Adult Circulatory Support
Short-Term In Vivo Preclinical Biocompatibility Evaluation of FW-II Axial Blood Pump in a Sheep Model
Chen, Haibo*†; Zhou, Jianye*; Sun, Hansong†; Tang, Yue†; Zhang, Yan†; Liu, Guangmao*; Hu, Shengshou*†
From the *Research Center for Cardiovascular Regenerative Medicine, the Ministry of Health; and †Department of Surgery, Cardiovascular Institute and Fuwai Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China.
Submitted for consideration August 2010; accepted for publication in revised form February 2011.
Reprint Requests: Shengshou Hu, MD, Department of Surgery, Chinese Academy of Medical Sciences, Peking Union Medical College, Fuwai Hospital and Cardiovascular Institute, 167 Bei li shi Road, Fuwai Da Jie, Xi Cheng District, Beijing 100037, People's Republic of China. Email: email@example.com.
We investigated the outcome of FW-II axial pump on healthy sheep (weight, 60–70 kg) for 2 weeks by perioperatively hematological and chemical tests, and circulating activated platelet and leukocyte-platelet aggregates measurements by flow cytometry assays. Complete necropsy and histopathological examinations and thorough pump inspection were performed at study termination for evidence of thrombi. In this experimental series, one sheep died of pulmonary edema, the other four sheep reached the scheduled endpoint of 14 days without device-related problems, and flow range was maintained at 2.5–4.0 L/min. The number of red blood cells and platelets decreased within first 3 days but increased gradually after the first postoperative week. In all animals, serum glutamic oxaloacetic transaminase increased significantly after surgery but gradually returned to normal limits within 2 weeks. Platelet activation, granulocyte-platelet aggregates, and monocyte-platelet aggregates reached the peak at postoperative day 2. Postexplant examinations indicated round thrombus in the hub areas of pumps. No evidence of ischemia or infarction was found in the explanted hearts, livers, spleens, kidneys, and brains of the five animals. Our results demonstrate that FW-II ventricular assist device (VAD) is a promising device for left ventricular (LV) support with moderate anticoagulation.
Over the past 5 years, the FW axial blood pump had been developed at Fu Wai Hospital; numerical computational fluid dynamics (CFD) analysis were used to develop the FW-II blood pump.1,2 After optimization of CFD analysis for FW axial blood pump, average shear distribution of impeller is obviously decreased. In vitro testing showed that temperature near the impeller increased within 3°C, durability time was 30 days long, and preliminary in vivo animal experiment demonstrated that hemodynamic effect achieved physiological requirements.3,4
Blood compatibility evaluation is necessary before future clinical use of any pumps, and governing regulatory requirements are usually used to test biocompatibility by most of device developers.5,6 However, these requirements do not necessarily reflect the more subtle effects of ventricular assist devices (VADs), such as chronic inflammation, immune dysfunction, and hormonal derangements.7–9 In recent years, degree of platelet activation and leukocyte-platelet aggregates by flow cytometric assays have been indicative of the level of thrombosis or thromboembolism after implantation of left ventricular assist device (LVAD) and reflected specific hemostatic alterations with the clinical use of cardiovascular devices.10–14 Therefore, in addtion to hemotological parameter, in this report we will investigate FW-II device biocompatibility by applying flow cytometric measurement of platelet activation and microaggregate formation, as well as postexplant device analysis to five blood pumps implanted in sheep for duration of 2 weeks.
Materials and Methods
Device Descriptions and Surgery
The FW-II axial blood pump is a miniaturized rotary blood pump with a diameter of 30 mm and a weight of 180 g (including direct current [DC] motor). The pump consists of a rotor driven by a brushless direct current motor, the housing of the rotor, the inflow inducer, the outflow diffuser, and the impeller (Figure 1).
Five healthy adult male sheep (weighing between 60 and 70 kg) were used in this study. After being anesthetized, sheep were placed in right lateral position, the chest was opened through a left thoracotomy, an inflow cannula was inserted into the apex of the left ventricular, and an outflow cannula was connected to the descending aorta using a bovine jugular vein graft. The pump was placed on the back of the sheep and connected to the cannulae using ½-inch Tygon tubing. Activated clotting time (ACT) was checked every 4–6 h after surgery and controlled at approximately 200–250 seconds by heparin. Heparin was discontinued when international normalized ratio (INR) was within the target therapeutic range. Postoperative animals received oral warfarin sodium to maintain an INR of 3.0–3.5 for the duration of the study. Pump-operating parameters (fixed rate rotational speed setting and pump flow) were recorded continuously with an ultrasonic flow meter (Transonic T101 and ½-inch flow probe; Transonic Systems, Inc. Ithaca, NY). Our Institutional Animal Care and Use Committee approved all protocols used in the present study.
Hematologic Parameter Tests
Blood was obtained by jugular vein puncture, and the minimum blood volume collected was 12 ml, with the remaining blood being used for following assays. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, urea and creatinine levels, as well as red blood cell (RBC) count, platelet count, white blood cell (WBC) count, and hematocrit values were tested at baseline (within 2 days before VAD implantation). These hematologic and biochemical tests were also performed at operative days 1, 2, 3, 5, 7, 10, 12, and 14. The free plasma hemoglobin was measured using plasma hemoglobin measuring kits (Sigma, St. Louis, MO).
The autopsy was performed at the day of termination of the trials. In each case, FW-II axial blood pump was explanted, disassembled, and photographed. The pump housing, impeller, inflow, and outflow grafts were carefully inspected and evaluated for fibrin formation or thrombus. Macroscopical and histological examination was performed on the heart, liver, kidneys, brain, and spleen, and tissue samples from these organs were fixed in formaldehyde and embedded in paraffin. Sections, 3–5 μm in thickness, were stained using hematoxylin and eosin for histological evaluation.
Animals and Devices
Postoperatively, all sheep recovered from anesthesia without complications and were extubated within the first 6 hours. Every sheep received benzylpenicillin for preventing infection, and no fever and weight loss were observed. None of the sheep developed anorexia, infection, or neurologic disorders. One sheep died of pulmonary edema at postoperative day 12, and the other four sheep reached the study endpoint of 14 days. The FW-II VADs functioned without technical problems throughout the entire course of each experiment. An average pump speed of 7,800 ± 361 RPM produced average flows of 2.6 ± 1.1 L/min. Table 1 summarizes device flow and beat rate for each experiment.
Hematologic and Biochemical Data
We demonstrated the results of hematological and chemical studies performed on blood samples. Results showed a transient postsurgical decrease of RBC count from a base level (5.4 ± 1.1 × 106/mm3) to 3.4 ± 0.9 × 106/mm3. After the first postimplantation week, the RBC count returned to the normal level (4.7 ± 1.1 × 106/mm3). Plasma-free hemoglobin (plfHb) concentration was predominantly 40 ± 30 mg/L at postoperative day 3 and then decreased significantly at postoperative day 14 (20 ± 10 mg/L). Postoperative AST increased significantly, reached the peak at postoperative day 1 (206.2 ± 33.8 IU/L), and returned to normal level at postoperative day 14 (52.7 ± 18.4 IU/L). Other blood indices (ALT, total bilirubin, urea nitrogen, and creatinine) retained unchanged pre- and postimplantation (Table 2).
Temporal Course of Circulating Activated Platelet and Leukocyte-Platelet Aggregates
Platelet activation, granulocyte platelet aggregates, and monocyte platelet aggregates were determined by flow cytometric assays with annexin V, CD41/61, and CD14 (Figure 2). Compared with preoperative values (5% ± 3%, 3% ± 1%, and 2% ± 1%), the number of platelet activation, granulocyte-platelet aggregates, and monocyte-platelet aggregates reached a peak (45.6% ± 2.7%, 42.4% ± 2.3%, and 39.8% ± 1.5%, respectively, p < 0.01) at postoperative day 2; they decreased significantly (32.5% ± 3.94%, 30.6% ± 2.7%, and 28.6% ± 2.69%, respectively, p < 0.05) at postoperative day 7 (Figure 3). Platelet activation and leukocyte platelet aggregates of the third sheep after implantation of FW-II VAD are significantly higher than those of other four animals and increased before the study terminated (Figure 4).
Pump Postexplant and Pathological Findings
The five pumps had mild thrombus formation in the hub area and weighed 0.006 ± 0.002 g and 0.008 ± 0.004 g in the front and rear hub, respectively (Figure 5) (Table 3). In the third sheep, linear thrombus was seen at the connections of the inflow to the pump. The inflow cannula in other four sheep and the outflow graft anastomosis in all sheep were unremarkable. There were no thrombi found in other components of FW-II axial blood pump (impeller, straightener, and diffuser). No evidence of ischemia or infarction was found in the explanted hearts, livers, spleens, kidneys, and brains of the third sheep by postmortem macroscopic and microscopic examination (Figure 6). For the other four sheep, no ischemia and infarction were observed either in major organs.
Compared with a pulsatile blood pump, the benefits of an axial flow VAD are its compact size, valveless design, and simple construction. As a result, a number of axial flow VADs, such as the MicroMed DeBakey VAD, the Jarvik2000, the HeartMate II, have been developed and achieved good clinical outcomes. In the past 5 years, our study group also focused on axial flow VADs. To develop an axial blood pump, minimal hemolysis must be accomplished. In our study, red cell count decreased significantly at postoperative day 3 but returned to normal level after 7 days of operation, and plasma-free hemoglobin is <40 mg/dl. Insufficient perfusion of thromboembolic damage to end organs significantly influences survival in LVAD patients.15 Impact of LVAD on end organs is also an important evaluation. According to biochemical test results from our study, renal and hepatic functions were not adversely affected. Although transient increases in glutamic oxaloacetic transaminase (GOT) levels were observed in all animals in the immediate postoperative period, this can be attributed to the inevitable trauma sustained during surgery. The GOT level gradually returned to within normal range in all animals within 1 week, and no other treatment was necessary.
Previous in vitro and numerical studies demonstrated that the high shear stress generated by rotary blood pump can induce platelet activation and increase the risk of thromboembolism.16 In our study, FW-II VAD also induced platelet activation and leukocyte-platelet aggregates, and returned to preoperative level within 14 days. The results obtained from this study help in identifying the underlying mechanisms that are responsible for the enhanced hemostatic response, hence the increased cardioembolic risk. A recognized feature of the complex interplay regulating the pathogenesis of thrombosis is the effect of blood flow-induced mechanical forces on platelets. These flow-induced forces trigger platelet activation, and this process commences with the secretion of procoagulant and self-stimulating substances from granules, which catalyze thrombin production.17 As a direct consequence of activation, the platelets undergo a change in shape, marked by pseudopod extension. This increases the strength of adhesion to exogenous surfaces and decreases the resistance to aggregation.
However, in one sheep with thrombosis in the inflow connection with blood pump, heart rate (HR), mean arterial blood pressure (MAP), and central venous pressure (CVP) were higher than other four sheep, this was related to thrombus which partially influenced hemodynamics. The number of platelet activation and leukocyte platelet aggregates is also significantly higher than other four sheep. Previous studies also had showed that animals with higher platelet activity consistently exhibited evidence of increased thromboembolism in terms of renal infarcts with respect to calves with uneventful VAD implantation periods.11 Elevated flow shear stresses that are present in rotary blood pump devices enhance the hemostatic response and their propensity to initiate thromboembolism.18,19 The interaction between leukocytes and platelets may be an important contributor to hemostasis in thrombosis.20
To develop an axial blood pump, antithrombogenicity evaluation was the focus during implantation of 2 weeks or longer durations. After the sheep with FW-II VAD support was euthanized, detailed pathological and histological examinations on the explanted axial blood pump are conducted, and we found mild thrombus formation in front and rear hub of FW-II axial blood pumps. However, no histologic changes were observed in major end organs, and there was also no clinical evidence of thrombolism. In most second-generation LVADs, heat generation on the bearings is known to denaturize proteins and can lead to thrombosis.21–25 In recent years, magnetic levational bearings VAD, such as PediPump which is a mixed flow and rotary VAD that uses passive magnetic radial bearings to support the impeller,26 may solve mechanical bearings thrombosis of FW-II axial blood pump.
In summary, the results obtained from this study provided very useful information with respect to the FW-II VAD-related changes in vivo after implantation. In our sheep model, the FW-II VAD system demonstrated excellent blood-handling characteristics and reliability for 14 days, both of which are crucial to the clinical success of any implantable LVAD. Annexin V, CD41/61, and CD14 may potentially be indicators for thrombolism.
Supported by grants from the 863 Program (2006AA02Z4D3) and the Eleventh Five-Year Plan, the National Development and Reform Commission (2006BAI01A09).
The authors acknowledge the staff of animal center at Fuwai Hospital.
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Copyright © 2011 by the American Society for Artificial Internal Organs
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