Mitigation effect of cell exclusion on blood damage in spiral groove bearings.

Cell exclusion in spiral groove bearing (SGB) excludes red blood cells from high shear regions in the bearing gaps and potentially reduce haemolysis in rotary blood pumps. However, this mechanobiological phenomenon has been observed in ultra-low blood haematocrit only, whether it can mitigate blood damage in a clinically-relevant blood haematocrit remains unknown. This study examined whether cell exclusion in a SGB alters haemolysis and/or high-molecular-weight von Willebrand factor (HMW vWF) multimer degradation. Citrated human blood was adjusted to 35 % haematocrit and exposed to a SGB (n = 6) and grooveless disc (n = 3, as a non-cell exclusion control) incorporated into a custom-built Couette test rig operating at 2000RPM for an hour; shearing gaps were 20, 30, and 40 µm. Haemolysis was assessed via spectrophotometry and HMW vWF multimer degradation was detected with gel electrophoresis and immunoblotting. Haemolysis caused by the SGB at gaps of 20, 30 and 40 µm were 10.6 ± 3.3, 9.6 ± 2.7 and 10.5 ± 3.9 mg/dL.hr compared to 23.3 ± 2.6, 12.8 ± 3.2, 9.8 ± 1.8 mg/dL.hr by grooveless disc. At the same shearing gap of 20 µm, there was a significant reduced in haemolysis (P = 0.0001) and better preserved in HMW vWF multimers (p < 0.05) when compared SGB to grooveless disc. The reduction in blood damage in the SGB compared to grooveless disc is indicative of cell exclusion occurred at the gap of 20 µm. This is the first experimental study to demonstrate that cell exclusion in a SGB mitigates the shear-induced blood damage in a clinically-relevant blood haematocrit of 35 %, which can be potentially utilised in future blood pump design.

Discrete responses of erythrocytes, platelets, and von Willebrand factor to shear.

Despite decades of technological advancements in blood-contacting medical devices, complications related to shear flow-induced blood trauma are still frequently observed in clinic. Blood trauma includes haemolysis, platelet activation, and degradation of High Molecular Weight von Willebrand Factor (HMW vWF) multimers, all of which are dependent on the exposure time and magnitude of shear stress. Specifically, accumulating evidence supports that when blood is exposed to shear stresses above a certain threshold, blood trauma ensues; however, it remains unclear how various constituents of blood are affected by discrete shears experimentally. The aim of this study was to expose blood to discrete shear stresses and evaluate blood trauma indices that reflect red cell, platelet, and vWF structure. Citrated human whole blood (n = 6) was collected and its haematocrit was adjusted to 30 ± 2% by adding either phosphate buffered saline (PBS) or polyvinylpyrrolidone (PVP). Viscosity of whole blood was adjusted to 3.0, 12.5, 22.5 and 37.5 mPa·s to yield stresses of 3, 6, 9, 12, 50, 90 and 150 Pa in a custom-developed shearing system. Blood samples were exposed to shear for 0, 300, 600 and 900 s. Haemolysis was measured using spectrophotometry, platelet activation using flow cytometry, and HMW vWF multimer degradation was quantified with gel electrophoresis and immunoblotting. For tolerance to 300, 600 and 900 s of exposure time, the critical threshold of haemolysis was reached after blood was exposed to 90 Pa for 600 s (P < 0.05), platelet activation and HMW vWF multimer degradation were 50 Pa for 600 s and 12 Pa for 300 s respectively (P < 0.05). Our experimental results provide simultaneous comparison of blood trauma indices and thus also the relation between shear duration and magnitude required to induce damage to red cells, platelets, and vWF. Our results also demonstrate that near-physiological shear stress (<12 Pa) is needed in order to completely avoid any form of blood trauma. Therefore, there is an urgent need to design low shear-flow medical devices in order to avoid blood trauma in this blood-contacting medical device field.

Evaluation of real-time thrombus detection method in a magnetically levitated centrifugal blood pump using a porcine left ventricular assist circulation model.

Pump thrombosis induces significant complications and requires timely detection. We proposed real-time monitoring of pump thrombus in a magnetically levitated centrifugal blood pump (mag-lev pump) without using additional sensors, by focusing on the changes in the displacement of the pump impeller. The phase difference between the current and displacement of the impeller increases with pump thrombus. This thrombus detection method was previously evaluated through simulated circuit experiments using porcine blood. Evaluation of real-time thrombus detection in a mag-lev blood pump was performed using a porcine left ventricular assist circulation model in this study. Acute animal experiments were performed five times using five Japanese domestic pigs. To create thrombogenic conditions, fibrinogen coating that induces thrombus formation in a short time was applied to the inner surfaces of the pump. An inflow and an outflow cannula were inserted into the apex of the left ventricle and the carotid artery, respectively, by a minimally invasive surgical procedure that allowed minimal bleeding and hypothermia. Pump flow was maintained at 1 L/min without anticoagulation. The vibrational frequency of the impeller (70 Hz) and its vibrational amplitude (30 µm) were kept constant. The thrombus was detected based on the fact that the phase difference between the impeller displacement and input current to the magnetic bearing increases when a thrombus is formed inside a pump. The experiment was terminated when the phase difference increased by over 1° from the lowest value or when the phase difference was at the lowest value 12 hours after commencing measurements. The phase difference increased by over 1° in three cases. The pump was stopped after 12 hours in two cases. Pump thrombi were found in the pump in three cases in which the phase difference increased by over 1°. No pump thrombus was found in the other two cases in which the phase difference did not increase. We succeeded in real-time thrombus monitoring of a mag-lev pump in acute animal experiments.

Shear-dependent platelet aggregation size.

Nonsurgical bleeding is the most frequent complication of left ventricular assist device (LVAD) support. Supraphysiologic shear rates generated in LVAD causes impaired platelet aggregation, which increases the risk of bleeding. The effect of shear rate on the formation size of platelet aggregates has never been reported experimentally, although platelet aggregation size can be considered to be directly relevant to bleeding complications. Therefore, this study investigated the impact of shear rate and exposure time on the formation size of platelet aggregates, which is vital in predicting bleeding in patients with an LVAD. Human platelet-poor plasma (containing von Willebrand factor, vWF) and fluorochrome-labeled platelets were subjected to a range of shear rates (0-10 000 s-1 ) for 0, 5, 10, and 15 minutes using a custom-built blood-shearing device. Formed sizes of platelet aggregates under a range of shear-controlled environment were visualized and measured using microscopy. The loss of high molecular weight (HMW) vWF multimers was quantified using gel electrophoresis and immunoblotting. An inhibition study was also performed to investigate the reduction in platelet aggregation size and HMW vWF multimers caused by either mechanical shear or enzymatic (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13-ADAMTS13, the von Willebrand factor protease) mechanism under low and high shear conditions (360 and 10 000 s-1 ). We found that the average size of platelet aggregates formed under physiological shear rates of 360-3000 s-1 (200-300 µm2 ) was significantly larger compared to those sheared at >6000 s-1 (50-100 µm2 ). Furthermore, HMW vWF multimers were reduced with increased shear rates. The inhibition study revealed that the reduction in platelet aggregation size and HWM vWF multimers were mainly associated with ADAMTS13. In conclusion, the threshold of shear rate must not exceed >6000 s-1 in order to maintain the optimal size of platelet aggregates to "plug off" the injury site and stop bleeding.

Verification of a thrombus induction method at the target point inside the blood pump using a fibrinogen coating for a thrombus detection study.

Although the magnetically levitated centrifugal blood pump (mag-lev pump) is considered superior to other pumps in antithrombogenicity, thrombotic complications are still reported. Research into thrombus detection inside a mag-lev pump is very important for solving this problem. Our research group has already proposed a method to detect a thrombus inside a mag-lev pump in real time without an additional sensor, which is named the impeller vibration method. To efficiently advance our research with reproducibility, a preconditioning method to induce thrombus inside the pump was thought to be necessary. Therefore, this study aimed to develop a preconditioning method that induces thrombus formation. To verify this method, in vitro experiments for thrombus detection were performed. A mag-lev pump developed at Tokyo Institute of Technology was used. A fibrinogen solution was coated on the inner surfaces of the bottom housing to induce thrombus formation at the target point inside the pump. The thrombus is detected by utilizing the phenomenon that the phase difference between the impeller displacement and input current to the magnetic bearing increases when a thrombus is formed inside a pump. Five hundred mL of porcine blood anticoagulated with heparin sodium was circulated in the mock circuit, and protamine sulfate was administered. Flow rate (1 L/min), impeller vibrational frequency (70 Hz), and vibrational amplitude (30 µm) were set to constant. The experiment was terminated when the phase difference increased by over 2° from the minimum value. The experiments were performed in fibrinogen-coated (group F, n = 5) and non-coated pumps (group N, n = 5). In group F, thrombus formation was observed at the fibrinogen-coated point of the housing. In contrast, a relatively small thrombus was observed in varying locations such as the housing or the impeller in group N. Thrombus formation time (the time from when the phase difference takes the minimum value to when the experiment is terminated) was different between the two groups. The mean time was significantly shorter in group F (44 ± 29 minutes) than in group N (143 ± 38 minutes; p = 0.0019). Therefore, a preconditioning method that induced thrombus formation at the target point inside a blood pump was successfully developed.

Detection of thrombosis in a magnetically levitated blood pump by vibrational excitation of the impeller.

The use of contactless support technology for the impeller has led to an increase in the durability of ventricular assist devices (VADs), and these have been in clinical use worldwide. However, pump thrombosis and stroke are still issues to be solved. We have developed a method for detecting the thrombosis in a magnetically levitated blood pump without the need for additional sensors or other equipment. In the proposed method, a sinusoidal current is applied to the electromagnets used for the magnetic bearing, resulting in vibration of the impeller. The phase difference between the current and displacement of the impeller increases with pump thrombosis. First, we describe the principle by which the pump thrombosis is detected. Pump thrombosis reduces the narrowest fluid gap in the pump and this gives rise to a change in the phase difference. Second, we report on experiments in which we changed the narrowest fluid gap using oriented polypropylene tape and showed that decreasing the narrowest fluid gap resulted in an increase in phase difference. For these experiments, the measurements were repeated three times for each condition. Third, we examine the relationship between the pump thrombosis and the phase difference evaluated by observations of the underside of the impeller when operating the pump with porcine blood. Since light was unable to penetrate the blood layer, the erythrocytes were removed for this observation. Only one observation was made. The results showed the phase difference rapidly increased at the same moment when the pump thrombosis was observed. This implies the proposed method has the potential to detect the early stages of pump thrombosis. Finally, in vitro experiments to detect thrombosis when using whole porcine blood in the pump were conducted. The experiment was carried out five times. To intentionally form a thrombus inside the pump, the activated clotting time was controlled to be less than 200 s. In every case, the phase difference increased by more than one degree after tens of minutes. Then, the pump was disassembled and a small amount of pump thrombosis was observed. We conclude that real-time diagnosis of pump thrombosis may be realized by measuring the phase difference without the need for additional sensors.

Mechanical antithrombogenic properties by vibrational excitation of the impeller in a magnetically levitated centrifugal blood pump.

Mechanical circulatory support devices have been used clinically for patients with heart failure for over 10 years. However, thrombus formation inside blood pumps remains a risk to patient life, causing pump failure and contributing to neurological damage through embolization. In this article, we propose a method for preventing thrombus formation by applying vibrational excitation to the impeller. We evaluate the ability of this method to enhance the antithrombogenic properties of a magnetically levitated centrifugal blood pump and ensure that the impeller vibration does not cause undue hemolysis. First, 3 vibrational conditions were compared using an isolated pump without a mock circulation loop; the vibrational excitation frequencies and amplitudes for the impeller were set to (a) 0 Hz-0 µm, (b) 70 Hz-10 µm, and (c) 300 Hz-2.5 µm. The motor torque was measured to detect thrombus formation and obtain blood coagulation time by calculating the derivative of the torque. Upon thrombus detection, the pump was stopped and thrombi size were evaluated. The results showed an increase in the blood coagulation time and a decrease in the rate of thrombus formation in pumps with the impeller vibration. Second, an in vitro hemolysis test was performed for each vibrational condition to determine the effect of impeller vibration on hemolysis. The results revealed that there was no significant difference in hemolysis levels between each condition. Finally, the selected vibration based on the above test results and the non-vibration as control were compared to investigate antithrombogenic properties under the continuous flow condition. The blood coagulation time and thrombi size were investigated. As a result, vibrational excitation of the impeller at a frequency of 300 Hz and amplitude of 2.5 µm was found to significantly lengthen clotting time, decreasing the rate of pump thrombus compared to the non-vibration condition. We indicate the potential of impeller vibration as a novel mechanical antithrombogenic mechanism for rotary blood pumps.

Development of an Intelligent Ventricular Assist Device with a Function of Sensorless Thrombus Detection.

Thrombus is one of the major problems in ventricular assist devices (VADs). However, method for detecting thrombus in early stage has not been established yet. In this study, we propose an intelligent function that the VAD itself can detect thrombus automatically and alert it to medical staffs. In the proposed method, thrombus formation inside a blood pump is detected by monitoring blood viscosity. This viscosity measurement is performed by using magnetic levitation system for the impeller. Hence, it can be implemented without any additional sensors or mechanisms in principle. For verification of the method, at first, we visualized inside of the pump during thrombus formation with measuring blood viscosity by using erythrocytes removed porcine blood. The result showed that the viscosity of the blood increased as blood coagulation progressed. Then, we conducted in vitro principle verification experiments with three different whole porcine blood. In all experiments, the measured blood viscosity increased and small thrombus was observed inside the pump. From these results, we confirmed that the proposed method has a possibility to detect and predict the thrombus in early stage.

Optical Dynamic Analysis of Thrombus Inside a Centrifugal Blood Pump During Extracorporeal Mechanical Circulatory Support in a Porcine Model.

Complications due to pump thrombus remain the weak point of mechanical circulatory support (MCS), such as the use of a left ventricular assist device (LVAD) or extracorporeal membrane oxygenation, leading to poor outcomes. Hyperspectral imaging (HSI) is an effective imaging method using a hyperspectral (HS) camera, which comprises a spectrophotometer and a charge-coupled device camera to discriminate thrombus from whole blood. Animal experiments were conducted to analyze dynamic imaging of thrombus inside a prototype of a hydrodynamically levitated centrifugal blood pump using an HSI system. Six pigs were divided into a venous circulation group (n = 3) and an arterial circulation group (n = 3). Inflow and outflow cannulae were inserted into the jugular veins in the venous circulation group. The latter simulated an LVAD application. To create thrombogenic conditions, pump flow was maintained at 1 L/min without anticoagulation. An image of the bottom surface of the pump was captured by the HS camera every 4 nm over the wavelength range of 608-752 nm. Real-time dynamic images of the inside of the pump were displayed on the monitor. Appearance of an area displaying thrombus was detected within 24 h after the start of the circulation in every experiment. This imaging system also succeeded in determining the origins of pump thrombus: from inside the pump in two cases, and from outside in four cases. Two main possible sources of pump thrombus originating outside the pump were identified on autopsy: wedge thrombus around the inflow cannula; and string-like thrombus at the junction between the pump inlet and circuit tube. The results of this study from close observation of the changing appearance of pump thrombus may contribute to improvements in the safety of extracorporeal MCS.

Plasma Skimming in a Spiral Groove Bearing of a Centrifugal Blood Pump.

Plasma skimming is a phenomenon in which discharge hematocrit is lower than feed hematocrit in microvessels. Plasma skimming has been investigated at a bearing gap in a spiral groove bearing (SGB), as this has the potential to prevent hemolysis in the SGB of a blood pump. However, it is not clear whether plasma skimming occurs in a blood pump with the SGB, because the hematocrit has not been obtained. The purpose of this study is to verify plasma skimming in an SGB of a centrifugal blood pump by developing a hematocrit measurement method in an SGB. Erythrocyte observation using a high-speed microscope and a bearing gap measurement using a laser confocal displacement meter was performed five times. In these tests, bovine blood as a working fluid was diluted with autologous plasma to adjust the hematocrit to 1.0%. A resistor was adjusted to achieve a pressure head of 100 mm Hg and a flow rate of 5.0 L/min at a rotational speed of 2800 rpm. Hematocrit on the ridge region in the SGB was measured using an image analysis based on motion image of erythrocytes, mean corpuscular volume, the measured bearing gap, and a cross-sectional area of erythrocyte. Mean hematocrit on the ridge region in the SGB was linearly reduced from 0.97 to 0.07% with the decreasing mean bearing gap from 38 to 21 µm when the rotational speed was changed from 2250 to 3000 rpm. A maximum plasma skimming efficiency of 93% was obtained with a gap of 21 µm. In conclusion, we succeeded in measuring the hematocrit on the ridge region in the SGB of the blood pump. Hematocrit decreased on the ridge region in the SGB and plasma skimming occurred with a bearing gap of less than 30 µm in the hydrodynamically levitated centrifugal blood pump.

Real-Time Observation of Thrombus Growth Process in an Impeller of a Hydrodynamically Levitated Centrifugal Blood Pump by Near-Infrared Hyperspectral Imaging.

Understanding the thrombus formation in cardiovascular devices such as rotary blood pumps is the most important issue in developing more hemocompatible devices. The objective of this study was to develop a hyperspectral imaging (HSI) method to visualize the thrombus growth process within a rotary blood pump and investigate the optical properties of the thrombus. An in vitro thrombogenic test was conducted using fresh porcine blood and a specially designed hydrodynamically levitated centrifugal blood pump with a transparent bottom. The pump rotating at 3000 rpm circulated the blood at 1.0 L/min. The bottom surface of the pump was illuminated with white light pulsed at the same frequency as the pump rotation, and the backward-scattered light was imaged using the HSI system. Using stroboscopic HSI and an image construction algorithm, dynamic spectral imaging at wavelengths ranging from 608 to 752 nm within the rotating pump was achieved. After completing the experiment, we collected the red thrombus formed in the pump impeller and quantified the thrombus hemoglobin concentration (Hbthrombus ). The spectrum changed around the center of the impeller, and the area of change expanded toward the impeller flow path. The shape corresponded approximately to the shape of the thrombus. The spectrum change indicated that the light scattering derived from red blood cells decreased. The Hbthrombus was 4.7 ± 1.3 g/dL versus a total hemoglobin of 13 ± 0.87 g/dL. The study revealed that Hbthrombus was reduced by the surrounding blood flow.

Evaluation of a Spiral Groove Geometry for Improvement of Hemolysis Level in a Hydrodynamically Levitated Centrifugal Blood Pump.

The purpose of this study is to evaluate a spiral groove geometry for a thrust bearing to improve the hemolysis level in a hydrodynamically levitated centrifugal blood pump. We compared three geometric models: (i) the groove width is the same as the ridge width at any given polar coordinate (conventional model); (ii) the groove width contracts inward from 9.7 to 0.5 mm (contraction model); and (iii) the groove width expands inward from 0.5 to 4.2 mm (expansion model). To evaluate the hemolysis level, an impeller levitation performance test and in vitro hemolysis test were conducted using a mock circulation loop. In these tests, the driving conditions were set at a pressure head of 200 mm Hg and a flow rate of 4.0 L/min. As a result of the impeller levitation performance test, the bottom bearing gaps of the contraction and conventional models were 88 and 25 µm, respectively. The impeller of the expansion model touched the bottom housing. In the hemolysis test, the relative normalized index of hemolysis (NIH) ratios of the contraction model in comparison with BPX-80 and HPM-15 were 0.6 and 0.9, respectively. In contrast, the relative NIH ratios of the conventional model in comparison with BPX-80 and HPM-15 were 9.6 and 13.7, respectively. We confirmed that the contraction model achieved a large bearing gap and improved the hemolysis level in a hydrodynamically levitated centrifugal blood pump.

Evaluation of erythrocyte flow at a bearing gap in a hydrodynamically levitated centrifugal blood pump.

We have developed a hydrodynamically levitated centrifugal blood pump for extracorporeal circulatory support. In the blood pump, a spiral groove bearing was adopted for a thrust bearing. In the spiral groove bearing, separation of erythrocytes and plasma by plasma skimming has been postulated to occur. However, it is not clarified that plasma skimming occurs in a spiral groove bearing. The purpose of this study is to verify whether plasma skimming occurs in the spiral groove bearing of a hydrodynamically levitated centrifugal blood pump. For evaluation of plasma skimming in the spiral groove bearing, an impeller levitation performance test using a laser focus displacement meter and a microscopic visualization test of erythrocyte flow using a high-speed microscope were conducted. Bovine blood diluted with autologous plasma to adjust hematocrit to 1.0% was used as a working fluid. Hematocrit on the ridge region in the spiral groove bearing was estimated using image analysis. As a result, hematocrits on the ridge region with gaps of 45 µm, 31 µm, and 25 µm were calculated as 1.0%, 0.6%, and 0.3%, respectively. Maximum skimming efficiency in this study was calculated as 70% with a gap of 25 µm. We confirmed that separation of erythrocyte and plasma occurred in the spiral groove bearing with decrease in bearing gap in a hydrodynamically levitated centrifugal blood pump.

Feasibility of the optical imaging of thrombus formation in a rotary blood pump by near-infrared light.

Blood coagulation is one of the primary concerns when using mechanical circulatory support devices such as blood pumps. Noninvasive detection and imaging of thrombus formation is useful not only for the development of more hemocompatible devices but also for the management of blood coagulation to avoid risk of infarction. The objective of this study is to investigate the use of near-infrared light for imaging of thrombus formation in a rotary blood pump. The optical properties of a thrombus at wavelengths ranging from 600 to 750 nm were analyzed using a hyperspectral imaging (HSI) system. A specially designed hydrodynamically levitated centrifugal blood pump with a visible bottom area was used. In vitro antithrombogenic testing was conducted five times with the pump using bovine whole blood in which the activated blood clotting time was adjusted to 200 s prior to the experiment. Two halogen lights were used for the light sources. The forward scattering through the pump and backward scattering on the pump bottom area were imaged using the HSI system. HSI showed an increase in forward scattering at wavelengths ranging from 670 to 750 nm in the location of thrombus formation. The time at which the thrombus began to form in the impeller rotating at 2780 rpm could be detected. The spectral difference between the whole blood and the thrombus was utilized to image thrombus formation. The results indicate the feasibility of dynamically detecting and imaging thrombus formation in a rotary blood pump.

Fluid dynamic design for low hemolysis in a hydrodynamically levitated centrifugal blood pump.

We have developed a hydrodynamically levitated centrifugal blood pump for extracorporeal circulatory support as a bridge to decision pump. The impeller is levitated using hydrodynamic bearings without any complicated control circuit or displacement sensor. However, the effect of the outer circumferential velocity and the bearing area on the hemolytic property has not been clarified, even if the bearing gap is same size. The purpose of this study is to experimentally evaluate the effect of the outer circumferential velocity and the bearing area in the bearing gaps on the hemolytic property in a hydrodynamically levitated centrifugal blood pump. We prepared three models for testing. These models have the same bearing gap size by adjusting the impeller levitation position. However, the outer circumferential velocity of the impeller and the bearing area in the minimum bearing gaps are different. The outer circumferential velocity of the impeller and the bearing area were assumed to be related to the maximum shear rate and the exposure time. For the evaluation, we conducted an impeller levitation performance test and an in vitro hemolysis test. As a result, the normalized index of hemolysis (NIH) was reduced from 0.084 g/100 L to 0.040 g/100 L corresponding to a reduction in the outer circumferential velocity and a reduction in the bearing area, even if the minimum bearing gaps were same size. We confirmed that, even if the bearing gap was same size under the stably levitated condition, the outer circumferential velocity and the bearing area should be decreased in order to improve the hemolytic property.