In vitro thrombogenicity evaluation of rotary blood pumps by thromboelastometry.

In vitro thrombogenicity tests for rotary blood pumps (RBPs) could benefit from assessing coagulation kinematics, as RBP design improves. In this feasibility study, we investigated if the method of thromboelastometry (TEM) is able to assess coagulation kinematics under the in vitro conditions of RBP tests. We conducted in vitro thrombogenicity tests (n=4) by placing Deltastream® DP3 pumps into test loops that were filled with 150 mL of slightly anti-coagulated porcine blood, adjusted to an activated clotting time (ACT) well below clinically recommended levels. Blood samples were taken at certain time points during the experiment until a continuous decrease in pump flow indicated major thrombus formation. Blood samples were analyzed for ACT, platelet count (PLT), and several TEM parameters. While visible thrombus formation was observed in three pumps, ACT indicated an ongoing activation of coagulation, PLT might have indicated platelet consumption. Unexpectedly, most TEM results gave no clear indications. Nonetheless, TEM clotting time obtained by non-anticoagulated and chemically non-activated whole blood (HEPNATEM-CT) appeared to be more sensitive for the activation of coagulation in vitro than ACT, which might be of interest for future pump tests. However, more research regarding standardization of thrombogenicity pump tests is urgently required.

The porcine abattoir blood model-Evaluation of platelet function for in-vitro hemocompatibility investigations.

BACKGROUND: The major obstacle of blood-contacting medical devices is insufficient hemocompatibility, particularly thrombogenicity and platelet activation. Pre-clinical in-vitro testing allows for the evaluation of adverse thrombogenicity-related events, but is limited, among others, by the availability and quantity of human blood donations. The use of animal blood is an accepted alternative for several tests; however, animal and particularly abattoir blood might present species-specific differences to human blood as well as elevated blood values, and pre-activated platelets due to stressed animals and non-standardized blood collection. MATERIAL & METHODS: To this end, we investigated porcine abattoir blood in comparison to human donor blood with the focus on platelet pre-activation and remaining activation potential. By means of light transmission aggregometry, aggregation kinetics of platelet rich plasma after stimulation with three different concentrations of each adenosine diphosphate (ADP) (5 µM, 10 µM, 20 µM) and collagen (2.5 µg/ml, 5 µg/ml, 10 µg/ml) were monitored. RESULTS: The activation with collagen revealed no significant differences in platelet behavior of the two species. In contrast, stimulation with ADP resulted in a lower maximum aggregation and a high disaggregation for porcine abattoir blood. The latter is a species-specific phenomenon of porcine platelets. Variations within each study cohort were comparable for human and abattoir pig. CONCLUSION: The similarities in platelet activation following collagen stimulation and the preservation of the porcine-specific reaction to ADP prove a general functionality of the abattoir blood. This finding provides a first step towards the complete validation of the porcine abattoir blood model.

Hemocompatibility Evaluation of Biomaterials-The Crucial Impact of Analyzed Area.

The hemocompatibility of blood-contacting medical devices remains one of the major challenges in medical device development. A common tool for the analysis of adherent and activated platelets on materials following in vitro tests is microscopy. Currently, most researchers develop their own routines, resulting in numerous different methods that are applied. The majority of those (semi-)manual methods analyze only a very small fraction of the material surface (<1%), which neglects the inhomogeneity of platelet distribution and makes results hardly comparable. Within this study, we examined the relation between the fraction of analyzed sample area and the platelet adhesion result. By means of image segmentation and machine learning algorithms, 103 100 microscopy images were analyzed automatically. We discovered a crucial impact of the analyzed surface fraction and thus a misrepresentation of a surface's platelet adhesion unless up to 40% of the sample surface is analyzed. These findings underline the necessity of standardization in the field of in vitro hemocompatibility tests and analyses in particular and provide a first basis to make future tests more reliable and comparable.

Perioperative onset of acquired von Willebrand syndrome: Comparison between HVAD, HeartMate II and on-pump coronary bypass surgery.

OBJECTIVES: Acquired von Willebrand syndrome (AvWS) is associated with postoperative bleeding complications in patients with continuous flow left ventricular assist devices (CF-LVADs). The aim of this study is to analyze the perioperative vWF profile comparing an axial pump (HMII) to a centrifugal pump (HVAD) regarding the correlation between perioperative occurrence of AvWS, early- and late-postoperative bleeding events. METHODS: From July 2013 until March 2015 blood samples of 33 patients (12 HMII/ 8 HVAD/ 13 controls) were prospectively collected at 12 different time points and analyzed for the vWF antigen (vWF:Ag), its activity (vWF:Ac) and the vWF:Ac/vWF:Ag-ratio (vWF:ratio). The follow up period for postoperative bleeding events was from July 2013 until July 2016. RESULTS: Postoperatively, there was no difference in the vWF-profile between HVAD and HMII groups. However, a subgroup of patients already had significantly lower vWF:ratios preoperatively. Postoperatively, both CF-LVAD groups presented significantly lower vWF:ratios compared to the control group. Bleeding events per patient-year did not differ between the two groups (HMII vs. HVAD: 0.67 vs. 0.85, p = 0.685). We detected a correlation between vWF:ratio <0.7at LVAD-start (r = -0.583, p = 0.006) or at the end of surgery (r = -0.461, p = 0.035) and the occurrence of pericardial tamponade. In the control group, the drop in both vWF:Ag and vWF:Ac recovered immediately postoperatively above preoperative values. CONCLUSION: A subgroup of patients with end-stage heart failure already suffers AvWS preoperatively. In both CF-LVAD groups, AvWS begins immediately after surgery. Intraoperative vWF:ratios <0.7 correlate with higher incidences of pericardial tamponade and re-operation. The presumably dilutive effect of the heart lung machine on vWF vanishes immediately at the end of surgery, possibly as part of an acute-phase response.

Coagulation Management in Jersey Calves: An ex vivo Study.

OBJECTIVE: Jersey calves are frequently used as an experimental animal model for in vivo testing of cardiac assist devices or orthopedic implants. In this ex vivo study, we analyzed the coagulation system of the Jersey calves and the potential of human-based coagulation management to circumvent perioperative bleeding complications during surgery. Experimental Procedure: Blood from 7 Jersey calves was subjected to standard laboratory tests and thromboelastometry analysis. An ex vivo model of dilutional coagulopathy was used to study the effects of fibrinogen or prothrombin complex concentrate supplementation. Fibrinolysis was induced with tissue plasminogen activator to identify potential therapeutic strategies involving tranexamic acid or aprotinin. Furthermore, anticoagulation strategies were evaluated by incubating the blood samples with dabigatran or rivaroxaban. RESULTS: Baseline values for thromboelastometry and standard laboratory parameters, including prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin III, and D-dimers, were established. Fifty percent diluted blood showed a statistically significant impairment of hemostasis. The parameters significantly improved after the administration of fibrinogen or prothrombin complex concentrate. Tranexamic acid and aprotinin ameliorated tissue plasminogen activator-induced fibrinolysis. Both dabigatran and rivaroxaban significantly prolonged the coagulation parameters. CONCLUSIONS: In this ex vivo study, coagulation factors, factor concentrate, antifibrinolytic reagents, and anticoagulants regularly used in the clinic positively impacted coagulation parameters in Jersey calf blood.

Micro-structuring of polycarbonate-urethane surfaces in order to reduce platelet activation and adhesion.

In the development of new hemocompatible biomaterials, surface modification appears to be a suitable method in order to reduce the thrombogenetic potential of such materials. In this study, polycarbonate-urethane (PCU) tubes with different surface microstructures to be used for aortic heart valve models were investigated with regard to the thrombogenicity. The surface structures were produced by using a centrifugal casting process for manufacturing PCU tubes with defined casting mold surfaces which are conferred to the PCU surface during the process. Tubes with different structures defined by altering groove widths were cut into films and investigated under dynamic flow conditions in contact with porcine blood. The analysis was carried out by laser scanning microscopy which allowed for counting various morphological types of platelets with regard to the grade of activation. The comparison between plain and shaped PCU samples showed that the surface topography led to a decline of the activation of the coagulation cascade and thus to the reduction of the fibrin synthesis. Comparing different types of structures revealed that smooth structures with a small groove width (d ~ 3 µm) showed less platelet activation as well as less adhesion in contrast to a distinct wave structure (d ~ 90 µm). These results prove surface modification of polymer biomaterials to be a suitable method for reducing thrombogenicity and hence give reason for further alterations and improvements.

Establishing a method for in vitro investigation of mechanical parameters causing acquired von Willebrand syndrome in ventricular assist devices.

Acquired von Willebrand Syndrome (AvWS) is known as a frequent bleeding complication in patients on ventricular assist device (VAD) support. Clinicians demand that the requirements for VADs with regard to hemocompatibility should also include low susceptibility for AvWS. Clinical AvWS diagnosis is known to be a complex, high-price, and time-consuming analysis. This article investigates an easy-to-handle, time-efficient, and inexpensive method for comparative AvWS investigations in vitro. Von Willebrand Factor activity level (vWF:Ac) and von Willebrand Factor antigen level (vWF:Ag) were chosen from the complete set of clinically established parameters. Blood plasma (human and porcine) was exposed to an inhomogeneous shear field in a shear-inducing test set up for up to 4 h. Plasma samples were drawn after different load periods and analyzed for vWF:Ac and vWF:Ag. vWF multimer analysis of selected samples were used as reference for determination of high molecular weight multimer (HMWM) loss. AvWS was detected after 20 min of shear load via vWF:Ac/vWF:Ag ratio and multimer analysis. A good correlation between the vWF:Ac/vWF:Ag ratio and HMWM loss (multimer analysis) was found for human plasma. AvWS characteristics of human and porcine plasma for analyzed samples were comparable. A correlation between vWF:Ac/vWF:Ag ratio and HMWM in porcine plasma could not be found. Results gained in this study indicate that vWF:Ac/vWF:Ag ratio is sensitive enough for comparative AvWS investigations in vitro with human blood. The applicability of the method suggested in this article for AvWS characterization in porcine blood needs to be investigated in further studies. The selection of analysis kits promises a less cost- and labor-intensive, time-consuming, and complex method for comparative AvWS investigations in vitro compared with AvWS diagnosis in patients.