Simultaneous Measurement of Thrombin Generation and Fibrin Formation in Whole Blood Applying Continuous Flow Is Predictive for the Amount of Blood Loss during/after Cardiothoracic Surgery

Background. Thrombin is the key enzyme converting fibrinogen into fibrin and assays based on the formation of thrombin and fibrin are currently often used separately both in research and clinical settings. Recently it was shown that thrombin generation and fibrin formation do not always go hand in hand as factor XIIa independently influences the crosslinking of fibrin and thereby the stability of the clot.

Objective. The development and clinical validation of an assay that is able to simultaneously measure the generation of thrombin and formation of fibrin in whole blood.

Methods and results. We have redesigned an air-bearing rheometer, based on the plate and cone viscositor, rendering it sensitive enough to measure fibrin formation based on the change in viscosity. A confocal fluorescent camera is placed into the rheometer, allowing the measurement of thrombin generation based on the conversion of a thrombin-sensitive substrate as in the original Calibrated Automated Thrombinography (CAT) method. Furthermore, the method allows the application of laminar flow conditions within the range of 100 s^-1 until 1200 s^-1. For all variables related to fibrin formation and thrombin generation, the intra- and inter-assay variation were below 10% in platelet-poor plasma (PPP). For both parameters endogenous thrombin potential (ETP) and peak we demonstrated an inverse relationship with increasing flow rates (100-1200 s^-1) in PPP. This was in line with fibrin formation as maximum clot strength (based on viscosity) was inversely related to an increase in flow rate. Furthermore, as expected, the addition of increasing concentrations of fibrinogen to defibrinated plasma was accompanied with an increase in ETP, peak and maximum clot strength. Our method proved to be sensitive enough to detect an increase of 0.1 mg/ml fibrinogen. Furthermore, in the presence of tissue plasminogen activator, it allows to study fibrinolysis. The system was clinically validated in 70 patients undergoing cardiothoracic surgery. Blood samples were taken pre-bypass before heparinisation. Besides thrombin generation and fibrin formation measurements, clinical parameters together with the blood loss and the amount of transfusion products were recorded until 24 hours after surgery. Thrombin generation parameters obtained with the specially designed rheometer correlated well with the traditional whole blood CAT, whereas fibrin formation parameters

correlated well with the fibrinogen content and the rotational thromboelastometry (ROTEM) parameters. Importantly, when patients were divided into two groups based on the median clot strength determined with the rheometer, a significant difference in perioperative and total blood loss was established.

Conclusion. We have technically and clinically validated a new method capable of simultaneously measuring thrombin generation and fibrin formation in plasma and whole blood under continuous flow. Our method proved to be sensitive enough to detect differences in fibrinogen content and revealed an effect of different flow rates on both thrombin generation and clot strength. We hereby have not only combined thrombin generation and fibrin formation in whole blood, but by introducing continuous flow, our method is also one step closer to physiology.Importantly,in contrast to the traditionally used methods, our test applied pre-operatively was found to be predictive for the amount of blood loss during and after cardiothoracic surgery.