Hemoglobin-based Oxygen Carriers Research Articles

Spin labelled hemoglobin-based oxygen carriers (HBOC): preparation and evaluation of in vivo / in vitro stability

Spin labelled hemoglobin-based oxygen carriers (HBOC): preparation and evaluation of in vivo / in vitro stability

Strategies for 'bloodless' surgery: the experience of cytoreductive surgery for peritoneal carcinomatosis in Jehovah's Witnesses.

Cytoreductive surgery (CRS) for the management of peritoneal carcinomatosis (PC) can involve significant blood loss which necessitates the transfusion of blood products. This poses a particular challenge in the Jehovah's Witnesses population whose beliefs do not permit the transfusion of blood products or blood-related derivatives. This report describes the experience of one institution performing CRS with hyperthermic intraperitoneal chemotherapy (HIPEC) for PC in Jehovah's Witnesses and perioperative management strategies employed to avoid blood transfusion. A review of literature and prospectively collated data of Jehovah's Witnesses patients who underwent extensive CRS for PC and HIPEC for PC. Four patients had CRS and HIPEC for PC. The median PC index score was 11 and complete cytoreduction was achieved in all cases. Primary tumours were ovarian (n = 1), colorectal (n = 2) and neuroendocrine tumour of gastrointestinal origin (n = 1). The median difference between preoperative and postoperative haemoglobin was 38 g/L (23-43 g/L). Strategies included acute normovolumaeic haemodilution and autotransfusion within a closed circuit, autotransfusion from cell salvage and provisions for possible use of a haemoglobin based oxygen carrier. Ancillary measures identified and implemented to minimize transfusion dependence included, but were not limited to, preoperative iron infusion, perioperative acute haemodilution and cell salvage, administration of tranexamic acid, prothrombinex and use of paediatric tubes for venepuncture. The review suggests CRS and HIPEC for extensive PC can be done safely in circumstances where transfusion of allogenic blood products is not permitted.

Intraosseous transfusion of hemoglobin vesicles in the treatment of hemorrhagic shock with collapsed vessels in a rabbit model.

Intravenous transfusion sometimes encounters difficulty under prehospital conditions when peripheral vessels are collapsed and inaccessible. We investigated whether the cellular type hemoglobin-based oxygen carriers (Hemoglobin Vesicles: HbVs) allow intraosseous administration into blood circulation for the resuscitation of rabbits with severe hemorrhagic shock. New Zealand white rabbits (2.5 kg average) were set in severe hemorrhagic shock [mean arterial pressure (MAP): 21 ± 2 mm Hg, Hb 5.1 ± 0.8 g/dL]. Immediately thereafter, 12 mL/kg of HbVs, 5% human serum albumin (HSA), autologous whole blood (WB), stored red blood cells (RBCs) or 36 mL/kg of Lactated Ringer's (LR) were intraosseously transfused, followed by an additional intraosseous transfusion with 8 mL/kg of HSA (following HbV, HSA or stored RBC transfusion), or WB or 24 mL/kg of LR (following LR transfusion), respectively. Intraosseous transfusion of HbVs increased MAP (48 ± 9 mm Hg) and improved hypohemoglobinemia (7.1 ± 0.6 g/dL) as well as WB or RBC transfusion. In contrast, neither HSA nor LR improved hemodynamics or Hb levels. Seven out of 10 rabbits receiving HbVs survived for 24 hours, while only one out of 10 rabbits receiving LR survived (WB and RBC; 100% survivals, HSA; 30% survival). Intraosseous infusion of HbVs might be an effective initial treatment to maintain hemodynamics during acute hemorrhagic shock. This approach could be used in emergency situations in which access to peripheral vessels is difficult.

Purification of Lumbricus terrestris erythrocruorin (LtEc) with anion exchange chromatography

The naturally extracellular hemoglobin (erythrocruorin) of the Canadian nightcrawler, Lumbricus terrestris (LtEc), is a unique oxygen transport protein that may be an effective substitute for donated human blood. Indeed, this ultra-high molecular weight (~3.6 MDa) hemoglobin has already been shown to avoid the side effects associated with previous hemoglobin-based oxygen carriers and its high thermal stability (Tm = 56°C) and resistance to heme oxidation (kox = 0.04 hr−1 × 103 at 20°C) allow it to be stored for long periods of time without refrigeration. However, before it can be tested in human clinical trials, an effective and scalable purification process for LtEc must be developed. We have previously purified LtEc for animal studies with tangential flow filtration (TFF), which allows rapid and scalable purification of LtEc based on its relatively large size, but that type of size-based purification may not be able to specifically remove some impurities and high MW (>500 kDa) contaminants like endotoxin (MW = ~1–4 MDa). Anion exchange (AEX) and immobilized metal affinity chromatography (IMAC) are two purification methods that have been previously used to purify mammalian hemoglobins, but they have not yet been used to purify large invertebrate hemoglobins like LtEc. Therefore, the goal of this study was to determine if AEX and IMAC resins could successfully purify LtEc from crude earthworm homogenate, while also preserving its macromolecular structure and function. Both processes were able to produce purified LtEc with low levels of endotoxin, but IMAC purification induced significantly higher levels of heme oxidation and subunit dissociation than AEX. In addition, the IMAC process required an additional desalting step to enable LtEc binding. In contrast, AEX produced highly pure LtEc that was not dissociated. LtEc purified by AEX also exhibits similar oxygen binding characteristics (P50 = 27.33 ± 1.82 mm Hg, n = 1.58 ± 0.17) to TFF-purified LtEc (P50 = 28.84 ± 0.40 mm Hg, n = 1.93 ± 0.02). Therefore, AEX appears to be the optimal method for LtEc purification.

A case study of 10 patients administered HBOC-201 in high doses over a prolonged period: outcomes during severe anemia when transfusion is not an option.

Hemoglobin-Based Oxygen Carriers (HBOCs) can act as an "oxygen bridge" in acute severe anemia when transfusion is indicated, but not possible. We present data on 10 Expanded Access (EA) patients treated with high cumulative doses of Hemopure (HBOC-201), to assess the ability of HBOC-201 to safely treat life threatening anemia in situations where high volumes of product were administered over an extended period of time. Inclusion in this study required that the patient receive at least 10 units of HBOC-201 between 2014 and 2017 under the FDA-sanctioned EA program. Depending on a patient's geographical location, treatment with HBOC-201 was obtained through either a single patient emergency Investigational New Drug (IND) application, or an intermediate size population IND. Of the 41 patients who were treated during this period, 10 patients received 10 or more units of the product. Data were obtained from medical records. Treatments with HBOC-201 started within 24 hours of signing consent and were administered at an average rate of 1.99 (SD 0.17) units per day over a mean of 8.2 days (SD 2.9), during which patients received on average 16.2 units (SD 5.7 units) of HBOC-201. The median pre-treatment nadir corpuscular hemoglobin (Hb) concentration was 3.3 (SD 0.9) g/dL and post-treatment Hemoglobin was 7.3 (SD 1.7) g/dL. Common side effects included methemoglobinemia, gastrointestinal symptoms, and hypertension. However, no product-related serious adverse events (SAEs) were noted. All patients survived. Administration of HBOC-201 over an extended period is a feasible and safe oxygen bridge for severely anemic patients who cannot be transfused with RBC.

Massive Transfusion Protocols for Pediatric Patients: Current Perspectives.

In adults, the use of balanced resuscitation and study of massive transfusion protocols have led to improved outcomes for patients and continues to be refined. In children, massive transfusion protocols require further development and study to assess efficacy. Standardization is needed as transfusions and activation of protocols still rely on physician discretion in most pediatric settings. Further research is required to define the pediatric trauma population that will benefit, when to activate these protocols and how to use adjuncts such as tranexamic acid or factor VII in resuscitation. In addition, future implementation of technology such as hemoglobin-based oxygen carriers to increase survival should be studied further in this subset of patients.

Improved Hemodynamic Recovery and 72-Hour Survival Following Low-Volume Resuscitation with a PEGylated Carboxyhemoglobin in a Rat Model of Severe Hemorrhagic Shock.

Hemorrhage is a leading cause of death from potentially survivable civilian and military trauma. As projected conflicts move from settings of tactical and logistical supremacy to hyper-dynamic tactical zones against peer and near-peer adversaries, protracted medical evacuation times are expected. Treatment at the point-of-injury is critical. Although crystalloids like Lactated Ringer's (LR) are ubiquitous, whole blood (WB) is the preferred resuscitation fluid following hemorrhage; however, logistical constraints limit the availability of WB in prehospital settings. Hemoglobin-based oxygen carriers (HBOCs) offer both hemodynamic support and oxygen-carrying capacity while avoiding logistical constraints of WB. We hypothesized that low-volume resuscitation of severe hemorrhagic shock with an HBOC (PEGylated carboxyhemoglobin, [PC]) would improve hemodynamic recovery and 72-hour survival; comparable to WB and superior to LR. A total of 21 anesthetized male Sprague-Dawley rats underwent severe hemorrhagic shock followed by randomly assigned low-volume resuscitation with LR, WB, or PC, and then recovered from anesthesia for up to 72-hour observation. Mean arterial pressure (MAP) was recorded continuously under anesthesia, and arterial blood gases were measured at baseline (BL), 60minutes post-hemorrhage (HS1h), and 24hours post-resuscitation (PR24h). Survival was presented on a Kaplan-Meier plot and significance determined with a log-rank test. Cardiovascular and blood gas data were assessed with one-way analysis of variance and post hoc analysis where appropriate. All measured cardiovascular and blood chemistry parameters were equivalent between groups at BL and HS1h. BL MAP values were 90±3, 86±1, and 89±2mmHg for LR, PC, and WB, respectively. Immediately following resuscitation, MAP values were 57±4, 74±5, and 62±3mmHg, with PC equivalent to WB and higher than LR (P<0.05). WB and LR were both lower than BL (P<0.0001), whereas PC was not (P=0.13). The PC group's survival to 72hours was 57%, which was not different from WB (43%) and higher than LR (14%; P<0.05). A single bolus infusion of PC produced superior survival and MAP response compared to LR, which is the standard fluid resuscitant carried by combat medics. PC was not different from WB in terms of survival and MAP, which is encouraging because its reduced logistical constraints make it viable for field deployment. These promising findings warrant further development and investigation of PC as a low-volume, early treatment for hemorrhagic shock in scenarios where blood products may not be available.

Resuscitation from Hemorrhagic Shock after Traumatic Brain Injury with Polymerized Hemoglobin

Hemorrhagic Shock (HS) after Traumatic Brain Injury (TBI) is common and lethal among military and civilians. Blood transfusion has been the resuscitation gold standard procedure for many years. However, only 3% of age‐eligible individuals donate blood, which limits blood availability and cannot meet current blood demands. This study was compared the efficacy to resuscitate from HS after TBI via infusion of Lactated Ringer’s solution (LR, electrolyte solution used to restore the loss of blood volume), fresh blood (Blood, autologous blood drawn during the hemorrhage), or Polymerized Hemoglobin [PolyHb, a Hemoglobin Based Oxygen Carrier (HBOC) that shows promise in increasing oxygenation]. Fifteen Wistar rats (350–400g) were instrumented with catheters in the femoral artery and vein and subjected to TBI through a pneumatic controlled cortical impact (CCI) (Leica Biosystems, Vista, CA). To induce TBI, a 5mm craniotomy over the right cerebral cortex was performed, and the scalp was impacted at a velocity of 5m/s and dwell time of 200ms. The scalp was subsequently closed and animals were given 10 min to stabilize before performing HS. HS was induced by blood withdrawal from the femoral artery catheter to achieve a mean arterial pressure (MAP) between 35–40mmHg, and the hypotensive state was maintained for 90 min before resuscitation. Animals were separated into 3 groups based on the resuscitation solution: Blood, LR, or PolyHb. Animals were monitored for an additional 120 min before euthanasia. Measurements were taken at baseline (BL), 90 min into HS (HS), and 30 min (R1) and 2 hours (R2) after resuscitation. The MAP, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were decreased equally in all groups during HS. Blood and PolyHb groups increased MAP compared to the LR group at R1. The MAP was not different between Blood and PolyHb at R2. The SBP was increased only for the Blood group as compared to the LR group at R1. Moreover, DBP was increased for both Blood and PolyHb compared to the LR group at R1 and R2. As expected, hematocrit decreased in all groups after HS, with only the Blood group recovering hematocrit after resuscitation. Total hemoglobin (tHb) decreased after HS, and increased after resuscitation for the Blood and PolyHb groups but not in the LR group. Oxygen saturation was decreased for the Blood and PolyHb groups compared to the LR group at R1 and R2. The oxygen saturation was increased on the Blood group compared to PolyHb group at R2. After HS all groups increased lactate, after R1 just Blood and LR group recovered lactate to BL levels, and PolyHb did not decreased lactate concentration at R2 compared to the blood group. In conclusion, our results show that PolyHb was effective to resuscitation from HS after TBI, restoring blood pressure similarly to Blood group and could be a good alternative to transfusion for field resuscitation from HS after TBI.Support or Funding InformationThis work was supported by the NIH Heart Lung and Blood Institute under Grant, R01‐HL126945, and the DOD DMRDP under Grants W81XWH‐18‐1‐0059, W81XWH‐17‐2‐0047

Artificial Blood: The History and Current Perspectives of Blood Substitutes.

Blood transfusions are one of the most common procedures performed in hospitalized patients. Yet, despite all of the measures taken to ensure the safety of the blood supply, there are known risks associated with transfusions, including infectious and noninfectious complications. Meanwhile, issues with blood product availability, the need for compatibility testing, and the storage and transport requirements of blood products, have presented challenges for the administration of blood transfusions. Additionally, there are individuals who do not accept blood transfusions (e.g., Jehovah’s Witnesses). Therefore, there is a need to develop alternative agents that can reliably and safely replace blood. However, although there have been many attempts to develop blood substitutes over the years, there are currently no such products available that have been approved by the United States Food and Drug Administration (FDA). However, a more-recently developed hemoglobin-based oxygen carrier has shown promise in early clinical trials and has achieved the status of “Orphan Drug” under the FDA.

Polydopamine-based surface modification of hemoglobin particles for stability enhancement of oxygen carriers

Templated assembly techniques have been extensively used to develop various types of hemoglobin (Hb) loaded particles with improved performance. However, several instability issues must still be solved, including Hb exposure, enhanced Hb auto-oxidation, and the relatively weak binding of Hb to cross-linkers. Herein, to meet the stability requirements for novel hemoglobin-based oxygen carriers (HBOCs), hemoglobin-polydopamine particles (Hb-PDA) were fabricated using a mild process that combines the co-precipitation of Hb and an inorganic template with the spontaneous adhesion of PDA. The Hb-PDA showed uniform size distribution, chemical integrity of both Hb and PDA, high biocompatibility, and robust oxygen delivery. Our results demonstrated that the use of polydopamine as a biocompatible coating material reduced Hb leakage from the particles under both static and flow conditions, thus mitigating the toxicity associated with free Hb and strengthening the stability of Hb particles. In addition, Hb-PDA reduced HUVEC (Human Umbilical Vein Cells) oxidative injury and scavenged 85% of the available hydroxyl radicals, exhibiting its potential to act as an antioxidant for encapsulated Hb. Hb-PDA therefore shows significant promise as a cell-like structurally and functionally stable HBOCs.

Microvascular and Systemic Impact of Resuscitation with PEGylated Carboxyhemoglobin-Based Oxygen Carrier or Hetastarch in a Rat Model of Transient Hemorrhagic Shock.

Hemorrhage is the leading cause of preventable, traumatic death. Currently, prehospital resuscitation fluids provide preload but not oxygen-carrying capacity-a critical blood function that mitigates microvascular ischemia and tissue hypoxia during hemorrhagic shock. Solutions containing polymerized hemoglobin have been associated with vasoactive and hypertensive events. A novel hemoglobin-based oxygen carrier, modified with PEGylation and CO moieties (PEG-COHb), may overcome these limitations. To evaluate the systemic and microcirculatory effects of PEG-COHb as compared with the 6% hetastarch in a rat model of hemorrhagic shock. Male Sprague Dawley rats (N = 20) were subjected to severe, controlled, hemorrhagic shock. Animals were randomized to 20% estimated blood-volume resuscitation with either 6% hetastarch or PEG-COHb. Continuous, invasive, cardiovascular measurements, and arterial blood gases were measured. Microcirculatory measurements of interstitial oxygenation (PISFO2) and vasoactivity helped model oxygen delivery in the spinotrapezius muscle using intravital and phosphorescence quenching microscopy. Hemorrhage reduced mean arterial pressure (MAP), arteriolar diameter, and PISFO2, and increased lactate 10-fold in both groups. Resuscitation with both PEG-COHb and hetastarch improved cardiovascular parameters. However, PEG-COHb treatment resulted in higher MAP (P < 0.001), improved PISFO2 (14 [PEG-COHb] vs. 5 [hetastarch] mmHg; P < 0.0001), lower lactate post-resuscitation (P < 0.01), and extended survival from 90 to 142 min (P < 0.001) as compared with the hetastarch group. PEG-COHb improved MAP PISFO2, lactate, and survival time as compared with 6% hetastarch resuscitation. Importantly, hypertension and vasoactivity were not detected in response to PEG-COHb resuscitation supporting further investigation of this resuscitation strategy.

Renal Protection Against Ischemia Reperfusion Injury: Hemoglobin-based Oxygen Carrier-201 Versus Blood as an Oxygen Carrier in Ex Vivo Subnormothermic Machine Perfusion.

The optimal method of oxygen delivery to donor kidneys during ex vivo machine perfusion has not been established. We have recently reported the beneficial effects of subnormothermic (22°C) blood perfusion in the preservation of porcine donation after circulatory death kidneys. Since using blood as a clinical perfusate has limitations, including matching availability and potential presence of pathogen, we sought to assess hemoglobin-based oxygen carrier (HBOC-201) in oxygen delivery to the kidney for renal protection. Pig kidneys (n = 5) were procured after 30 minutes of warm in situ ischemia by cross-clamping the renal arteries. Organs were flushed with histidine tryptophan ketoglutarate solution and subjected to static cold storage or pulsatile perfusion with an RM3 pump at 22°C for 4 hours with HBOC-201 and blood. Thereafter, kidneys were reperfused with normothermic (37°C) oxygenated blood for 4 hours. Blood and urine were subjected to biochemical analysis. Total urine output, urinary protein, albumin/creatinine ratio, flow rate, resistance were measured. Acute tubular necrosis, apoptosis, urinary kidney damage markers, neutrophil gelatinase-associated lipocalin 1, and interleukin 6 were also assessed. HBOC-201 achieved tissues oxygen saturation equivalent to blood. Furthermore, upon reperfusion, HBOC-201 treated kidneys had similar renal blood flow and function compared with blood-treated kidneys. Histologically, HBOC-201 and blood-perfused kidneys had vastly reduced acute tubular necrosis scores and degrees of terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining versus kidneys treated with cold storage. Urinary damage markers and IL6 levels were similarly reduced by both blood and HBOC-201. HBOC-201 is an excellent alternative to blood as an oxygen-carrying molecule in an ex vivo subnormothermic machine perfusion platform in kidneys.

Rational Mutagenesis to Engineer Heme Stability in Recombinant Human Hemoglobin to Design Potential Hemoglobin Based Oxygen Carrier

Certain life-saving medical conditions like surgery, war wound and trauma necessitate blood transfusions. The transfusion process is associated with several risks and side effects. Heterologous expression of human hemoglobin for developing HBOCs seems to be an alternative to blood transfusion. However, such hemoglobins suffer quite a few disadvantages as described in Varnado et al. A major problem that prevents wider use of HBOCs is the dissociation of “heme” from its protein matrix. The released heme causes serious renal cytotoxicity which needs to be minimized. We successfully engineered heme stability into myoglobin by covalent linkage with a side chain from the apoprotein mimicking a cyanobacterial globin.Efforts are on in translating the heme stability in human hemoglobin. Based on rational mutagenesis, we were indeed able to engineer heme stability to an extent into recombinant human hemoglobin. Specific mutations in the α- and β- chains individually caused significant increase (∼3-4 fold) in the hemin retention. These mutations did not show any major influence on the structural stability and heme coordination of the protein. Moreover, engineered rHb mutants exhibited autooxidation rates similar to the wild type protein. Such preliminary success in engineering heme stability holds promise for the production of stable recombinant HBOCs.

Synthesis and characterisation of aqueous haemoglobin-based microcapsules coated by genipin-cross-linked albumin

Bovine serum albumin (BSA)-coated haemoglobin (Hb)-microcapsules prepared by co-precipitation of Hb and MnCO3 may present an alternative type of artificial blood substitute. Prepared microcapsules were analysed by Scanning electron microscopy (SEM) and Respirometry, cytotoxicity was evaluated by addition of microcapsules to murine fibroblast-derived cell line L929 (American Type Culture Collection, NCTC clone 929 of strain L). The capsules come along with a mean diameter of approximately 0.6 μm and a mean volume of 1.13 × 10−19 L, thus an average human red blood cell with a volume of 9 × 10−14 L is about 800,000 times bigger. Hb-microcapsules are fully regenerable by ascorbic acid and maintain oxygen affinity because oxygen is able to pass the BSA wall of the capsules and thereby binding to the ferrous iron of the haemoglobin entity. Therefore, these microcapsules present a suitable type of potential artificial haemoglobin-based oxygen carrier (HbOC).

Ex-Vivo Normothermic Limb Perfusion With a Hemoglobin-Based Oxygen Carrier Perfusate.

Ex-vivo normothermic limb perfusion (EVNLP) has been proven to preserve limb viability better than standard cold storage. Perfusates containing packed red blood cells (pRBC) improve outcomes when compared to acellular perfusates. Limitations of pRBC-based perfusion include limited availability, need for cross match, mechanical hemolysis, and activation of pro-inflammatory proteins. Hemoglobin-based oxygen carrier (HBOC)-201 (Hemopure) is a solution of polymerized bovine hemoglobin, characterized by low immunogenicity, no risk of hemolytic reaction, and enhanced convective and diffusive oxygen delivery. This is a preliminary study on the feasibility of EVNLP using HBOC-201 as an oxygen carrier. Three porcine forelimb perfusions were performed using an established EVNLP model and an HBOC-201-based perfusate. The perfusion circuit included a roller pump, oxygenator, heat exchanger, and reservoir. Electrolytes, limb temperature, weight, compartment pressure, nerve conduction, and perfusion indicated by indocyanine green angiography and infra-red thermography were monitored. Histological evaluation was performed with hematoxylin and eosin and electron microscopy. Three limbs were perfused for 21.3±2.1hours. Muscle contractility was preserved for 10.6±2.4hours. Better preservation of the mitochondrial ultrastructure was evident at 12hours in contrast to crystallization and destruction features in the cold-storage controls. An HBOC-201-EVNLP produced outcomes similar to RBC-EVNLP with preservation of muscle contractility and mitochondrial structure.

Protective effects of HBOC on pulmonary vascular leakage after haemorrhagic shock and the underlying mechanisms

Volume resuscitation is an important early treatment for haemorrhagic shock. Haemoglobin-based oxygen carrier (HBOC) can expand the volume and provide oxygen for tissues. Vascular leakage is common complication in the process of haemorrhagic shock and resuscitation. The aim of this study was to observe the effects of HBOC (a bovine-derived, cross-linked tetramer haemoglobin oxygen-carrying solution, 0.5 g/L) on vascular leakage in rats after haemorrhagic shock. A haemorrhagic shock rat model and hypoxic vascular endothelial cells (VECs) were used. The role of intercellular junctions and endothelial glycocalyx in the protective effects of HBOC and the relationship with mitochondrial function were analysed. After haemorrhagic shock, the pulmonary vascular permeability to FITC-BSA, Evans Blue was increased, endothelial glycocalyx was destroyed and the expression of intercellular junction proteins was decreased. After haemorrhagic shock, a small volume of HBOC solution (6 ml/kg) protected pulmonary vascular permeability, increased structural thickness of endothelial glycocalyx, the levels of its components and increased expression levels of the intercellular junction proteins ZO-1, VE-cadherin and occludin. Moreover, HBOC significantly increased oxygen delivery and consumption in rats, improved VEC mitochondrial function and structure. In conclusion, HBOC mitigates endothelial leakage by protecting endothelial glycocalyx and intercellular junctions through improving mitochondrial function and tissue oxygen delivery.

Increased and safe utilization of high-risk donor livers for transplantation after ex situ resuscitation and assessment using sequential hypo- and normothermic machine perfusion

Background: Despite persistent donor organ shortage, a high number of donor livers is currently not used for transplantation. We aimed to increase the number of transplantable livers by resuscitating and assessing hepatobiliary viability of initially declined high-risk livers using a protocol of end-ischemic sequential ex situ hypothermic and normothermic machine perfusion. Method: In this prospective clinical trial, all nationwide declined livers were eligible for inclusion (Netherlands Trial Registry NTR5972). The protocol consisted of one hour hypothermic oxygenated perfusion (10°C) for resuscitation, one hour of controlled oxygenated rewarming, and subsequent normothermic machine perfusion (NMP) for viability testing. A perfusion fluid containing a hemoglobin-based oxygen carrier was used for all temperature phases. During the first 150 min of NMP, viability of liver and biliary tree was assessed, using the following criteria: perfusate lactate <1.7mmol/L, pH 7.35-7.45, cumulative bile production >10mL and biliary pH>7.45. Livers meeting these criteria were secondary accepted for transplantation. All recipients gave written informed consent. Primary endpoint was safety and feasibility, as reflected by a 3-months graft survival rate of at least 80%. Results: Between August 2017 and October 2018, 16 livers underwent machine perfusion after an average of 288 (241-480) min of static cold preservation. All livers were derived from donation after circulatory death donors, with a median age of 63 (range 42-82) years. During NMP, all livers cleared lactate and produced sufficient bile volume, but in 5 cases biliary pH remained <7.45. The 11 (69%) livers that met all viability criteria were successfully transplanted, increasing the number of deceased donor liver transplants by 20%. Patient and with graft survival at 3 months was 100%. Conclusion: Sequential hypo- and normothermic machine perfusion enabled resuscitation and selection of initially declined high-risk donor livers. This method offered a valuable tool to safely increase the number of transplantable livers by 20%.

Systemic and microvascular comparison of Lactated Ringer’s solution, VIR-HBOC, and alpha-alpha crosslinked haemoglobin-based oxygen carrier in a rat 10% topload model

Medical support for traumatic haemorrhage is lacking for far-forward combat units. VIR-HBOC (haemoglobin-based oxygen carrier) is a novel biological therapeutic under development as a field-stable resuscitation fluid. HBOCs have a long history of complications, chief among them is vasoconstrictive hypertension, which must be resolved before efficacy testing. As such, VIR-HBOC was compared against Lactated Ringers (LRS; vehicle) and a cross-linked haemoglobin (ααHb; a known vasoactive HBOC) in a rat topload model. Twenty-three male, Sprague Dawley rats were randomly assigned to receive a 10% infusion (estimated total blood volume) of one test article while normotensive and under anaesthesia. Cardiovascular, blood chemistry and oximetry, microvascular arteriolar diameters, and interstitial tissue oxygenation parameters were measured. Circulatory half-life was calculated by plasma total haemoglobin. Treatment with ααHb caused immediate increases in mean arterial pressure compared to LRS and VIR-HBOC groups, and corresponding arteriolar vasoconstriction (p < .05), which did not occur for LRS or VIR-HBOC. Circulatory half-lives for VIR-HBOC and ααHb were calculated as 340 and 157 min, respectively. This first report of VIR-HBOC showed no evidence of a hypertensive or vasoactive effect. It was well-tolerated over the eight-hour time course of this topload model, which warrants further investigation in studies of haemorrhagic shock.

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality.

In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function. Site-specific PEGylation at the naturally reactive thiol on human hemoglobin (βCys93) alters hemoglobin oxygen binding affinity and increases its autooxidation rate. In order to avoid this issue, new reactive thiol residues were therefore engineered at sites distant to the heme group and the α/β dimer/dimer interface. The two mutants were βCys93Ala/αAla19Cys and βCys93Ala/βAla13Cys. Gel electrophoresis, size exclusion chromatography and mass spectrometry revealed efficient PEGylation at both αAla19Cys and βAla13Cys, with over 80% of the thiols PEGylated in the case of αAla19Cys. For both mutants there was no significant effect on the oxygen affinity or the cooperativity of oxygen binding. PEGylation at αAla19Cys had the additional benefit of decreasing the rates of autoxidation and heme release, properties that have been considered contributory factors to the adverse clinical side effects exhibited by previous hemoglobin based oxygen carriers. PEGylation at αAla19Cys may therefore be a useful component of future clinical products.

Alternatives to Transfusion

We discuss two main categories of blood substitutes: perfluorocarbons and hemoglobin-based oxygen carriers. We provide a review of the notable products developed in both categories and include their attributes as well as their setbacks. We contribute a case report tothe growing literature of the successful use of Sanguinate. We find that artificial oxygen carriers are an attractive field of research because of the practical limitations and the multitude of potential complications that surround human blood transfusions.