Device‐induced platelet dysfunction in mechanically assisted circulation increases the risks of thrombosis and bleeding

Thrombotic and bleeding complications are the major obstacles for expanding mechanical circulatory support (MCS) beyond the current use. While providing the needed hemodynamic support, those devices can induce damage to blood, particularly to platelets. In this study, we investigated device‐induced alteration of three major platelet surface receptors, von Willebrand factor (VWF) and associated hemostatic functions relevant to thrombosis and bleeding. Fresh human whole blood was circulated in an extracorporeal circuit with a clinical rotary blood pump (CentriMag, Abbott, Chicago, IL, USA) under the clinically relevant operating condition for 4 hours. Blood samples were examined every hour for glycoprotein (GP) IIb/IIIa activation and receptor loss of GPVI and GPIbα on the platelet surface with flow cytometry. Soluble P‐selectin in hourly collected blood samples was measured by enzyme linked immunosorbent assay to characterize platelet activation. Adhesion of device‐injured platelets to fibrinogen, collagen, and VWF was quantified with fluorescent microscopy. Device‐induced damage to VWF was characterized with western blotting. The CentriMag blood pump induced progressive platelet activation with blood circulating time. Particularly, GPIIb/IIIa activation increased from 1.1% (Base) to 11% (4 hours) and soluble P‐selectin concentration increased from 14.1 ng/mL (Base) to 26.5 ng/mL (4 hours). Those device‐activated platelets exhibited increased adhesion capacity to fibrinogen. Concurrently, the CentriMag blood pump caused progressive platelet receptor loss (GPVI and GPIbα) with blood circulating time. Specifically, MFI of the GPVI and GPIbα receptors decreased by 17.2% and 16.1% for the 4‐hours sample compared to the baseline samples, respectively. The device‐injured platelets exhibited reduced adhesion capacities to collagen and VWF. The high molecular weight multimers (HMWM) of VWF in the blood disappeared within the first hour of the circulation. Thereafter the multimeric patterns of VWF were stable. The change in the VWF multimeric pattern was different from the progressive structural and functional changes of platelets with the circulation time. This study suggested that the CentriMag blood pump could induce two opposite effects on platelets and associated hemostatic functions under a clinically relevant operating condition. The device‐altered hemostatic function may contribute to thrombosis and bleeding simultaneously as occurring in patients supported by a rotary blood pump. Device‐induced damage of platelets may be an important cause for bleeding in patients supported with rotary blood pump MCS systems relative to device‐induced loss of HMWM‐VWF.     

Impact of high mechanical shear stress and oxygenator membrane surface on blood damage relevant to thrombosis and bleeding in a pediatric ECMO circuit

The roles of the large membrane surface of the oxygenator and the high mechanical shear stress (HMSS) of the pump in the extracorporeal membrane oxygenation (ECMO) circuit were examined under a pediatric support setting. A clinical centrifugal pump and a pediatric oxygenator were used to construct the ECMO circuit. An identical circuit without the oxygenator was constructed for comparison. Fresh human blood was circulated in the two circuits for 4 hours under the identical pump speed and flow. Blood samples were collected hourly for blood damage assessment, including platelet activation, generation of platelet-derived microparticles (PDMP), losses of key platelet hemostasis receptors (glycoprotein (GP) Ibα (GPIbα) and GPVI), and high molecular weight multimers (HMWM) of von Willebrand factor (VWF) and plasma free hemoglobin (PFH). Platelet adhesion on fibrinogen, VWF, and collagen was further examined. The levels of platelet activation and generation of PDMP and PFH exhibited an increasing trend with circulation time while the expression levels of GPIbα and GPVI receptors on the platelet surface decreased. Correspondingly, the platelets in the blood samples exhibited increased adhesion capacity to fibrinogen and decreased adhesion capacities on VWF and collagen with circulation time. Loss of HMWM of VWF occurred in both circuits. No statistically significant differences were found in all the measured parameters for blood damage and platelet adhesion function between the two circuits. The results indicate that HMSS from the pump played a dominant role in blood damage associated with ECMO and the impact of the large surface of the oxygenator on blood damage was insignificant.     

Paradoxical Effect of Nonphysiological Shear Stress on Platelets and von Willebrand Factor

Blood can become hypercoagulable by shear‐induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25 Pa, 125 Pa) with an exposure time of 0.5 s, generated by using a novel blood‐shearing device. Platelet activation (P‐selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear‐induced vWF damage was characterized with Western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWMs) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis, while the reduction in platelet receptors and the loss of HMWM‐vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices.     

The role of PI3K/Akt signaling pathway in non‐physiological shear stress‐induced platelet activation

The PI3K/Akt signaling pathway has been implicated in playing an important role in platelet activation during hemostasis and thrombosis involving platelet‐matrix interaction and platelet aggregation. Its role in non‐physiological shear stress (NPSS)‐induced platelet activation relevant to high‐shear blood contacting medical devices (BCMDs) is unclear. In the context of blood cells flowing in BCMDs, platelets are subjected to NPSS (>100 Pa) with very short exposure time (<1 s). In this study, we investigated whether NPSS with short exposure time induces platelet activation through the PI3K/Akt signaling pathway. Healthy donor blood treated with or without PI3K inhibitor was subjected to NPSS (150 Pa) with short exposure time (0.5 s). Platelet activation indicated by the surface P‐selectin expression and activated glycoprotein (GP) IIb/IIIa was quantified using flow cytometry. The phosphorylation of Akt, activation of the PI3K signaling, was characterized by western blotting. Changes in adhesion behavior of NPSS‐sheared platelets on fibrinogen, collagen, and von Willebrand factor (vWF) were quantified with fluorescent microscopy by perfusing the NPSS‐sheared and PI3K inhibitor‐treated blood through fibrinogen, collagen, and vWF‐coated microcapillary tubes. The results showed that the PI3K/Akt signaling was involved with both NPSS‐induced platelet activation and platelet‐matrix interaction. NPSS‐sheared platelets exhibited exacerbated platelet adhesion on fibrinogen, but had diminished platelet adhesion on collagen and vWF. The inhibition of PI3K signaling reduced P‐selectin expression and GPIIb/IIIa activation with suppressed Akt phosphorylation and abolished NPSS‐enhanced platelet adhesion on fibrinogen in NPSS‐sheared blood. The inhibition of PI3K signaling can attenuate the adhesion of unsheared platelets (baseline) on collagen and vWF, while had no impact on adhesion of NPSS‐sheared platelets on collagen and vWF. This study confirmed the important role of PI3K/Akt signaling pathway in NPSS‐induced platelet activation. The finding of this study suggests that blocking PI3K/Akt signaling pathway could be a potential method to treat thrombosis in patients implanted with BCMDs.     

Computational and experimental evaluation of the fluid dynamics and hemocompatibility of the CentriMag blood pump

The CentriMag centrifugal blood pump is a newly developed ventricular assist device based on magnetically levitated bearingless rotor technology. A combined computational and experimental study was conducted to characterize the hemodynamic and hemocompatibility performances of this novel blood pump. Both the three-dimensional flow features of the CentriMag blood pump and its hemolytic characteristics were analyzed using computational fluid dynamics (CFD)-based modeling. The hydraulic pump performance and hemolysis level were quantified experimentally. The CFD simulation demonstrated a clean and streamlined flow field in the main components of the CentriMag blood pump. The predicted results by hemolysis model indicated no significant high shear stress regions in the pump. A comparison of CFD predictions and experimental results showed good agreements. The relatively large gap passages (1.5 mm) between the outer rotor walls and the lower housing cavity walls provide a very good surface washing through a secondary flow path while the shear stresses in the secondary flow paths are reduced, resulting in a low rate of hemolysis ([Normalized Index of Hemolysis] NIH = 0.0029 +/- 0.006) without a decrease of the pump's hydrodynamic performance (pressure head: 352 mm Hg at a flow rate of 5.0 L/min and a rotational speed of 4,000 rpm).     

The CentriMag Centrifugal Blood Pump as a Benchmark for In Vitro Testing of Hemocompatibility in Implantable Ventricular Assist Devices

Implantable ventricular assist devices (VADs) have proven efficient in advanced heart failure patients as a bridge‐to‐transplant or destination therapy. However, VAD usage often leads to infection, bleeding, and thrombosis, side effects attributable to the damage to blood cells and plasma proteins. Measuring hemolysis alone does not provide sufficient information to understand total blood damage, and research exploring the impact of currently available pumps on a wider range of blood cell types and plasma proteins such as von Willebrand factor (vWF) is required to further our understanding of safer pump design. The extracorporeal CentriMag (Thoratec Corporation, Pleasanton, CA, USA) has a hemolysis profile within published standards of normalized index of hemolysis levels of less than 0.01 g/100 L at 100 mm Hg but the effect on leukocytes, vWF multimers, and platelets is unknown. Here, the CentriMag was tested using bovine blood (n = 15) under constant hemodynamic conditions in comparison with a static control for total blood cell counts, hemolysis, leukocyte death, vWF multimers, microparticles, platelet activation, and apoptosis. The CentriMag decreased the levels of healthy leukocytes (P < 0.006), induced leukocyte microparticles (P < 10^?5), and the level of high molecular weight of vWF multimers was significantly reduced in the CentriMag (P < 10^?5) all compared with the static treatment after 6 h in vitro testing. Despite the leukocyte damage, microparticle formation, and cleavage of vWF multimers, these results show that the CentriMag is a hemocompatible pump which could be used as a standard in blood damage assays to inform the design of new implantable blood pumps.     

Evaluation of centrifugal blood pumps in term of hemodynamic performance using simulated neonatal and pediatric ECMO circuits

The objective of this translational study was to evaluate the FDA-approved PediMag, CentriMag, and RotaFlow centrifugal blood pumps in terms of hemodynamic performance using simulated neonatal and pediatric extracorporeal membrane oxygenation (ECMO) circuits with different sizes of arterial and venous cannulae. Cost of disposable pump heads was another important variable for this particular study. The experimental circuit was composed of one of the centrifugal pump heads, a polymethylpentene membrane oxygenator, neonatal and pediatric arterial/venous cannulae, and 1/4-inch ID tubing. Circuits were primed with lactated Ringer’s solution and packed human red blood cells (hematocrit 35%). Trials were conducted at 36°C using the three pump heads and different cannulae (arterial/venous cannulae: 8 Fr/18 Fr, 10 Fr/20 Fr, and 12 Fr/22 Fr) at various flow rates (200–2400 mL/min, 200 mL/min increments) and rotational speeds. Pseudo patient pressure was 60 mm Hg. Real-time pressure and flow data were recorded for analysis. The RotaFlow pump had a higher pressure head and flow range compared with the PediMag and CentriMag pumps at the same rotational speed and identical experimental settings (P < 0.001). The PediMag pump had lower flow output than others (P < 0.001). Small-caliber arterial cannulae and higher flow rates predictably created higher circuit pressures and pressure drops. There was no significant difference in hemodynamic energy delivered to the pseudo patient with each of the three pumps. The arterial cannula had the highest pressure drop and hemodynamic energy loss in the circuit when compared to the oxygenator and arterial tubing. The RotaFlow centrifugal pump had a significantly better hemodynamic performance when compared to the PediMag and CentriMag blood pumps at identical experimental conditions in simulated neonatal and pediatric ECMO settings. In addition, the cost of the RotaFlow pump head ($400) is 20 to 30-fold less than the other centrifugal pumps [CentriMag ($12 000) or PediMag ($8000)] that were evaluated in this translational study.     

Controlling the flow balance: In vitro characterization of a pulsatile total artificial heart in preload and afterload sensitivity

The objective of this study is to identify the preload and afterload sensitivity of the ReinHeart TAH 2.0. For adequate left-right flow balance, the concept of a reduced right stroke volume (by about 10%) and active adaption of the right diastole duration are evaluated concerning the controllability of the flow balance. This study used an active mock circulation loop to test a wide range of preload and afterload conditions. Preload sensitivity was tested at atrial pressures (APs) between 4 and 20 mm Hg. Left afterload was varied in a range of 60-140 mm Hg mean aortic pressure (MAP), right afterload was simulated between 15 and 40 mm Hg. Four scenarios were developed to verify that the flow difference fully covers the defined target range of 0-1.5 L/min. Although a positive correlation between inlet pressure and flow is identified for the right pump chamber, the left pump chamber already fills completely at an inlet pressure of 8-10 mm Hg. With increasing afterload, both the left and right flow decrease. A positive flow balance (left flow exceeds right flow) is achieved over the full range of tested afterloads. At high APs, the flow difference is limited to a maximum of 0.7 L/min. The controllability of flow balance was successfully evaluated in four scenarios, revealing that a positive flow difference can be achieved over the full range of MAPs. Under physiological test conditions, the linear relationship between flow and heart rate was confirmed, ensuring good controllability of the TAH.     

Development of rear‐impeller axial flow blood pump for realization of axial flow blood pump installed at aortic valve position

In this study, rear‐impeller axial flow blood pumps (RIAFBP) were developed to realize a trans‐valve axial ventricular assist device (VAD) which consists of the latter blood pump and a polymer monomembrane aortic valve, such as the jellyfish valve. The motor of the RIAFBP is installed in the left ventricle, and its impeller is placed at the aortic valve position. In the prototype RIAFBP, the rotation of the motor is sustained by polyethylene bushings. The RIAFBP has a length of 50 mm and diameter of 19.6 mm. The miniature RIAFBP has the same construction as that of the prototype; however, it employs a ceramic bearing and fin bearing to improve endurance and to reduce blood stagnation. The miniature RIAFBP has a length of 63 mm and diameter of 12 mm. Both RIAFBPs were examined by an in vitro experiment using a 33% glycerin solution. The prototype RIAFBP achieved a maximum pump outflow of 8.5 L/min against a pump head of 100 mm Hg at a rotational speed of 12 000 rpm. The miniature RIAFBP achieved 7 L/min against a pump head of 70 mm Hg at a rotational speed of 21 600 rpm. In conclusion, the miniature RIAFBP has enough pump performance to realize the trans‐valve axial VAD.     

Blood Trauma Testing of CentriMag and RotaFlow Centrifugal Flow Devices: A Pilot Study

Mechanical circulatory assist devices that provide temporary support in heart failure patients are needed to enable recovery or provide a bridge to decision. Minimizing risk of blood damage (i.e., hemolysis) with these devices is critical, especially if the length of support needs to be extended. Hematologic responses of the RotaFlow (Maquet) and CentriMag (Thoratec) temporary support devices were characterized in an in vitro feasibility study. Paired static mock flow loops primed with fresh bovine blood (700 mL, hematocrit [Hct] = 25 ± 3%, heparin titrated for activated clotting time >300 s) pooled from a single‐source donor were used to test hematologic responses to RotaFlow (n = 2) and CentriMag (n = 2) simultaneously. Pump differential pressures, temperature, and flow were maintained at 250 ± 10 mm Hg, 25 ± 2°C, and 4.2 ± 0.25 L/min, respectively. Blood samples (3 mL) were collected at 0, 60, 120, 180, 240, 300, and 360 min after starting pumps in accordance with recommended Food and Drug Administration and American Society for Testing and Materials guidelines. The CentriMag operated at a higher average pump speed (3425 rpm) than the RotaFlow (3000 rpm) while maintaining similar constant flow rates (4.2 L/min). Hematologic indicators of blood trauma (hemoglobin, Hct, platelet count, plasma free hemoglobin, and white blood cell) for all measured time points as well as normalized and modified indices of hemolysis were similar (RotaFlow: normalized index of hemolysis [NIH] = 0.021 ± 0.003 g/100 L, modified index of hemolysis [MIH] = 3.28 ± 0.52 mg/mg compared to CentriMag: NIH = 0.041 ± 0.010 g/100 L, MIH = 6.08 ± 1.45 mg/mg). In this feasibility study, the blood trauma performance of the RotaFlow was similar or better than the CentriMag device under clinically equivalent, worst‐case test conditions. The RotaFlow device may be a more cost‐effective alternative to the CentriMag.     

FW-Ⅱ型轴流泵短期辅助抗体内血栓形成性能评价

目的 对优化设计FW-Ⅱ轴流泵短期辅助的抗血栓性能进行评价,为后期临床应用提供依据.方法 5只60～70 kg成年小尾寒羊,建立心室-泵-降主动脉旁路循环模型.FW-Ⅱ轴流泵辅助循环2周,分别在术前和置入泵术后1、2、3、7、10、14天抽取静脉血,应用流式细胞仪检测Annexin V,CD41/61及CD14量化外周血中活化血小板及白细胞-血小板聚集;检测不同转速下血小板活化及白细胞-血小板聚集体.实验结束后观察轴流泵各组成部分有无血栓形成,取材观察心、脑、脾、肾大体和镜下病理表现.结果 同术前相比较,置入轴流泵术后实验动物血小板活化及白细胞-血小板聚集体形成显著增加,第2天达到高峰,7天内维持较高水平,后逐渐降低,但术后14天仍高于术前水平.泵转速8000转/min时活化血小板和白细胞-血小板聚集体数最低.实验结束后拆分泵,轴流泵轴根部有少量血栓形成,叶轮、泵管及前、后导叶无血栓形成,心、脑、脾、肾等主要脏器大体和镜下观察均无血栓形成及缺血梗死表现.结论 FW-Ⅱ型泵抗血栓性能良好,可用于短期循环辅助.特定转速下血小板活化和白细胞-血小板聚集体值达到最低.

Abstract：

Objective To evaluate in vivo antithrombosis property of optimized FW-Ⅱ axial blood pump and provides evidence for future clinical use.Methods A left ventricle-pump-descending aorta bypass model was established in five healthy sheep (60-70 kg) and the circulation of these sheep was assisted by FW-Ⅱ axial blood pump for 2 weeks.In preoperative and postoperative day 1,2,3,7,10 and 14,blood was drawn from the jugular vein to examine platelet activation and leukocyte-platelet aggregation respectively quantified with Annexin V,CD41/61 and CD14-PE by flow cytometry assays.Immediately after termination of the experiment,FW-Ⅱ axial blood pumps were explanted and each part was inspected for thrombus formation.Macroscopic and histological examinations were checked on heart,brain,kidney and spleen,respectively for thrombosis.Results Compared with preoperative baseline,the number of platelet activation and leukocyte-platelet aggregation reached a peak at postoperative day 2,it retained a high level within 7 days,then gradually decreased,but was still higher than preoperative level at dayl4.According to rotating speed,the number of platelet activation and platelet-leukocyte aggregation were lowest at the speed of 8000 r/min Minus thrombus were found in the front and rear hub of the pump rotor,and there was no thrombus at other components (flow straighter,impeller and pump housing).There were no ischemia and infarction evidences in macroscopic and histological examination of the heart,brain,kidney and spleen.Conclusion FW-II axial blood pump can be used to assist left ventricular circulation for 2 weeks with a satisfactory antithrombosis property.The level of platelet activation and leukocyte-platelet aggregation can be reduced to a lowest level at an optimized pump rotating speed.     

Development of a Hyperactive Primary Hemostatic System During Off‐Pump Lung Transplantation Resulting From an Unbalance Between von Willebrand Factor and Its Cleaving Protease ADAMTS13

An unbalance between the platelet‐adhesive protein von Willebrand factor (VWF) and its cleaving protease ADAMTS13 is a risk factor for thrombosis. Here, we assessed levels and functionality of VWF and ADAMTS13 in patients undergoing off‐pump lung transplantation. We analyzed plasma of 10 patients and distinguished lung transplantation‐specific effects from those generally accompanying open‐chest surgeries by comparing results with 11 patients undergoing off‐pump coronary bypass graft (CABG) surgery. Forty healthy volunteers were included for reference values. VWF antigen levels as well as the VWF ristocetin cofactor activity/VWF antigen ratio increased during lung transplantation and after CABG surgery. An increase in VWF propeptide levels was paralleled by a decrease in ADAMTS13 activity. This was more pronounced during lung transplantation. Similarly, the capacity of plasma to support platelet aggregation under shear flow conditions in vitro was more increased during lung transplantation. The proportion of high molecular weight VWF multimers was elevated in both groups without evidence for ultra‐large VWF. VWF's collagen binding activity remained unchanged. In conclusion, a hyperactive primary hemostatic system develops during lung transplantation resulting both from a pronounced (functional) increase of the VWF molecule and decrease of ADAMTS13. This may increase the risk of platelet thrombosis within the allograft.     

The effects of arterial pressure during normothermic kidney perfusion

Background

Ex vivo normothermic perfusion (EVNP) can reverse some of the detrimental effects of ischemic injury. However, in kidneys with warm and cold ischemic injury the optimal perfusion pressure remains undetermined. The aim of this study was to evaluate the effects of two different arterial pressures during EVNP.

Methods

Porcine kidneys underwent static cold storage for 23 h followed by 1 h of EVNP using leukocyte depleted blood at a mean arterial pressure of either 55 or 75 mm Hg. After this, kidneys were reperfused for 3 h to assess renal function and injury. This was compared with a control group that underwent 24 h cold storage.

Results

During EVNP, kidneys perfused at 75 mm Hg had a higher renal blood flow, increased oxygen consumption (median 59.9 mL/min/g (range 30.1–78.6] versus 31.8 [8.2–53.8] mL/min/g; P = 0.026), and produced more urine (P = 0.002) than kidneys perfused at 55 mm Hg. During ex vivo reperfusion, renal blood flow was significantly higher in the 75 mm Hg and 55 mm Hg groups compared with the control (area under the curve median 75 mm Hg 462 [228–745], 55 mm Hg 454 [254–923] versus control 262 [215–442] mL/min/100g.h; P = 0.040). There was a significant loss of renal function and increase in tubular injury in the 55 mm Hg group kidneys (P = 0.001, 0.007). Levels of endothelin 1 were significantly reduced in the 75 mm Hg group (P = 0.026).

Conclusions

A mean arterial pressure of 75 mm Hg during EVNP resulted in less tubular damage and less endothelial injury during ex vivo reperfusion compared with kidneys perfused at 55 mm Hg.     

Conventional or minimized cardiopulmonary bypass support during coronary artery bypass grafting? – An analysis by means of perfusion and body mass index

The use of minimized cardiopulmonary bypass support to reduce the side effects of extracorporeal circulation is still contradictorily discussed. This study compares perfusion operated by conventional (CCPB) and minimized (MCPB) cardiopulmonary bypass support during coronary artery bypass grafting (CABG). This study includes the data of 5164 patients treated at our department between 2004 and 2014. Tissue perfusion during cardiopulmonary bypass support and cardiac arrest was assessed by means of body mass index, hemodilution, blood pressure with corresponding pump flow and venous oxygen saturation, serum lactate, and serum pH. Hemodilution was more pronounced after CCPB: hemoglobin had dropped to 4.47 ± 0.142 g/dL after CCPB and to 2.77 ± 0.148 g/dL after MCPB (P = 0.0022). Despite the higher pump flow in conventional circuits (4.86–4.95 L/min vs. 4.1–4.18 L/min), mean blood pressure was higher during minimized bypass support (53 ± 10 vs. 56 ± 13 mm Hg [aortic clamping], 57 ± 9 vs. 61 ± 12 mm Hg [34°C], 55 ± 9 vs.59 ± 11 mm Hg [aortic clamp removal], P < 0.0001) at all time points. Venous oxygen saturation remained on comparable levels of >70% during both conventional and minimized cardiopulmonary bypass support. The increase in serum lactate was more pronounced after CCPB (8.98 ± 1.28 vs. 3.66 ± 1.25 mg/dL, P = 0.0079), corresponding to a decrease in serum pH to acidotic levels (7.33 ± 0.06 vs. 7.35 ± 0.06, P < 0.0001). These effects were evident in all BMI ranges. Minimized cardiopulmonary bypass support provides efficient perfusion in all BMI ranges and is thus equivalent to conventional circuits.     

Hemodynamic Evaluation of Avalon Elite Bi‐Caval Dual Lumen Cannulas and Femoral Arterial Cannulas

Translational research is a useful tool to provide scientific evidence for cannula selection during extracorporeal life support (ECLS). The objective of this study was to evaluate four Avalon Elite bi‐caval dual lumen cannulas and nine femoral arterial cannulas in terms of flow range, circuit pressure, pressure drop, and hemodynamic energy transmission in a simulated adult ECLS model. A veno‐venous ECLS circuit was used to evaluate four Avalon Elite bi‐caval dual lumen cannulas (20, 23, 27, and 31 Fr), and a veno‐arterial ECLS circuit was used to evaluate nine femoral arterial cannulas (15, 17, 19, 21, and 23 Fr). The two circuits included a Rotaflow centrifugal pump, a Quadrox‐D adult oxygenator, and 3/8 in ID tubing for arterial and venous lines. The circuits were primed with lactated Ringer’s solution and packed human red blood cells (hematocrit 40%). Trials were conducted at rotational speeds from 1000 to 5000 RPM (250 rpm increments) for each Avalon cannula, and at different flow rates (0.5–7 L/min) for each femoral arterial cannula. Real‐time pressure and flow data were recorded for analysis. Small caliber cannulas created higher circuit pressures, higher pressure drops and higher M‐numbers compared with large ones. The inflow side of Avalon dual lumen cannula had a significantly higher pressure drop than the outflow side (inflow vs. outflow: 20 Fr‐100.2 vs. 49.2 mm Hg at 1.1 L/min, 23 Fr‐93.7 vs. 41.4 mm Hg at 1.6 L/min, 27 Fr‐102.3 vs. 42.8 mm Hg at 2.6 L/min, 31 Fr‐98.1 vs. 44.7 mm Hg at 3.8 L/min). There was more hemodynamic energy lost in the veno‐arterial ECLS circuit using small cannulas compared to larger ones (17 Fr vs. 19 Fr vs. 21 Fr at 4 L/min—Medtronic: 71.0 vs. 64.8 vs. 60.9%; Maquet: 71.4 vs. 65.6 vs. 62.0%). Medtronic femoral arterial cannulas had lower pressure drops (Medtronic vs. Maquet at 4 L/min: 17 Fr‐121.7 vs. 125.0 mm Hg, 19 Fr‐71.2 vs. 73.7 mm Hg, 21 Fr‐42.9 vs. 47.4 mm Hg) and hemodynamic energy losses (Medtronic vs. Maquet at 4 L/min: 17 Fr‐43.6 vs. 44.4%, 19 Fr‐31.0 vs. 31.4%, 21 Fr‐20.8 vs. 22.4%) at high flow rates when compared with the Maquet cannulae. The results for this study provided valuable hemodynamic characteristics of all evaluated adult cannulas with human blood in order to guide ECLS cannula selection in clinical practice. Use of larger cannulas are suggested for VV‐ and VA‐ECLS.     

Activation of porcine endothelium in response to xenogeneic serum causes thrombosis independently of platelet activation

BACKGROUND: Endothelial cell activation and microvascular thrombosis are hallmarks of hyperacute xenograft rejection. However, the molecular determinants of platelet-endothelial interaction and thrombus formation are poorly understood. This study investigated whether: (i) xenogeneic human serum (HS), as a source of xenoreactive antibodies and complement, activates porcine aortic endothelial cells (PAEC) to promote thrombus formation under high shear stress; (ii) the endothelial adhesive proteins vitronectin receptor and P-selectin are involved in the von Willebrand factor (VWF)-platelet interaction during the thrombotic process under flow; (iii) reactive oxygen species (ROS) are activated by complement and served as intracellular signals for adhesive protein up-regulation. METHODS: The PAEC were pre-exposed for 90 min in static conditions to medium plus 10, 20, and 50% HS or 20% porcine serum (PS), as control, then cells were perfused at 50 dynes/cm2 in a parallel plate flow chamber with human blood and area occupied by thrombi was measured. The role of complement in HS-induced thrombus formation was assessed by incubating PAEC with 20% HS in the presence of soluble complement receptor type 1 (sCR1) before blood perfusion. The effect of platelet activation was assessed using human blood treated or not with ADP and then flowed over PAEC pre-exposed to 20% HS or 20% PS as control. To identify the endothelial adhesive proteins involved in thrombus formation PAEC treated with 20% HS were then incubated with anti-vitronectin receptor antibody, anti-P-selectin antibody or P-selectin glycoprotein ligand-1 (PSGL-1), the soluble ligand of P-selectin, before the adhesion assay. Confocal microscopy was used to detect changes in endothelial adhesive protein expression. VWF interaction with platelet receptors GPIb and alphaIIbbeta3 was assessed adding aurin tricarboxylic acid (ATA) and anti-alphaIIbbeta3 antibody to blood before perfusion. The ROS involvement in xenogeneic serum-induced thrombus formation was determined studying the intracellular production of hydrogen peroxide (H2O2). The effect of antioxidants and metal chelators on HS-induced thrombus formation was evaluated treating PAEC with pyrrolidine dithiocarbamate (PDTC) or 1,3-dimethyl-2-thiourea (DMTU) before and during incubation with 20% HS followed by blood perfusion. The effect of antioxidants and sCR1 on ROS generation was investigated treating PAEC with PDTC or DMTU before and during incubation with 20% HS. Intracellular ROS generation was measured by fluorescence spectroscopy using the probe dihydrorhodamine 123 (DHR-123). RESULTS: Human serum but not PS caused thrombus formation on PAEC under high shear stress. Blockade of complement activation by sCR1 prevented xenogeneic serum-induced thrombus formation. Activated platelets did not promote thrombus formation on resting endothelium, and did not further increase platelet deposition on xenogeneic serum-treated PAEC. Vitronectin receptor and P-selectin were up-regulated on the endothelial surface by HS. Their functional blockade by specific antibodies prevented platelet deposition and thrombus formation. H2O2 production significantly increased when PAEC were exposed to the xenogeneic condition. Antioxidants and sCR1 completely prevented thrombus formation by reducing excessive ROS production and the expression of vitronectin receptor and P-selectin. CONCLUSIONS: Xenogeneic complement induces endothelial cell activation and thrombosis which is independent of platelet activation. Complement deposition elicits a rapid generation of ROS that lead to overexpression of endothelial adhesive molecules instrumental for platelet deposition.     

Ovine Leukocyte Microparticles Generated by the CentriMag Ventricular Assist Device In Vitro

Ventricular assist devices (VADs) are a life‐saving form of mechanical circulatory support in heart failure patients. However, VADs have not yet reached their full potential due to the associated side effects (thrombosis, bleeding, infection) related to the activation and damage of blood cells and proteins caused by mechanical stress and foreign materials. Studies of the effects of VADs on leukocytes are limited, yet leukocyte activation and damage including microparticle generation can influence both thrombosis and infection rates. Therefore, the aim was to develop a multicolor flow cytometry assessment of leukocyte microparticles (LMPs) using ovine blood and the CentriMag VAD as a model for shear stress. Ovine blood was pumped for 6 h in the CentriMag and regular samples analyzed for hemolysis, complete blood counts and LMP by flow cytometry during three different pump operating conditions (low flow, standard, high speed). The high speed condition caused significant increases in plasma‐free hemoglobin; decreases in total leukocytes, granulocytes, monocytes, and platelets; increases in CD45⁺ LMPs as well as two novel LMP populations: CD11b^bright/HLA‐DR^? and CD11b^dull/HLA‐DR⁺, both of which were CD14^?/CD21^?. CD11b^bright/HLA‐DR^? LMPs appeared to respond to an increase in shear magnitude whereas the CD11b^dull/HLA‐DR⁺ LMPs significantly increased in all pumping conditions. We propose that these two populations are released from granulocytes and T cells, respectively, but further research is needed to better characterize these two populations.     

Induced Cell Trauma During in Vitro Perfusion: A Comparison Between Two Different Perfusion Systems

The purpose of this study was to compare blood cell activation during in vitro long-term perfusion using 2 parallel in vitro extracorporeal membrane oxygenation (ECMO) systems. We compared two substantially different perfusion systems, an assistance respiratoire extra corporelle (AREC) system on one hand, containing an AREC pump, silicon tubing, and a hollow-fiber oxygenator, and a centrifugal pump system, on the other hand, containing a Biomedicus centrifugal pump, PVC tubing, and a membrane oxygenator. We measured the platelet count using an automated blood cell counter. Platelet activation was evaluated using a flow cytometric technique for the platelet membrane expression of glycoproteins and ELISA for the plasma concentration of β-thromboglobulin (β-TG), a platelet specific protein released into the blood upon platelet activation. The neutrophil count was assayed using an automated blood cell counter and the plasma concentration of cytokines using an ELISA kit. A significant difference between the two systems was observed in terms of the platelet membrane expression of glycoprotein (GP)Ib (p = 0. 0001) and GPIIb/IIIa (p = 0.0037), indicating a lower degree of platelet activation in the AREC system. The concentration of neutrophils was significantly lower in the centrifugal system (p = 0.002) compared to the AREC system. The neutrophil membrane expression of CD11b was significantly lower (p = 0.0067) in the AREC system, indicating a lower degree of neutrophil activation compared to the centrifugal pump system. A significantly lower degree of hemolysis, as expressed by plasma hemoglobin, was observed in the AREC pump system (p = 0.0491). In conclusion, lower degrees of the platelet membrane expression of GPIb and GPIIb/IIIa and of the neutrophil membrane expression of CD11b were observed in the AREC system, indicating a lower degree of platelet and neutrophil activation in this system. No significant difference between the two systems as to the plasma concentration of interleukin (IL)-1β, IL-6, or IL-8 could be recorded. Further studies are warranted to specify the role of each individual component of the two systems.     

Evaluation of Two Femoral Arterial Cannulae With Conventional Non‐Pulsatile and Alternative Pulsatile Flow in a Simulated Adult ECLS Circuit

The objective of this study is to evaluate the hemodynamic characteristics of two femoral arterial cannulae in terms of circuit pressure, pressure drop, and hemodynamic energy transmission under non‐pulsatile and pulsatile modes in a simulated adult extracorporeal life support (ECLS) system. The ECLS circuit consisted of i‐cor diagonal pump and console (Xenios AG, Heilbronn, Germany), an iLA membrane ventilator (Xenios AG), an 18 Fr or 16 Fr femoral arterial cannula (Xenios AG), and a 23/25 Fr Estech remote access perfusion (RAP) femoral venous cannula (San Ramon, CA, USA). The circuit was primed with lactated Ringer’s solution and packed red blood cells to achieve a hematocrit of 35%. All trials were conducted at room temperature with flow rates of 1–4 L/min (1 L/min increments). The pulsatile flow settings were set at pulsatile frequency of 75 bpm and pulsatile amplitudes of 1000–4000 rpm (1000 rpm increments). Flow and pressure data were collected using a custom data acquisition system. Total hemodynamic energy (THE) is calculated by multiplying the ratio between the area under the hemodynamic power curve (∫flow × pressure dt) and the area under the pump flow curve (∫flow dt) by 1332. The pressure drop across the arterial cannula increased with increasing flow rate and decreasing cannula size. The pressure drops of 18 Fr and 16 Fr cannulae were 19.4–24.5 and 38.4–45.3 mm Hg at 1 L/min, 55.2–56.8 and 110.9–118.3 mm Hg at 2 L/min, 94.1–105.1 and 209.7–215.1 mm Hg at 3 L/min, and 169.2–172.6 and 376.4 mm Hg at 4 L/min, respectively. Pulsatile flow created more hemodynamic energy than non‐pulsatile flow, especially at lower flow rates. The percentages of THE loss across 18 Fr and 16 Fr cannula were 16.0–18.7 and 27.5–30.8% at 1 L/min, 35.1–35.7 and 52.3–53.8% at 2 L/min, 48.3–50.3 and 67.3–68.4% at 3 L/min and 62.9–63.1 and 79.0% at 4 L/min. The hemodynamic performance of the arterial cannula should be evaluated before use in clinical practice. The pressure drops and percentages of THE loss across two cannulae tested using human blood were higher compared to the manufacturer’s data tested using water. The cannula size should be chosen to match the expected flow rate. In addition, this novel i‐cor ECLS system can provide non‐pulsatile and ECG‐synchronized pulsatile flow without significantly increasing the cannula pressure drop and hemodynamic energy loss.     

Platelet protection in coronary artery surgery: benefits of heparin-coated circuits and high-dose aprotinin therapy.

OBJECTIVE: To examine the extent of platelet activation during extracorporeal circulation by using the combination of heparin-coated oxygenation systems and high-dose aprotinin therapy, and to examine the affinity and thereby the protective capacity of aprotinin to the glycoprotein (GP) receptors of the platelet membrane. DESIGN: Experimental in vitro study. SETTING: Research laboratory of a university hospital. PARTICIPANTS: Thirty-two volunteers (blood donors). MEASUREMENTS AND MAIN RESULTS: Thirty-two oxygenation circuits of the same construction series (16 heparin-coated and 16 noncoated) were investigated in a closed system of a heart-lung machine model with fresh human whole blood. In each of these two groups, eight circuits with and eight without a high-dose aprotinin application (250 kallikrein inhibitory units [KIU]/mL) were investigated. In all four groups, the number of platelets declined continuously during the 90-minute recirculation period. Group I (no heparin coating, no aprotinin) showed the greatest reduction; group IV (heparin coating, aprotinin) had a significantly smaller decrease in platelet number (p < 0.01). Platelet factor 4 (PF-4) levels, released from the alpha-granule, were in inverse proportion to the platelet loss. After 90 minutes of recirculation, the PF-4 values increased to 615.8% +/- 559.5% and 237.2% +/- 179.0% of the initial value for groups I and IV, respectively (p < 0.01). Affinity chromatography and immunoblotting techniques were used to evaluate the affinity of aprotinin for the GP receptors of the platelet membrane. The affinity appeared in the following order: GPIIb < GPIIIa < GPIb. CONCLUSION: Heparin-coated oxygenation systems and additional aprotinin caused significantly less platelet damage in an in vitro cardiopulmonary bypass model. Chromatographic and immunologic methods could prove aprotinin's affinity for the platelet receptor proteins GPIb and GPIIb-IIIa and therefore its probable role in diminishing the triggering of the platelet activation cascade.