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.

Extracorporeal Membrane Oxygenation-Induced Hemolysis: An In Vitro Study to Appraise Causative Factors.

In vitro hemolysis testing is commonly used to determine hemocompatibility of ExtraCorporeal Membrane Oxygenation (ECMO). However, poor reproducibility remains a challenging problem, due to several unidentified influencing factors. The present study investigated potential factors, such as flow rates, the use of anticoagulants, and gender of blood donors, which could play a role in hemolysis. Fresh human whole blood was anticoagulated with either citrate (n = 6) or heparin (n = 12; 6 female and 6 male blood donors). Blood was then circulated for 360 min at 4 L/min or 1.5 L/min. Regardless of flow rate conditions, hemolysis remained unchanged over time in citrated blood, but significantly increased after 240 min circulation in heparinized blood (p ≤ 0.01). The ratio of the normalized index of hemolysis (NIH) of heparinized blood to citrated blood was 11.7-fold higher at 4 L/min and 16.5-fold higher at 1.5 L/min. The difference in hemolysis between 1.5 L/min and 4 L/min concurred with findings of previous literature. In addition, the ratio of NIH of male heparinized blood to female was 1.7-fold higher at 4 L/min and 2.2-fold higher at 1.5 L/min. Our preliminary results suggested that the choice of anticoagulant and blood donor gender could be critical factors in hemolysis studies, and should be taken into account to improve testing reliability during ECMO.

Ex vivo assessment of erythrocyte tolerance to the HeartWare ventricular assist device operated in three discrete configurations.

Despite technological advances in ventricular assist devices (VADs) to treat end-stage heart failure, hemocompatibility remains a constant concern, with supraphysiological shear stresses an unavoidable reality with clinical use. Given that impeller rotational speed is related to the instantaneous shear within the pump housing, it is plausible that the modulation of pump speed may regulate peak mechanical shear stresses and thus ameliorate blood damage. The present study investigated the hemocompatibility of the HeartWare HVAD in three configurations typical of clinical applications: standard systemic support left VAD (LVAD), pediatric support LVAD, and pulmonary support right VAD (RVAD) conditions. Two ex vivo mock circulation blood loops were constructed using explanted HVADs, in which pump speed and external loop resistance were manipulated to reflect the flow rates and differential pressures reported in configurations for standard adult LVAD (at 3150 rev⸱min-1 ), pediatric LVAD (at 2400 rev⸱min-1 ), and adult RVAD (at 1900 rev⸱min-1 ). Using bovine blood, the mock circulation blood loops were tested at 37°C over a period of 6 hours (consistent with ASTM F1841-97) and compared with static control. Hemocompatibility assessments were conducted for each test condition, examining hematology, hemolysis (absolute and normalized index), osmotic fragility, and blood viscosity. Regardless of configuration, continuous exposure of blood to the VAD over the 6-hour period significantly altered hematological and rheological blood parameters, and induced increased hemolysis when compared with a static control sample. Comparison of the three operational VAD configurations identified that the adult LVAD condition-associated with the highest pump speed, flow rate, and differential pressure across the pump-resulted in increased normalized hemolysis index (NIH; 0.07) when compared with the lower pump speed "off-label" counterparts (NIH of 0.04 in pediatric LVAD and 0.01 in adult RVAD configurations). After normalizing blood residence times between configurations, pump speed was identified as the primary determinant of accumulated blood damage; plausibly, blood damage could be limited by restricting pump speed to the minimum required to support matched cardiac output, but not beyond.

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.

Effect of ex vivo extracorporeal membrane oxygenation flow dynamics on immune response.

BACKGROUND: Extracorporeal membrane oxygenation is a life-saving support for heart and/or lung failure patients. Despite technological advancement, abnormal physiology persists and has been associated with subsequent adverse events. These include thrombosis, bleeding, systemic inflammatory response syndrome and infection. However, the underlying mechanisms are yet to be elucidated. We aimed to investigate whether the different flow dynamics of extracorporeal membrane oxygenation would alter immune responses, specifically the overall inflammatory response, leukocyte numbers and activation/adhesion surface antigen expression. METHODS: An ex vivo model was used with human whole blood circulating at 37°C for 6 hours at high (4 L/minute) or low (1.5 L/minute) flow dynamics, with serial blood samples taken for analysis. RESULTS: During high flow, production of interleukin-1ß (p < 0.0001), interleukin-6 (p = 0.0075), tumour necrosis factor-α (p = 0.0013), myeloperoxidase (p < 0.0001) and neutrophil elastase (p < 0.0001) were significantly elevated over time compared to low flow, in particular at 6 hours. While the remaining assessments exhibited minute changes between flow dynamics, a consistent trend of modulation in leukocyte subset numbers and phenotype was observed at 6 hours. CONCLUSION: We conclude that prolonged circulation at high flow triggers a prominent pro-inflammatory cytokine response and activates neutrophil granule release, but further research is needed to better characterize the effect of flow during extracorporeal membrane oxygenation.

In Vitro Hemocompatibility Evaluation of Ventricular Assist Devices in Pediatric Flow Conditions: A Benchmark Study.

Development of pediatric ventricular assist devices (VADs) has significantly lagged behind that of adult devices. This frustrating reality is reflected by the fact that the Berlin Heart EXCOR VAD is currently the only approved pediatric-specific device in the USA. An alternative option is an off-label use of adult continuous-flow VADs, such as HeartMate II (HMII), which inevitably causes patient-device size mismatch in small children. We sought to conduct in vitro hemocompatibility testing in a pediatric flow condition, with a specific aim to provide benchmark values for future pediatric device development. Given the aforementioned fact that both pulsatile and continuous-flow devices are being used in the pediatric population, we opted to test both types of devices in the present study. The EXCOR and HMII blood pumps were tested using bovine blood under constant hemodynamic conditions (flow rate, Q = 2.5 ± 0.25L/min; differential pressure across the pump, ΔP = 68 ± 5mm Hg). Hemolysis was measured by Harboe assay. There was a steady increase in plasma free hemoglobin during in vitro testing, with a statistically significant difference between 5 and 360 min for both EXCOR (P < 0.0001) and HMII (P < 0.001). However, the degree of an increase in plasma free hemoglobin was more significant with HMII (P < 0.001). Normalized index of hemolysis for EXCOR and HMII were 0.003 ± 0.0026g/100 L and 0.085 ± 0.0119g/100 L, respectively. There was also a steady increase in platelet activation detected by CAPP2A antibody using flow cytometry, with a statistically significant difference between 5 and 360 min for both devices (P < 0.05). The degree of an increase in platelet activation was similar between the two devices (P = 0.218). High molecular weight von Willebrand factor (HMW vWF) multimer degradation measured by immunoblotting was evident for both devices, however, it was more pronounced with the EXCOR. EXCOR blood samples from all three time points (120, 240, and 360 min) were significantly different from the baseline (5 min), whereas only 360 min samples had a significant difference from the baseline with the HMII. In conclusion, we have observed similarities and differences in hemocompatibility profiles between the EXCOR and HMII, both of which are commonly used in the pediatric population. We anticipate the benchmark values in the present study will facilitate future pediatric VAD development.

The effect of shear stress on the size, structure, and function of human von Willebrand factor.

Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long-term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear-dependent effect on the functional activity of vWF. Human platelet-poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices.