Quantitative analysis of heme and hemoglobin for the detection of intravascular hemolysis.

BACKGROUND: Intravascular hemolysis is associated with massive release of hemoglobin and consequently labile heme into the blood, resulting in prothrombotic and proinflammatory events in patients. Though heme is well-known to participate in these adverse effects, it is not monitored. Instead, haptoglobin and hemoglobin serve as clinical biomarkers. The quantification of labile heme together with hemoglobin, however, should be considered in clinical diagnosis as well, to obtain a complete picture of the hemolytic state in patients. So far, quantification techniques for labile heme were not yet systematically analyzed and compared for their clinical application potential, especially in the presence of hemoglobin. RESULTS: Two commercial assays (Heme Assay Kit®, Hemin Assay Kit®) and five common approaches (pyridine hemochromogen assay, apo-horseradish peroxidase-based assay, UV/Vis spectroscopy, HPLC, mass spectrometry) were analyzed concerning their linearity, accuracy, and precision, as well as their ability to distinguish between hemoglobin-bound heme and labile heme. Further, techniques for the quantification of hemoglobin (Harboe method, SLS method, Hemastix®) were included to study their selectivity for hemoglobin and potential interference by the presence of labile heme. Both, indirect and direct approaches were suitable for the determination of a wide concentration of heme (∼0.02-45 µM) and hemoglobin (∼0.002-17 µM). A clear distinction between hemoglobin-bound heme and labile heme with one method was not possible. Thus, a novel combined approach is presented and applied to human and porcine plasma samples for the determination of hemoglobin and labile heme. SIGNIFICANCE: Our results demonstrate the need to develop improved techniques to differentiate labile and protein-bound heme for early detection of intravascular hemolysis. Here, we present a novel strategy by combining two spectroscopic methods, which is most reliable as an easy-to-use tool for the determination of hemoglobin and heme levels in plasma samples for the diagnosis of intravascular hemolysis and in basic biomedical research.

Post-Mortem Extracorporeal Membrane Oxygenation Perfusion Rat Model: A Feasibility Study.

The development of biomedical soft- or hardware frequently includes testing in animals. However, large efforts have been made to reduce the number of animal experiments, according to the 3Rs principle. Simultaneously, a significant number of surplus animals are euthanized without scientific necessity. The primary aim of this study was to establish a post-mortem rat perfusion model using extracorporeal membrane oxygenation (ECMO) in surplus rat cadavers and generate first post vivo results concerning the oxygenation performance of a recently developed ECMO membrane oxygenator. Four rats were euthanized and connected post-mortem to a venous-arterial ECMO circulation for up to eight hours. Angiographic perfusion proofs, blood gas analyses and blood oxygenation calculations were performed. The mean preparation time for the ECMO system was 791 ± 29 s and sufficient organ perfusion could be maintained for 463 ± 26 min, proofed via angiographic imaging and a mean femoral arterial pressure of 43 ± 17 mmHg. A stable partial oxygen pressure, a 73% rise in arterial oxygen concentration and an exponentially increasing oxygen extraction ratio up to 4.75 times were shown. Considering the 3Rs, the established post-mortal ECMO perfusion rat model using surplus animals represents a promising alternative to models using live animals. Given the preserved organ perfusion, its use could be conceivable for various biomedical device testing.

Novel Size-Variable Dedicated Rodent Oxygenator for ECLS Animal Models-Introduction of the "RatOx" Oxygenator and Preliminary In Vitro Results.

The overall survival rate of extracorporeal life support (ECLS) remains at 60%. Research and development has been slow, in part due to the lack of sophisticated experimental models. This publication introduces a dedicated rodent oxygenator ("RatOx") and presents preliminary in vitro classification tests. The RatOx has an adaptable fiber module size for various rodent models. Gas transfer performances over the fiber module for different blood flows and fiber module sizes were tested according to DIN EN ISO 7199. At the maximum possible amount of effective fiber surface area and a blood flow of 100 mL/min, the oxygenator performance was tested to a maximum of 6.27 mL O2/min and 8.2 mL CO2/min, respectively. The priming volume for the largest fiber module is 5.4 mL, while the smallest possible configuration with a single fiber mat layer has a priming volume of 1.1 mL. The novel RatOx ECLS system has been evaluated in vitro and has demonstrated a high degree of compliance with all pre-defined functional criteria for rodent-sized animal models. We intend for the RatOx to become a standard testing platform for scientific studies on ECLS therapy and technology.

Refurbishment of Extracorporeal Life Support Oxygenators in the Context of In Vitro Testing.

Refurbishing single use extracorporeal membrane oxygenation (ECMO) oxygenators for in vitro research applications is common. However, the refurbishment protocols that are established in respective laboratories have never been evaluated. In the present study, we aim at proving the relevance of a well-designed refurbishing protocol by quantifying the burden of repeatedly reused oxygenators. We used the same three oxygenators in 5 days of 6 hours whole blood experiments. During each experiment day, the performance of the oxygenators was measured through the evaluation of gas transfer. Between experiment days, each oxygenator was refurbished applying three alternative refurbishment protocols based on purified water, pepsin and citric acid, and hydrogen peroxide solutions, respectively. After the last experiment day, we disassembled the oxygenators for visual inspection of the fiber mats. The refurbishment protocol based on purified water showed strong degeneration with a 40-50 %-performance drop and clearly visible debris on the fiber mats. Hydrogen peroxide performed better; nevertheless, it suffered a 20% decrease in gas transfer as well as clearly visible debris. Pepsin/citric acid performed best in the field, but also suffered from 10% performance loss and very few, but visible debris. The study showed the relevance of a well-suited and well-designed refurbishment protocol. The distinct debris on the fiber mats also suggests that reusing oxygenators is ill-advised for many experiment series, especially regarding hemocompatibility and in vivo testing. Most of all, this study revealed the relevance of stating the status of test oxygenators and, if refurbished, comment on the implemented refurbishment protocol in detail.

Oxygenation performance assessment of an artificial lung in different central anatomic configurations.

OBJECTIVES: Aim of this work was to characterize possible central anatomical configurations in which a future artificial lung (AL) could be connected, in terms of oxygenation performance. METHODS: Pulmonary and systemic circulations were simulated using a numerical and an in vitro approach. The in vitro simulation was carried out in a mock loop in three phases: (1) normal lung, (2) pulmonary shunt (50% and 100%), and (3) oxygenator support in three anatomical configurations: right atrium-pulmonary artery (RA-PA), pulmonary artery-left atrium (PA-LA), and aorta-left atrium (Ao-LA). The numerical simulation was performed for the oxygenator support phase. The oxygen saturation (SO2) of the arterial blood was plotted over time for two percentages of pulmonary shunt and three blood flow rates through the oxygenator. RESULTS: During the pulmonary shunt phase, SO2 reached a steady state value (of 68% for a 50% shunt and of nearly 0% for a 100% shunt) 20 min after the shunt was set. During the oxygenator support phase, physiological values of SO2 were reached for RA-PA and PA-LA, in case of a 50% pulmonary shunt. For the same conditions, Ao-LA could reach a maximum SO2 of nearly 60%. Numerical results were congruous to the in vitro simulation ones. CONCLUSIONS: Both in vitro and numerical simulations were able to properly characterize oxygenation properties of a future AL depending on its placement. Different anatomical configurations perform differently in terms of oxygenation. Right to right and right to left connections perform better than left to left ones.

Inflow from a Cardiopulmonary Assist System to the Pulmonary Artery and Its Implications for Local Hemodynamics-a Computational Fluid Dynamics Study.

When returning blood to the pulmonary artery (PA), the inflow jet interferes with local hemodynamics. We investigated the consequences for several connection scenarios using transient computational fluid dynamics simulations. The PA was derived from CT data. Three aspects were varied: graft flow rate, anastomosis location, and inflow jet path length from anastomosis site to impingement on the PA wall. Lateral anastomosis locations caused abnormal flow distribution between the left and right PA. The central location provided near-physiological distribution but induced higher wall shear stress (WSS). All effects were most pronounced at high graft flows. A central location is beneficial regarding flow distribution, but the resulting high WSS might promote detachment of local thromboembolisms or influence the autonomic nervous innervation. Lateral locations, depending on jet path length, result in lower WSS at the cost of an unfavorable flow distribution that could promote pulmonary vasculature changes. Case-specific decisions and further research are necessary.

Minimally Invasive Central Cannulation for Extracorporeal Life Support: The Uniportal and Subxiphoid Approach.

OBJECTIVE: Extracorporeal life support (ECLS) for circulatory and/or respiratory failure is improving. Currently, invasive sternotomies or rib-spreading thoracotomies are used for central cannulation of the heart and great vessels. Although peripheral cannulation of the extremities is often used, this approach may result in immobility and unintentional dislodgement. Less invasive methods for central cannulation are needed to achieve long-term ECLS. The objective of this study was to develop 2 different minimally invasive approaches for central thoracic cannulation. METHODS: Porcine hearts were positioned in a plastic thoracic model. An endoscopic camera and multiple endoscopic instruments were used. Both access points, uniportal (lateral) and subxiphoidal, were simulatively investigated. A novel cannulation method using purse string sutures, a custom-made endoscopic puncture tool, guidewires, and dilator-assisted cannulas was developed. Simulations were tested in a closed circuit regarding leak tightness. RESULTS: The uniportal approach allowed a cannulation of the aorta, inferior vena cava, right atrium, and main pulmonary artery. Cannulation of the right branches of the pulmonary artery and vein was also possible. From the subxiphoid approach, cannulation of the aorta, main pulmonary artery, and both atria were possible. Subsequent evaluation and leakage tests revealed no damage to the surrounding structures and tightly sealed cannulation sites. The uniportal approach was also successfully performed in a human cadaver to connect the aorta and right atrium with cannulas from the subxiphoidal space. CONCLUSIONS: Both uniportal and subxiphoid central cannulation of potential sites for ECLS were feasible. This study encourages further investigation and potential clinical translation of minimally invasive central organ support.

In Vitro and In Vivo Feasibility Study for a Portable VV-ECMO and ECCO2R System.

Extracorporeal membrane oxygenation (ECMO) is an established rescue therapy for patients with chronic respiratory failure waiting for lung transplantation (LTx). The therapy inherent immobilization may result in fatigue, consecutive deteriorated prognosis, and even lost eligibility for transplantation. We conducted a feasibility study on a novel system designed for the deployment of a portable ECMO device, enabling the physical exercise of awake patients prior to LTx. The system comprises a novel oxygenator with a directly connected blood pump, a double-lumen cannula, gas blender and supply, as well as control and energy management. In vitro experiments included tests regarding performance, efficiency, and blood damage. A reduced system was tested in vivo for feasibility using a novel large animal model. Six anesthetized pigs were first positioned in supine position, followed by a 45° angle, simulating an upright position of the patients. We monitored performance and vital parameters. All in vitro experiments showed good performance for the respective subsystems and the integrated system. The acute in vivo trials of 8 h duration confirmed the results. The novel portable ECMO-system enables adequate oxygenation and decarboxylation sufficient for, e.g., the physical exercise of designated LTx-recipients. These results are promising and suggest further preclinical studies on safety and efficacy to facilitate translation into clinical application.