In vitro thrombogenicity evaluation of rotary blood pumps by thromboelastometry.

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

Veno-venous extracorporeal blood phototherapy increases the rate of carbon monoxide (CO) elimination in CO-poisoned pigs.

BACKGROUND AND OBJECTIVES: Carbon monoxide (CO) inhalation is the leading cause of poison-related deaths in the United States. CO binds to hemoglobin (Hb), displaces oxygen, and reduces oxygen delivery to tissues. The optimal treatment for CO poisoning in patients with normal lung function is the administration of hyperbaric oxygen (HBO). However, hyperbaric chambers are only available in medical centers with specialized equipment, resulting in delayed therapy. Visible light dissociates CO from Hb with minimal effect on oxygen binding. In a previous study, we combined a membrane oxygenator with phototherapy at 623 nm to produce a "mini" photo-ECMO (extracorporeal membrane oxygenation) device, which improved CO elimination and survival in CO-poisoned rats. The objective of this study was to develop a larger photo-ECMO device ("maxi" photo-ECMO) and to test its ability to remove CO from a porcine model of CO poisoning. STUDY DESIGN/MATERIALS AND METHODS: The "maxi" photo-ECMO device and the photo-ECMO system (six maxi photo-ECMO devices assembled in parallel), were tested in an in vitro circuit of CO poisoning. To assess the ability of the photo-ECMO device and the photo-ECMO system to remove CO from CO-poisoned blood in vitro, the half-life of COHb (COHb-t1/2 ), as well as the percent COHb reduction in a single blood pass through the device, were assessed. In the in vivo studies, we assessed the COHb-t1/2 in a CO-poisoned pig under three conditions: (1) While the pig breathed 100% oxygen through the endotracheal tube; (2) while the pig was connected to the photo-ECMO system with no light exposure; and (3) while the pig was connected to the photo-ECMO system, which was exposed to red light. RESULTS: The photo-ECMO device was able to fully oxygenate the blood after a single pass through the device. Compared to ventilation with 100% oxygen alone, illumination with red light together with 100% oxygen was twice as efficient in removing CO from blood. Changes in gas flow rates did not alter CO elimination in one pass through the device. Increases in irradiance up to 214 mW/cm2 were associated with an increased rate of CO elimination. The photo-ECMO device was effective over a range of blood flow rates and with higher blood flow rates, more CO was eliminated. A photo-ECMO system composed of six photo-ECMO devices removed CO faster from CO-poisoned blood than a single photo-ECMO device. In a CO-poisoned pig, the photo-ECMO system increased the rate of CO elimination without significantly increasing the animal's body temperature or causing hemodynamic instability. CONCLUSION: In this study, we developed a photo-ECMO system and demonstrated its ability to remove CO from CO-poisoned 45-kg pigs. Technical modifications of the photo-ECMO system, including the development of a compact, portable device, will permit treatment of patients with CO poisoning at the scene of their poisoning, during transit to a local emergency room, and in hospitals that lack HBO facilities.

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

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

Ghost Cells for Mechanical Circulatory Support In Vitro Testing: A Novel Large Volume Production.

The aim of this work is to establish a large volume batch production system to produce sufficient volumes of ghost cells to facilitate hemolysis testing of mechanical circulatory support devices. A volume of more than 405 mL with a hematocrit of at least 28% is required to perform in vitro hemolysis testing of mechanical circulatory support devices according to international standards. The established ghost cell production method performed at the institute is limited to 3.1 mL of concentrated cells, that is, cells with 100% hematocrit, due to predominantly manual process steps. Through semi-automation of the existing method by using the large volume batch production system, productivity is increased 60-fold to 188 mL while almost doubling process efficiency to 23.5%. Time-consuming manual work such as pipetting is now supported by sensor-based process engineering. With the help of the large volume batch production system, the objective of producing large quantities of ghost cells is successfully achieved. Thus, this work lays the foundation for spatially resolved hemolysis evaluation of mechanical circulatory support devices in combination with the small-scale fluorescent hemolysis detection method.

How Computational Modeling can Help to Predict Gas Transfer in Artificial Lungs Early in the Design Process.

Wearable extracorporeal membrane oxygenation (ECMO) circuits may soon become a viable alternative to conventional ECMO treatment. Common device-induced complications, however, such as blood trauma and oxygenator thrombosis, must first be addressed to improve long-term reliability, since ambulatory patients cannot be monitored as closely as intensive care patients. Additionally, an efficient use of the membrane surface can reduce the size of the devices, priming volume, and weight to achieve portability. Both challenges are linked to the hemodynamics in the fiber bundle. While experimental test methods can often only provide global and time-averaged information, computational fluid dynamics (CFD) can give insight into local flow dynamics and gas transfer before building the first laboratory prototype. In this study, we applied our previously introduced micro-scale CFD model to the full fiber bundle of a small oxygenator for gas transfer prediction. Three randomized geometries as well as a staggered and in-line configuration were modeled and simulated with Ansys CFX. Three small laboratory oxygenator prototypes were built by stacking fiber segments unidirectionally with spacers between consecutive segments. The devices were tested in vitro for gas transfer with porcine blood in accordance with ISO 7199. The error of the predicted averaged CFD oxygen saturations of the random 1, 2, and 3 configurations relative to the averaged in-vitro data (over all samples and devices) was 2.4%, 4.6%, 3.1%, and 3.0% for blood flow rates of 100, 200, 300, and 400 ml/min, respectively. While our micro-scale CFD model was successfully applied to a small oxygenator with unidirectional fibers, the application to clinically relevant oxygenators will remain challenging due to the complex flow distribution in the fiber bundle and high computational costs. However, we will outline our future research priorities and discuss how an extended mass transfer correlation model implemented into CFD might enable an a priori prediction of gas transfer in full size oxygenators.

Technical Indicators to Evaluate the Degree of Large Clot Formation Inside the Membrane Fiber Bundle of an Oxygenator in an In Vitro Setup.

The most common technical complication during ECMO is clot formation. A large clot inside a membrane oxygenator reduces effective membrane surface area and therefore gas transfer capabilities, and restricts blood flow through the device, resulting in an increased membrane oxygenator pressure drop (dpMO). The reasons for thrombotic events are manifold and highly patient specific. Thrombus formation inside the oxygenator during ECMO is usually unpredictable and remains an unsolved problem. Clot sizes and positions are well documented in literature for the Maquet Quadrox-i Adult oxygenator based on CT data extracted from devices after patient treatment. Based on this data, the present study was designed to investigate the effects of large clots on purely technical parameters, for example, dpMO and gas transfer. Therefore, medical grade silicone was injected into the fiber bundle of the devices to replicate large clot positions and sizes. A total of six devices were tested in vitro with silicone clot volumes of 0, 30, 40, 50, 65, and 85 mL in accordance with ISO 7199. Gas transfer was measured by sampling blood pre and post device, as well as by sampling the exhaust gas at the devices' outlet at blood flow rates of 0.5, 2.5, and 5.0 L/min. Pre and post device pressure was monitored to calculate the dpMO at the different blood flow rates. The dpMO was found to be a reliable parameter to indicate a large clot only in already advanced "clotting stages." The CO2 concentration in the exhaust gas, however, was found to be sensitive to even small clot sizes and at low blood flows. Exhaust gas CO2 concentration can be monitored continuously and without any risks for the patient during ECMO therapy to provide additional information on the endurance of the oxygenator. This may help detect a clot formation and growth inside a membrane oxygenator during ECMO even if the increase in dpMO remains moderate.

Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes.

In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.

Septic porcine blood does not further activate coagulation during in vitro membrane oxygenation.

Objectives: For patients with a severe acute respiratory distress syndrome (ARDS), extracorporeal membrane oxygenation (ECMO) represents a life-saving measure. Frequently, patients with severe ARDS also show signs of severe sepsis. As blood contact with the membrane oxygenator surface leads to adverse effects due to insufficient biocompatibility partly caused by activation of platelets, coagulation factors and leucocytes, we hypothesized that these adverse effects would be amplified if septic blood in a preactivated state came into contact with the membrane oxygenator. Methods: In a previously established in vitro 12-h ECMO test system (mock loop), we used septic or healthy domestic pig blood to analyse coagulation and inflammatory parameters. Sepsis was induced by a caecal ligation and puncture model in pigs. Results: At the beginning of the mock loop experiments, the septic blood showed significantly increased thrombin-antithrombin complexes (76.9 vs 27.7 µg/l), D-dimers (1.2 vs 0.3 mg/l) and fibrinogen concentration (1.8 vs 1.5 g/l), as well as elevated extrinsic coagulation activity (shorter EXTEM-CT: 44.2 vs 57 s) and higher lactate (3.4 vs 1.5 mmol/l) and cytokine levels (interleukin-6: 827 vs 31 pg/ml) when compared with the blood from healthy animals. Despite the preactivated status of the septic blood, no further increase of coagulation activity, inflammatory response or increased oxygenator resistance was observed in comparison to the control experiments. Conclusion: Septic porcine blood was not further activated due to the contact with an oxygenator, and no increased clot formation or biocompatibility problems were observed.

Gas exchange efficiency of an oxygenator with integrated pulsatile displacement blood pump for neonatal patients.

Oxygenators have been used in neonatal extracorporeal membrane oxygenation (ECMO) since the 1970s. The need to develop a more effective oxygenator for this patient cohort exists due to their size and blood volume limitations. This study sought to validate the next design iteration of a novel oxygenator for neonatal ECMO with an integrated pulsatile displacement pump, thereby superseding an additional blood pump. Pulsating blood flow within the oxygenator is generated by synchronized active air flow expansion and contraction of integrated silicone pump tubes and hose pinching valves located at the oxygenator inlet and outlet. The current redesign improved upon previous prototypes by optimizing silicone pump tube distribution within the oxygenator fiber bundle; introduction of an oval shaped inner fiber bundle core, and housing; and a higher fiber packing density, all of which in combination reduced the priming volume by about 50% (50 to 27 mL and 41 to 20 mL, respectively). Gas exchange efficiency was tested for two new oxygenators manufactured with different fiber materials: one with coating and one with smaller pore size, both capable of long-term use (OXYPLUS® and CELGARD®). Results demonstrated that the oxygen transfer for both oxygenators was 5.3-24.7 mlO2/min for blood flow ranges of 100-500 mlblood/min. Carbon dioxide transfer for both oxygenators was 3.7-26.3 mlCO2/min for the same blood flow range. These preliminary results validated the oxygenator redesign by demonstrating an increase in packing density and thus in gas transfer, an increase in pumping capacity and a reduction in priming volume.

A novel approach in extracorporeal circulation: individual, integrated, and interactive heart-lung assist (I3-Assist).

Extracorporeal life support (ECLS) is a well-established technique for the treatment of different cardiac and pulmonary diseases, e.g., congenital heart disease and acute respiratory distress syndrome. Additionally, severely ill patients who cannot be weaned from the heart-lung machine directly after surgery have to be put on ECLS for further therapy. Although both systems include identical components, a seamless transition is not possible yet. The adaption of the circuit to the patients' size and demand is limited owing to the components available. The project I³-Assist aims at a novel concept for extracorporeal circulation. To better match the patient's therapeutic demand of support, an individual number of one-size oxygenators and heat exchangers will be combined. A seamless transition between cardiopulmonary bypass and ECLS will be possible as well as the exchange of components during therapy to enhance circuit maintenance throughout long-term support. Until today, a novel oxygenator and heat exchanger along with a simplified manufacturing protocol have been established. The first layouts of the unit to allow the spill- and bubble-free connection and disconnection of modules as well as improved cannulas and a rotational pump are investigated using computational fluid dynamics. Tests were performed according to current guidelines in vitro and in vivo. The test results show the feasibility and potential of the concept.

Development of a rabbit animal model for miniaturized heart-lung machines.

The utilization of a heart-lung machine (HLM) for the correction of congenital heart defects can lead to various complications, which can culminate in multiorgan failure and death. To reduce the considerable risk of complications, we developed a miniaturized, highly integrated HLM (MiniHLM) for use in infants and children. For the purpose of testing the MiniHLM, we developed a new rabbit animal model. In all, surgery was performed on 32 rabbits. In the first series, 13 New Zealand white rabbits were placed on cardiopulmonary bypass (CPB) for 1 hour with the use of an initial version of the MiniHLM. In the second series, we operated on 19 Chinchilla Bastard rabbits using the further developed MiniHLM 02 or the Dideco Kids D100 system. While several adjustments had to be made to the operating protocol in the first series in order to lower the mortality rate, 15 of the 19 rabbits were successfully weaned from the HLM in the second series. Blood tests pertaining to hemolysis and the expression of inflammation were performed. In addition, tissue samples were taken from the right atrial auricle for the purpose of investigating the expression of inflammatory parameters. The newly developed MiniHLM prototype was tested successfully in an animal model in terms of technical function, hemolysis, and the expression of inflammation. On account of the comparability of their blood values, as well as their anatomy, Chinchilla Bastard rabbits serve as excellent models for the testing of CPB and support systems for infants and children that do not require the administration of foreign blood.

A newly developed miniaturized heart-lung machine--expression of inflammation in a small animal model.

Cardiopulmonary bypass may cause severe inflammatory reactions and multiorgan failure, especially in premature and low-weight infants. This is due in part to the large area of contact with extrinsic surfaces and the essential addition of foreign blood. Thus, we developed a new miniaturized heart-lung machine (MiniHLM) with a total static priming volume of 102mL (including arterial and venous lines) and tested it in a small animal model. Seven Chinchilla Bastard rabbits were perfused with the MiniHLM (dynamic priming volume 127mL). Seven animals serving as a control were perfused using Dideco Kids and a Stöckert roller pump (modified dynamic priming volume 149mL). The rabbits were anesthetized and sternotomized, followed by cannulation of the aorta and the right atrium. The aorta was clamped for 1h. Blood for examination of inflammation (TNF-α, IL-1ß, IL-6, IL-8, and IL-10) and blood gas analysis were taken before skin incision, 5min before opening of the aorta, 15min after opening of the aorta, and 4 h after the initiation of cardiopulmonary bypass. The parameters of inflammation were expressed by means of the comparative C(T) method (ΔΔC(T) method). After gradual reduction of perfusion with the HLM, the heart was decannulated, and the sternum was closed. All rabbits were successfully weaned from cardiopulmonary bypass. Blood gas analysis was unremarkable in all cases. Foreign blood was not administered. Although statistical significance was not achieved, there was a reduced expression of inflammatory markers in the MiniHLM group. The newly developed MiniHLM prototype was tested successfully in a small animal model in terms of technical function and expression of inflammation. Upcoming tests with the industrially manufactured MiniHLM may reveal the advantages of the MiniHLM in comparison with the conventional HLM.

The Aachen miniaturized heart-lung machine--first results in a small animal model.

Congenital heart surgery most often incorporates extracorporeal circulation. Due to foreign surface contact and the administration of foreign blood in many children, inflammatory response and hemolysis are important matters of debate. This is particularly an issue in premature and low birth-weight newborns. Taking these considerations into account, the Aachen miniaturized heart-lung machine (MiniHLM) with a total static priming volume of 102 mL (including tubing) was developed and tested in a small animal model. Fourteen female Chinchilla Bastard rabbits were operated on using two different kinds of circuits. In eight animals, a conventional HLM with Dideco Kids oxygenator and Stöckert roller pump (Sorin group, Milan, Italy) was used, and the Aachen MiniHLM was employed in six animals. Outcome parameters were hemolysis and blood gas analysis including lactate. The rabbits were anesthetized, and a standard median sternotomy was performed. The ascending aorta and the right atrium were cannulated. After initiating cardiopulmonary bypass, the aorta was cross-clamped, and cardiac arrest was induced by blood cardioplegia. Blood samples for hemolysis and blood gas analysis were drawn before, during, and after cardiopulmonary bypass. After 1 h aortic clamp time, all animals were weaned from cardiopulmonary bypass. Blood gas analysis revealed adequate oxygenation and perfusion during cardiopulmonary bypass, irrespective of the employed perfusion system. The use of the Aachen MiniHLM resulted in a statistically significant reduced decrease in fibrinogen during cardiopulmonary bypass. A trend revealing a reduced increase in free hemoglobin during bypass in the MiniHLM group could also be observed. This newly developed Aachen MiniHLM with low priming volume, reduced hemolysis, and excellent gas transfer (O(2) and CO(2)) may reduce circuit-induced complications during heart surgery in neonates.

Development of a miniaturized heart-lung machine for neonates with congenital heart defect.

Predominantly, standard adult heart lung machines are used for pediatric cardiac surgery, only with individually downsized components. Downsizing is limited, e.g., by the required gas exchange surface. To diminish complications, we developed a new miniaturized heart lung machine (MiniHLM) for neonates, with significantly reduced priming volume and blood contact surface by integration of all major system components in one single device. In particular, a rotary blood pump is centrically integrated into the oxygenator and the cardiotomy reservoir with integrated heat exchanger is directly connected. Thus, tubing is only necessary between patient and MiniHLM. A total priming volume of 102 ml could be achieved for the entire extracorporeal circuit (including arterial/venous line), in contrast to the currently smallest device on the market with 213 ml. In first animal experiments with female New Zealand rabbits, the MiniHLM guaranteed both a sufficient gas exchange and an adequate blood flow; 12 rabbits could successfully be weaned off after 1 hour of aortic clamp time. The first in vitro and in vivo tests confirm the concept of the MiniHLM. Its low priming volume and blood contact surface may significantly reduce complications during heart surgery in neonates.