# Dynamic and Quantitative Assessment of Blood Coagulation Status with an Oscillatory Rheometer
## 3 Results
### Titration of Various Proatamine Concentrations to a Fixed Heparin Concentration  
The actual heparin concentration during surgeries is often not known and has to be determined e.g., via back titration with protamine. The aim of this experiment was to investigate if the oscillatory rheometer can measure the back titration of various protamine concentrations (0.5, 1.0, 1.5, 2.0 and 3.0 IU/mL) to neutralise a fixed heparin concentration (2 IU/mL). As control, citrated blood without any heparin was activated by Thromborel (Figure 4, magenta lines). The lower concentrations of protamine (0.5 and 1.0 IU/mL, black and red lines, respectively) were too weak to neutralise heparin and thus the shear moduli shifted to the right indicating a delayed coagulation. However, the other concentrations of protamine (1.5-3.0 IU/mL) showed similar shear moduli as the non-heparinised control. Taking a closer look at the CT values provided by the oscillatory rheometer and KC 1A, there was again no significant difference between both devices (Table 4; Appendix A Figure A9). However, only the equimolar concentrations of heparin and protamine resulted in comparable CT values to the control, for all other protamine concentrations, there was a significant difference in CT values within the assay system (Kinexus Pro or KC 1A). Here, the KC 1A showed a slightly better differentiation between protamine concentrations, as only the equimolar concentration was not significantly different from the control. The oscillatory rheometer measurements showed non-significant CT values for the equimolar concentration (2 IU/mL) and 1.5 IU/mL heparin. Furthermore, the linear slopes of viscous and elastic shear moduli were all significantly different from the control measurements without heparin (Appendix A Figure A10a), indicating similarly to Figure A8 that there was a slight difference in coagulation between samples that was not obvious in the CT value. A surplus of heparin led to slightly flatter linear slopes (m (G\({}^{\prime}\)), m (G\({}^{\prime\prime}\))) compared to the mean control, but a shortage of protamine decreased the slopes by a far greater amount. The equimolar heparin-protamine concentration and 2 IU/mL heparin with 1.5 IU/mL protamine provided almost the same result as the control measurements. The elastic but not the viscous shear moduli at 300 s were significantly different from the control without heparin (Appendix A Figure A10b).  
Figure 4: Back titration of heparin with protamine. Citrated blood was heparinised (2.0 IU/mL) and simultaneously antagonised with various concentrations of protamine before activation with Thromborel. Citrated blood without heparin activated by Thromborel served as control. The effect of the inhibitor heparin was reversed by various concentrations of protamine (1.5-3.0 IU/mL). Both, 0.5 and 1.0 IU/mL protamine were too weak to reverse the effect of heparin. The lowest protamine concentration of 0.5 IU/mL has a strongly delayed clotting time (CT), a flatter slope and a lower elastic and viscous shear moduli compared to the other measurements.  
If there is a surplus of protamine, the clotting time is prolonged, the linear slopes and the viscous and elastic shear moduli were lower as in the control measurement. These first experiments indicate that the measurement of the current heparin level by the titration method with protamine can be determined with rheological methods that would normally be performed in clinical practice with the Haemostasis Management system (HMS), but further studies are necessary to confirm these promising results.