terumo^® Corporation, Tokyo, Japan) syringe over a 10 s period. In order to assess for contact activation of the autologous blood from the plastic re-infusion bag, additional blood was sampled from the 750 ml unprocessed residual blood in the collection bag, and from the CPB circuit itself. All samples underwent standardised laboratory and TEG coagulation studies, which included plasma based laboratory assays of coagulation i.e. aPTT, PT, thrombin time (TT), D-dimer, and fibrinogen and platelet count, and measurements of the viscoelastic properties of whole blood clot formation using TEG. This included measurement of R-time, K-time, alpha angle, and maximum amplitude.