Hemoglobin-based Oxygen Carriers Research Articles

Structural Stability and Biophysical Properties of the Mega-Protein Erythrocruorin Are Regulated by Polyethylene Glycol Surface Coverage.

A wide variety of hemoglobin-based oxygen carriers (HBOCs) have been designed for use as red blood cell (RBC) substitutes in transfusion medicine, ex vivo organ perfusion, oxygen delivery to hypoxic tissues, and a myriad of other applications. However, hemoglobin (Hb) derived from annelids (erythrocruorins [Ecs]) comprise a natural class of HBOC, since they are larger in size (30 nm in diameter) and contain more heme groups per molecule (144 heme groups) compared to human Hb (hHb; 5 nm in diameter and 4 heme groups). The larger size of Ec compared to hHb reduces tissue extravasation from the vascular space, thus, reducing vasoconstriction, systemic hypertension, and tissue oxidative injury when used as an RBC substitute. In addition, prior research has shown that Ecs possess slower auto-oxidation rates than hHb at physiological temperature, thus, making them attractive candidates for use as RBC substitutes. Unfortunately, it was also observed that Ecs have a much lower circulatory half-life in vivo compared to other HBOCs. Hence, conjugating polyethylene glycol (PEG) to the surface of Ec was proposed as a simple strategy to increase Ec circulatory half-life. Therefore, in order to inform future in vivo studies with PEGylated Ec, we decided to investigate the structural stability and biophysical properties of variable PEG surface coverage on Ec compared to native Ec. We observed an increase in PEG-Ec diameter and molecular weight (MW) and changes to the quaternary structure, secondary structure, and surface hydrophobicity after PEGylation. There was also an increase in oxygen binding affinity, reduction in oxygen offloading rate, and increase in auto-oxidation rate for increasing PEGylation ratios. Weak dissociation of Ec was also observed after dense PEGylation caused by steric repulsion of the conjugated PEG chains. Hence, we determined an optimum Ec PEGylation ratio that resulted in a substantial size and MW increase along with preservation of oxygen binding properties. In future studies, these materials will be tested in animal models to evaluate pharmacodynamics, pharmacokinetics, tissue oxygenation, microcirculatory responses, and overall safety.

Quality by design approach identifies critical parameters driving oxygen delivery performance in vitro for perfluorocarbon based artificial oxygen carriers

Perfluorocarbons (PFCs) exhibiting high solubility for oxygen are attractive materials as artificial oxygen carriers (AOC), an alternative to whole blood or Haemoglobin-based oxygen carriers (HBOCs). PFC-based AOCs, however, met clinical translation roadblocks due to product quality control challenges. To overcome these issues, we present an adaptation of Quality by Design (QbD) practices to optimization of PFC nanoemulsions (PFC-NEs) as AOCs. QbD elements including quality risk management, design of experiments (DoE), and multivariate data analysis facilitated the identification of composition and process parameters that strongly impacted PFC colloidal stability and oxygen transport function. Resulting quantitative relationships indicated a composition-driven tradeoff between stability and oxygen transport. It was found that PFC content was most predictive of in vitro oxygen release, but the PFC type (perfluoro-15-crown-5-ether, PCE or perfluorooctyl bromide, PFOB) had no effect on oxygen release. Furthermore, we found, under constant processing conditions, all PFC-NEs, comprised of varied PFC and hydrocarbon content, exhibited narrow droplet size range (100–150 nm) and narrow size distribution. Representative PFOB-NE maintained colloidal attributes upon manufacturing on larger scale (100 mL). QbD approach offers unique insights into PFC AOC performance, which will overcome current product development challenges and accelerate clinical translation.

Ex situ Normothermic Split Liver Machine Perfusion: Protocol for Robust Comparative Controls in Liver Function Assessment Suitable for Evaluation of Novel Therapeutic Interventions in the Pre-clinical Setting.

Background: Ex situ donor liver machine perfusion is a promising tool to assess organ viability prior to transplantation and platform to investigate novel therapeutic interventions. However, the wide variability in donor and graft characteristics between individual donor livers limits the comparability of results. We investigated the hypothesis that the development of a split liver ex situ machine perfusion protocol provides the ideal comparative controls in the investigation of machine perfusion techniques and therapeutic interventions, thus leading to more comparable results.Methods: Four discarded human donor livers were surgically split following identification and separation of right and left inflow and outflow vessels. Each lobe, on separate perfusion machines, was subjected to normothermic perfusion using an artificial hemoglobin-based oxygen carrier solution for 6 h. Metabolic parameters as well as hepatic artery and portal vein perfusion parameters monitored.Results: Trends in hepatic artery and portal vein flows showed a general increase in both lobes throughout each perfusion experiment, even when normalized for tissue weight. Progressive decreases in perfusate lactate and glucose levels exhibited comparable trends in between lobes.Conclusion: Our results demonstrate comparability between right and left lobes when simultaneously subjected to normothermic machine perfusion. In the pre-clinical setting, this model provides the ideal comparative controls in the investigation of therapeutic interventions.

Blood transfusion and alternatives in Jehovah's Witness patients.

More than 8.5 million people in the world observe the Jehovah's Witness faith, and require unique consideration for perioperative blood management as they generally refuse transfusion of blood and blood products. This review addresses a collaborative approach to each patient throughout the perioperative arena. The principles of this approach include optimization of hemoglobin levels preoperatively, attention to blood-salvaging methods intraoperatively, and minimization of blood draws postoperatively. In addition, we review the technologies currently in development as transfusion alternatives, including hemoglobin-based oxygen carriers. Progress has been made recently in the field of synthetic blood alternatives and hemoglobin-based oxygen carriers, which may lead to improved outcomes in this patient population. Utilization of multiple prevention and mitigation strategies to optimize oxygen supply and decrease oxygen demand will lead to decreased incidence of critical anemia and subsequent improved mortality in Jehovah's Witness patients.

Function of Hemoglobin-Based Oxygen Carriers: Determination of Methemoglobin Content by Spectral Extinction Measurements.

Suspensions of hemoglobin microparticles (HbMPs) are promising tools as oxygen therapeutics. For the approval of clinical studies extensive characterization of these HbMPs with a size of about 750 nm is required regarding physical properties, function, pharmaco-kinetics and toxicology. The standard absorbance measurements in blood gas analyzers require dissolution of red blood cells which does not work for HbMP. Therefore, we have developed a robust and rapid optical method for the quality and functionality control of HbMPs. It allows simultaneous determination of the portion of the two states of hemoglobin oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb) as well as the content of methemoglobin (metHb). Based on the measurement of collimated transmission spectra between 300 nm and 800 nm, the average extinction cross section of HbMPs is derived. A numerical method is applied to determine the composition of the HbMPs based on their wavelength-dependent refractive index (RI), which is a superposition of the three different states of Hb. Thus, light-scattering properties, including extinction cross sections can be simulated for different compositions and sizes. By comparison to measured spectra, the relative concentrations of oxyHb, deoxyHb, metHb are accessible. For validation of the optically determined composition of the HbMPs, we used X-ray fluorescence spectrometry for the ratio of Fe(II) (oxyHb/deoxyHb) and Fe(III) (metHb). High accuracy density measurements served to access heme-free proteins, size was determined by dynamic light scattering and analytical centrifugation and the shape of the HbMPs was visualized by electron and atomic force microscopy.

The anti-tumoral effects of the oxygen carrier YQ23 in a triple-negative breast cancer syngeneic model.

BackgroundTriple-negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and high mortality rate. In the search for effective therapeutic options, preclinical studies have suggested using systemic oxygenation to inhibit tumor growth and metastasis in various cancer models, including TNBC, by weakening the hypoxia-A2A adenosine receptors (A2AR)-driven immunosuppression in the tumor microenvironment (TME). In our present study, a hemoglobin-based oxygen carrier (HBOC) “YQ23” was tested for its role in modulating the TME and tumor inhibition.MethodsA syngeneic TNBC mouse model was established by inoculating 4T1 cells subcutaneously in BALB/c mice. Tumor (~100 mm3) bearing mice were treated either with saline or YQ23 (400 mg/kg) i.v. once weekly. To prove the immune-regulatory role of YQ23, CD4+ and CD8+ cells were depleted from a group of mice prior to treatment. Tumor growth was monitored for four weeks while xenografts were isolated at the end of the treatment for ex vivo immunohistological examination.ResultsYQ23 significantly inhibited the tumor growth, and this suppressive effect was abolished by depleting the host immune cells. Immunohistochemical staining of xenograft sections showed YQ23 reduced the level of hypoxia and adenosine producing ecto-enzyme CD73. Although there was no significant difference in the make up of the intra-tumoral immune populations, we observed a down-regulation of the immune checkpoint PD-1. In concordance with the weakened immunosuppression, the inflammatory cytokine interferon γ and cytolytic granzyme B were upregulated.ConclusionsYQ23 treatment may be a potential therapeutic strategy to modulate the TME in TNBC.

Known and Unknown Unknowns in Making Erythrocyte Transfusion Decisions.

Known and Unknown Unknowns in Making Erythrocyte Transfusion Decisions.

A Clinical Comparison of Two Different Oxygen Carriers for Combined Hypothermic and Normothermic Machine Perfusion of High-risk Donor Livers

Purpose: Ex-situ normothermic machine perfusion (NMP) is increasingly used for pretransplant viability assessment of high-risk donor livers. When applied after static cold storage (‘back-to-base’), a short period of hypothermic oxygenated machine perfusion (HMP) prior to NMP reduces ischemia-reperfusion injury during NMP. Excellent results have been reported after combined HMP and NMP, using a single perfusion solution containing an hemoglobin-based oxygen carrier (HBOC). We aimed to determine whether similar results can be obtained with a perfusion solution containing red blood cells (RBC) instead of HBOC. Methods: In a prospective observational cohort study, sequential HMP and NMP was applied in 50 nationwide discarded donor livers in the Netherlands. The first 18 procedures were part of a prospective clinical trial with a HBOC-based perfusion solution for both HMP and NMP (www.trialregister.nl; NTR5972). The subsequent 32 procedures were performed using Belzer Machine Perfusion Solution (MPS) for HMP, followed by an RBC-based perfusion solution for NMP. Results: A total of 50 consecutive HMP-NMP procedures were included. All but two livers were derived from donation after circulatory death donors, with a median donor risk index of 2.83 (IQR 2.51-3.09) and median donor age of 63 years (IQR 53-71). After viability assessment during NMP, 12 livers in the HBOC-group were transplanted versus 18 in the RBC-group (utilization rate 67% versus 55%, P=0.42). One-year graft and patient survival were 92% and 100% in the HBOC-group versus 94% and 100% in the RBC-group, resp. (P=0.96 and P=1.00). Post-transplant cholangiopathy occurred in one patient (3%). There were no differences in other post-transplant outcomes among the two groups. Conclusion: Ex-situ machine perfusion using sequential HMP-NMP for resuscitation and viability assessment of high-risk human donor livers results in excellent transplant outcomes, irrespective of the type of oxygen carrier used.

Oxygen Transport during Ex Situ Machine Perfusion of Donor Livers Using Red Blood Cells or Artificial Oxygen Carriers

Oxygenated ex situ machine perfusion of donor livers is an alternative for static cold preservation that can be performed at temperatures from 0 °C to 37 °C. Organ metabolism depends on oxygen to produce adenosine triphosphate and temperatures below 37 °C reduce the metabolic rate and oxygen requirements. The transport and delivery of oxygen in machine perfusion are key determinants in preserving organ viability and cellular function. Oxygen delivery is more challenging than carbon dioxide removal, and oxygenation of the perfusion fluid is temperature dependent. The maximal oxygen content of water-based solutions is inversely related to the temperature, while cellular oxygen demand correlates positively with temperature. Machine perfusion above 20 °C will therefore require an oxygen carrier to enable sufficient oxygen delivery to the liver. Human red blood cells are the most physiological oxygen carriers. Alternative artificial oxygen transporters are hemoglobin-based oxygen carriers, perfluorocarbons, and an extracellular oxygen carrier derived from a marine invertebrate. We describe the principles of oxygen transport, delivery, and consumption in machine perfusion for donor livers using different oxygen carrier-based perfusion solutions and we discuss the properties, advantages, and disadvantages of these carriers and their use.

559: Effectiveness of OxyVita Novel Hemoglobin-Based Oxygen Carrier (HBOC) in Treatment of CO Poisoning

559: Effectiveness of OxyVita Novel Hemoglobin-Based Oxygen Carrier (HBOC) in Treatment of CO Poisoning

Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry.

Methemoglobin (MetHb) is a hemoglobin (Hb) derivative with the heme iron in ferric state (Fe3+), unable to deliver oxygen. Quantification of methemoglobin is a very important diagnostic parameter in hypoxia. Recently, novel hemoglobin microparticles (Hb-MP) with a narrow size distribution around 700 nm, consisting of cross-linked Hb were proposed as artificial oxygen carriers. The cross-linking of Hb by glutaraldehyde (GA) generates a certain amount of MetHb. Due to the strong light scattering, quantitative determination of MetHb in Hb-MP suspensions by common spectrophotometry is not possible. Here, we demonstrate that 1H2O NMR relaxometry is a perfect tool for direct measurement of total Hb and MetHb concentrations in Hb-MP samples. The longitudinal relaxation rate 1/T1 shows a linear increase with increasing MetHb concentration, whereas the transverse relaxation rate 1/T2 linearly increases with the total Hb concentration. In both linear regressions the determination coefficient (R2) is higher than 0.99. The method does not require time-consuming pretreatment or digestion of the particles and is not impaired by light scattering. Therefore, it can be established as the method of choice for the quality control of Hb-MP and similar hemoglobin-based oxygen carriers in the future.

The need for an artificial oxygen carrier for disasters and pandemics, including COVID-19.

R.B. Weiskopf has consulted for sponsors of hemoglobin-based oxygen carriers in the past, but has not received any compensation from these entities in the past 3 years. M.F.M. James has consulted with sponsors of hemoglobin-based oxygen carriers in the past but has received no financial compensation for these activities at any time. P. Ness has consulted for sponsors of hemoglobin-based oxygen carriers in the past, but has not received any compensation from these entities in the past 3 years. E. Glassberg, N. Guinn, and A.E. Pusateri declare no conflicts of interest.

Synthesis of Hemoglobin-Based Oxygen Carrier Nanoparticles By Desolvation Precipitation.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) present an alternative to red blood cells (RBCs) when blood is not available. However, the most widely used synthesis techniques have fundamental flaws, which may have contributed toward disappointing clinical application. Polymerized Hb contains a heterogeneous distribution of particle size and shape, while Hb encapsulation inside liposomes results in high lipid burden and low Hb content. Meanwhile, there are a variety of other nanoparticle synthetic techniques which, having found success as drug delivery vehicles, may be well suited to function as an HBOC. We synthesized desolvated Hb nanoparticles (Hb-dNPs) with diameters of approximately 250 nm by the controlled precipitation of Hb with ethanol. Oxidized dextran was found to be an effective surface stabilizing agent that maintained particle integrity. In vitro biophysical characterization showed a high-affinity oxygen delivery profile (P50 = 7.72 mm Hg), suggesting a potential for therapeutic use and opening a new avenue for HBOC research.

Resuscitation After Hemorrhagic Shock in the Microcirculation: Targeting Optimal Oxygen Delivery in the Design of Artificial Blood Substitutes.

Microcirculatory preservation is essential for patient recovery from hemorrhagic shock. In hemorrhagic shock, microcirculatory flow and pressure are greatly reduced, creating an oxygen debt that may eventually become irreversible. During shock, tissues become hypoxic, cellular respiration turns to anaerobic metabolism, and the microcirculation rapidly begins to fail. This condition requires immediate fluid resuscitation to promote tissue reperfusion. The choice of fluid for resuscitation is whole blood; however, this may not be readily available and, on a larger scale, may be globally insufficient. Thus, extensive research on viable alternatives to blood has been undertaken in an effort to develop a clinically deployable blood substitute. This has not, as of yet, achieved fruition, in part due to an incomplete understanding of the complexities of the function of blood in the microcirculation. Hemodynamic resuscitation is acknowledged to be contingent on a number of factors other than volume expansion. The circulation of whole blood is carefully regulated to optimize oxygen delivery to the tissues via shear stress modulation through blood viscosity, inherent oxygen-carrying capacity, cell-free layer variation, and myogenic response, among other variables. Although plasma expanders can address a number of these issues, hemoglobin-based oxygen carriers (HBOCs) introduce a method of replenishing the intrinsic oxygen-carrying capacity of blood. There continue to be a number of issues related to HBOCs, but recent advances in the next-generation HBOCs show promise in the preservation of microcirculatory function and limiting toxicities. The development of HBOCs is now focused on viscosity and the degree of microvascular shear stress achieved in order to optimize vasoactive and oxygen delivery responses by leveraging the restoration and maintenance of physiological responses to blood flow in the microcirculation. Blood substitutes with higher viscous properties tend to improve oxygen delivery compared to those with lower viscosities. This review details current concepts in blood substitutes, particularly as they relate to trauma/hemorrhagic shock, with a specific focus on their complex interactions in the microcirculation.

The Critical Choice of Animal Models in Nanomedicine Safety Assessment: A Lesson Learned From Hemoglobin-Based Oxygen Carriers.

Intravenous injection of nanopharmaceuticals can induce severe hypersensitivity reactions (HSRs) resulting in anaphylactoid shock in a small percentage of patients, a phenomenon explicitly reproducible in pigs. However, there is a debate in the literature on whether the pig model of HSRs can be used as a safety test for the prediction of severe adverse reactions in humans. Given the importance of using appropriate animal models for toxicity/safety testing, the choice of the right species and model is a critical decision. In order to facilitate the decision process and to expand the relevant information regarding the pig or no pig dilemma, this review examines an ill-fated clinical development program conducted by Baxter Corporation in the United States 24 years ago, when HemeAssist, an αα (diaspirin) crosslinked hemoglobin-based O2 carrier (HBOC) was tested in trauma patients. The study showed increased mortality in the treatment group relative to controls and had to be stopped. This disappointing result had far-reaching consequences and contributed to the setback in blood substitute research ever since. Importantly, the increased mortality of trauma patients was predicted in pig experiments conducted by US Army scientists, yet they were considered irrelevant to humans. Here we draw attention to that the underlying cause of hemoglobin-induced aggravation of hemorrhagic shock and severe HSRs have a common pathomechanism: cardiovascular distress due to vasoconstrictive effects of hemoglobin (Hb) and reactogenic nanomedicines, manifested, among others, in pulmonary hypertension. The main difference is that in the case of Hb this effect is due to NO-binding, while nanomedicines can trigger the release of proinflammatory mediators. Because of the higher sensitivity of cloven-hoof animals to this kind of cardiopulmonary distress compared to rodents, these reactions can be better reproduced in pigs than in murine or rat models. When deciding on the battery of tests and the appropriate models to identify the potential hazard for nanomedicine-induced severe HSR, the pros and cons of the various species must be considered carefully.

Characterization and Biosafety Evaluation of Hemoglobin-Based Oxygen Carriers Coated with Polydopamine.

Hemoglobin-polydopamine particles (Hb-PDA) have shown high stability, with polydopamine (PDA) serving as a protective layer and antioxidant. However, the effects of the PDA coating on the properties and in vivo biosafety of Hb-PDA remain unclear. This work was conducted to characterize Hb-PDA and evaluate its biosafety. Hb-PDA exhibited negative surface charge and their infusion did not cause blood immunotoxicity or significant tissue injury. Hb-PDA were not phagocyted after co-incubation with macrophages for 3 h. Moreover, the particles showed the highest accumulation in the lungs, and a prolonged retention in major organs. It was also found that the particles were cleared by macrophages in splenic tissue and Kupffer cells in hepatic tissue. In summary, this research showed that Hb-PDA has high dispersion stability, low in vivo toxicity, and extended retention, illustrating its potency as a biosafe oxygen carrier.

Polymerized human hemoglobin facilitated modulation of tumor oxygenation is dependent on tumor oxygenation status and oxygen affinity of the hemoglobin-based oxygen carrier

Administration of hemoglobin-based oxygen carriers (HBOCs) into the systemic circulation is a potential strategy to relieve solid tumor hypoxia in order to increase the effectiveness of chemotherapeutics. Previous computational analysis indicated that the oxygen (O2) status of the tumor and HBOC O2 affinity may play a role in increased O2 delivery to the tumor. However, no study has experimentally investigated how low- and high-affinity HBOCs would perform in normoxic and hypoxic tumors. In this study, we examined how the HBOC, polymerized human hemoglobin (PolyhHb), in the relaxed (R) or tense (T) quaternary state modulates O2 delivery to hypoxic (FME) and normoxic (LOX) human melanoma xenografts in a murine window chamber model. We examined microcirculatory fluid flow via video shearing optical microscopy, and O2 distributions via phosphorescence quenching microscopy. Additionally, we examined how weekly infusion of a 20% top-load dose of PolyhHb influences growth rate, vascularization, and regional blood flow in the FME and LOX tumor xenografts. Infusion of low-affinity T-state PolyhHb led to increased tissue oxygenation, decreased blood flow, decreased tumor growth, and decreased vascularization in hypoxic tumors. However, infusion of both T-state and R-state PolyhHbs led to worse outcomes in normoxic tumors. Of particular concern was the high-affinity R-state PolyhHb, which led to no improvement in hypoxic tumors and significantly worsened outcomes in normoxic tumors. Taken together, the results of this study indicate that the tumor O2 status is a primary determinant of the potency and outcomes of infused PolyhHb.

Model-based evaluation of the microhemodynamic effects of PEGylated HBOC molecules in the rat brain cortex: a laser speckle imaging study

Hemoglobin-based oxygen carriers (HBOCs) were developed with the aim of substituting transfusions in emergency events. However, they exhibit adverse events, such as nitric oxide (NO) scavenging, vasoactivity, enhanced platelet aggregation, presently hampering their clinical application. The impact of two prototypical PEGylated HBOCs, Euro-PEG-Hb and PEG-HbO2, endowed by different oxygen affinities and hydrodynamic volumes, was assessed on the cerebrocortical parenchymal microhemodynamics, and extravasation through the blood-brain-barrier (BBB) by laser speckle contrast imaging (LSCI) method and near-infrared (NIR) imaging, respectively. By evaluating voxel-wise cerebrocortical red blood cell velocity, non-invasively for its mean kernel-wise value ( ), and model-derived kernel-wise predictions for microregional tissue hematocrit, THt, and fractional change in hematocrit-corrected vascular resistance, R', as measures of potential adverse effects (enhanced platelet aggregation and vasoactivity, respectively) we found i) no significant difference between tested HBOCs in the systemic and microregional parameters, and in the relative spatial dispersion of THt, and R' as additional measures of HBOC-related adverse effects, and ii) no extravasation through BBB by Euro-PEG-Hb. We conclude that Euro-PEG-Hb does not exhibit adverse effects in the brain microcirculation that could be directly attributed to NO scavenging.

Neuroprotective effects of combination therapy of regional cold perfusion and hemoglobin-based oxygen carrier administration on rat transient cerebral ischemia

Regional cold perfusion and hemoglobin-based oxygen carrier administration both exert neuroprotective effects against cerebral ischemia reperfusion injury. We herein investigated whether the combination of these two therapies leads to stronger neuroprotective effects. Combination therapy was performed with the regional perfusion of cold HemoAct, a core-shell structured hemoglobin-albumin cluster, in a rat transient middle cerebral artery occlusion model. The effects of combination therapy, the intra-arterial administration of 10 °C HemoAct (10H) initiated at the onset of reperfusion, were compared with those of monotherapies, the intra-arterial administration of 10 °C saline (10S) and 37 °C HemoAct (37H), and an untreated control under the condition of 2-hour ischemia/24-hour reperfusion. The durability of therapeutic effects and the therapeutic time window of combination therapy were assessed based on comparisons with the 10H and control groups. Significantly better neurological findings and smaller infarct volumes were observed in the three treated (10S, 37H, and 10H) groups than in the control group. Among the 3 treated groups, only the 10H group showed significant improvements over the control group in the other items examined, including cerebral blood flow reduction, brain edema, and protein extravasation. The significant therapeutic effects of combination therapy on neurological disabilities and infarct volumes were confirmed at least until 7 days after reperfusion. Furthermore, combination therapy ameliorated neurological disabilities and hemorrhagic transformation in rats subjected to 4- and 5-hour ischemia/24-hour reperfusion. Since therapeutic effects may be expected until at least 5 h of complete ischemia and reperfusion, this combination therapy is a promising neuroprotective strategy against severe ischemic stroke.

Corrigendum to: Ex-Vivo Normothermic Limb Perfusion With a Hemoglobin-Based Oxygen Carrier (HBOC) Perfusate.

Corrigendum to: Ex-Vivo Normothermic Limb Perfusion With a Hemoglobin-Based Oxygen Carrier (HBOC) Perfusate.