HBOC perfusion fluid provided sufficient oxygen delivery for livers to perform metabolic functions that indicate their viability (Figure 2). Active liver metabolism was also confirmed by the progressive storage of glycogen in hepatocytes (Figure 3). There was progressive regeneration of ATP stores over the course of the perfusion, and there were no differences between the RBC and HBOC groups (Table 4 and Figure 2, panel H). There was an increase in perfusate levels of 8-OH-dG, an established marker of oxidative stress, although this appeared to plateau after the first 2 hours of perfusion and again, there were no differences between the RBC and HBOC perfused groups (Figure 2, panel G). There was a significantly higher oxygen extraction observed in the HBOC group compared with the RBC group, and this difference was apparent throughout the course of the perfusion (Figure 2, panel F).
The viable livers in the Hemopure group had a similar histological appearance to those perfused with packed RBC (not shown) with the majority of hepatocytes showing normal morphology with an intact hepatocyte plate/sinusoidal lining (Figure 3a.A-D). After perfusion with Hemopure, the vasculature appeared to contain a pink-staining solution (3a.E) which was not present after RBC-based perfusions and which appeared to be flushed out effectively with 2 L 10% dextrose at the end of the perfusion process (Figure 3a.F). Extrahepatic bile ducts perfused with Hemopure maintained normal morphology (Figure 3a.C) with a largely intact surface epithelium, viable epithelial lining of the deep peribiliary glands and no loss of stromal nuclei, arterial medial nuclei or evidence of thrombosis. Within both groups, the livers deemed viable (based on perfusion characteristics) demonstrated an increase in glycogen storage (Table 5, Figure 3b) or maintained high glycogen stores during perfusion, whereas those which were deemed nonviable failed to restore glycogen reserves (Table 5). PAS stain was unaffected by the presence of Hemopure. Importantly, there was no histological evidence of damage caused by Hemopure infusion and livers that were viable according to our criteria, had similar histological features in both RBC and HBOC-infused groups. Although we do not use the scoring system at our center, when we compared the 2 groups histologically using our own system or Suzuki’s criteria for IRI,36 there were no observable differences.
HSEC and BEC did not increase intracellular ROS production during in vitro IRI when cultured in standard media (Figure 4a). Human hepatocytes demonstrated increased ROS accumulation when exposed to hypoxia that was accentuated during hypoxia reoxygenation (H-R) as we have previously demonstrated.31 When human hepatocytes, HSEC or BEC, were cultured in Hemopure-containing media, there was no significant increase in intracellular ROS production during normoxia, hypoxia or H-R. These results demonstrate that Hemopure does not increase ROS accumulation in isolated primary liver cells during in vitro IRI.
Our previous work has shown that increases in intracellular ROS increase cell death in parenchymal liver cells primarily via apoptosis but also necrosis.31 As Figure 4b demonstrates, when human hepatocytes, HSEC or BEC, were cultured in Hemopure during normoxia, hypoxia or H-R there was no increase in apoptosis relative to cells cultured in standard media. There was no increase in necrosis in human hepatocytes, HSEC or BEC during in vitro IRI when cultured with Hemopure (data not shown). Hemopure therefore shows no increase in cytotoxicity in primary human liver cells during IRI.
Organ machine perfusion is becoming an increasingly attractive preservation method since experimental studies have demonstrated it mitigate IRI and potentially improve allograft function.8 In particular, data from early clinical transplant series using normothermic machine perfused grafts show promising results and provide a potential means of overcoming the critical shortage of donor organs.6,11,12,37,38 Regardless of perfusion temperature, it is generally accepted that oxygenation of the perfusate is advantageous.4 Here we show for the first time that Hemopure, an acellular oxygen carrier, has the potential to replace packed red cells as the oxygen carrier of choice in a human NMP-L model. This study was primarily designed to assess the feasibility of Hemopure to replace RBC in a model of viability testing using NMP-L. This is not a model of true reperfusion given that we do not use whole blood and there is no immune cell population (other than resident immune cells) in the perfusate, however, including a subsequent reperfusion step to simulate a clinical transplantation would not have added any additional information to the chosen study endpoint.
In the present experiments, we observed increased oxygen consumption in the HBOC liver group. We believe this to be a result of the physiological and rheological properties of Hemopure. As previously described, the oxygen dissociation curve of Hemopure lies to the right of corpuscular hemoglobin (with a p50 of 40 mm Hg) and therefore gives up oxygen to tissues more readily.38,39 This difference was more pronounced at the initial phase of the perfusion, before the liver core and the perfusate temperature reaching 37°C, during which Hb-O2 affinity would normally be increased, giving oxygen to tissues less freely. Across all temperatures therefore, Hemopure will give up more oxygen to tissues than corpuscular hemoglobin. Additional properties such as a molecular diameter approximately 1/1000th the diameter of a red-blood cell and the fact Hemopure is a less viscous fluid, result in a more homogenous perfusion39 and facilitate the diffusive transport of oxygen in the microcirculation improving tissue oxygenation. Low-viscosity preservation fluids may protect against the development of posttransplant biliary complications however this aspect of NMP-L requires further research.40-42
There is mounting evidence that circulating resident leukocytes, or those few that may be present in blood products, can activate proinflammatory signaling pathways that accentuate organ damage.45 Although a leukocyte filter decreases the amount of circulating leukocytes, cells trapped in the filter can still potentially trigger proinflammatory cascades that activate ROS, damage-associated molecular pattern molecules and cytokine production, impacting upon organ quality.45-47 Damage-associated molecular pattern molecule filters are starting to be trialed in some perfusion research settings. Third-party blood can also sensitize the organ recipient and although its significance in preventing liver damage will require further research, replacing RBC with HBOC has would avoid these phenomena.48
Some models do not require the use of third-party blood products. At present, for normothermic perfusion of the heart, the organ recovery team obtains whole donor blood at the time of organ procurement.11 However, this approach is not feasible for all potentially recovered donor organs as the blood volume to prime the perfusion devices would be more than the circulatory volume of the donor. The logistics of retrieving donor blood is also unfavorable because it causes severe hypotension and leads to a delay in cross clamping the aorta.
Although third-party blood provides good results for NMP-L, there are several reasons why finding alternatives may be an important development for the clinical adoption of machine perfusion in the future. The obvious reason is to avoid any unnecessary blood usage—a scarce resource and vital for major surgical procedures or other therapeutic interventions. Complying with ethical and legislative regulations, acquiring approval for third-party blood to be used in NMP-L research is a lengthy process. Using an acellular oxygen carrier would avoid this and overcome other challenges that are associated with the use of blood products such as traceability. Additionally, HBOCs do not require cross-matching and have a long shelf-life at room temperature. In our experience, this prevented delays and minimized the cold ischemic time which is a key factor when attempting to use extended criteria donor livers.
Whereas our research team has focused on viability testing and maximizing organ utilization in a model of NMP-L, others have pursued subnormothermic23 or hypothermic in a bid to improve the long-term results in organs from DCD donors.4,9 The optimal temperature for liver perfusion remains a matter of continuing discussion and an area of intensive research.49-51 Hemopure can deliver oxygen within the wide range of the conventionally used machine perfusion temperatures (10-37°C), currently being trialed in clinical and research settings.23,50
NMP-L can also be used to improve logistics through significantly extending the organ preservation time.5 Hemolysis caused by sheer stress from the device pump and circuit tubing decreases the perfusate oxygen carrying capacity over time and is currently one of the main limiting factors for further extension of organ preservation times.52 The hemolysed RBC debris, although eventually cleared by the liver, may adversely disrupt the hepatic microcirculation, particularly in the peribiliary vascular plexus during the NMP-L procedure. A purified acellular fluid theoretically avoids these limitations. The half-life of Hemopure is 16 hours and recurring anemia in the clinical setting is usually noted 24 hours after administration. We have not observed any degradation of the product however its stability beyond 6 hours of perfusion is yet to be tested.53 One of the constraints for wider and faster implementation of the NMP technology into the organ preservation pathway is its high cost. Hemopure costs more per unit than RBCs; however, this cost could be offset by the numerous advantages its use offers.54
There has been a longstanding interest in developing an efficient and safe alternative to donor blood. Several products have been tested mainly in the preclinical setting with promising results although they have not been adopted into routine clinical practice.20,55 Despite negative reports from a meta-analysis examining the use of 5 different HBOC products, Hemopure has demonstrated clinical efficacy in trials investigating its use in general, urological, orthopedic, vascular and cardiac surgery, though it demonstrated some side effects, most commonly hypertension and bradycardia.21,56-59 Liver machine perfusion with Hemopure ex situ avoids the potential complexities of systemic in vivo interactions and their potential side effects. Histological assessment also showed that flushing the liver at the end of NMP-L effectively removes Hemopure from the liver, so only a very small volume (if any) would reach the recipient circulation.
The main limitation of our study is being unable to assess the effect of true reperfusion during transplantation as the livers were not transplanted. We also chose not to simulate the reperfusion effect by NMP-L with whole blood containing immune cell populations. This model, however, provided reassurance that Hemopure does not cause any apparent histological damage, and it is able to deliver enough oxygen to fully support human liver metabolism at normothermic condition. Such confirmation was necessary before evaluating Hemopure in a clinical transplant setting.
In conclusion, this study suggests that Hemopure-based perfusion fluid is a feasible alternative to the blood-based solution currently used for NMP-L. Hemopure may be logistically, rheologically, and immunologically superior to packed red cells when used in a normothermic perfusion model. Our findings warrant further HBOC-based machine perfusion fluid testing in a pilot clinical trial.
This study was funded by QEHB Charities (Liver Foundation) at University Hospitals Birmingham NHS Foundation Trust. The Hemopure fluid was kindly provided free of charge by Zaf Zafirelis from HbO2 Therapeutics. The Liver Assist device used for this project was provided by the Organ Assist company. Mr. Bhogal is funded by the Academy of Medical Sciences. The authors gratefully acknowledge the generous project support provided by all the team members of the Liver Unit at Queen Elizabeth Hospital Birmingham. The authors thank Dr. Gary Reynolds of the Centre for Liver Research for tissue and histological processing, and Ms. Bridget Gunson for her assistance in obtaining the regulatory approval for the study. Finally, the authors would like to thank their organ donors, their families and the NHSBT network for allowing them to perform this work.
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