Review Articles: Narrative Review Article

Blood Substitutes and Oxygen Therapeutics: A Review

Jahr, Jonathan S. MD, FASA^*; Guinn, Nicole R MD^†; Lowery, David R. MD^‡,§; Shore-Lesserson, Linda MD, FAHA, FASE^¶; Shander, Aryeh MD, FCCM, FCCP, FASA^#,**

Author Information

From the ^*David Geffen School of Medicine at University of California Los Angeles, Ronald Reagan UCLA Medical Center, Los Angeles, California

^†Department of Anesthesiology, Center for Blood Conservation Duke University Medical Center, Durham, North Carolina

^‡US Military, San Antonio, Texas

^§Department of Anesthesiology, Uniformed Services University of the Health Sciences, San Antonio Military Medical Center, San Antonio, Texas

^‖Zucker School of Medicine at Hofstra/Northwell, Manhasset, New York

^¶Cardiovascular Anesthesiology, Northshore University Hospital, Manhasset, New York

^#Department of Anesthesiology, Critical Care and Hyperbaric Medicine, Englewood Hospital and Medical Center, Englewood, New Jersey

^**TeamHealth Research Institute, Englewood Hospital and Medical Center, Englewood, New Jersey

Accepted for publication October 23, 2018.

Funding: Institutional and/or departmental.

Conflicts of Interest: See Disclosures at the end of the article.

The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the US Government.

J. S. Jahr and N. R. Guinn contributed equally and share first authorship.

Reprints will not be available from the authors.

Address correspondence to Jonathan S. Jahr, MD, FASA, David Geffen School of Medicine at University of California Los Angeles, Ronald Reagan UCLA Medical Center, 757 Westwood Plaza, Suite 3325, Los Angeles, CA 90095. Address e-mail to [email protected].

Anesthesia & Analgesia 132(1):p 119-129, January 2021. | DOI: 10.1213/ANE.0000000000003957

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Abstract

Allogeneic blood is transfused to treat severe anemia and major blood loss. Our quest for a true “replacement for blood” has led us in many different directions in experimentation and development. In scenarios and patients for whom blood is not an option, the search for a blood substitute has been ongoing, with a few products showing promise in this niche. Many products have been introduced that improve tissue oxygenation and reduce the risk of critical ischemia, but clinical trials have not yielded a commercial product that equals allogeneic erythrocytes without significant adverse effects.^¹ Rather than looking for a product that is either equivalent or superior to allogeneic erythrocytes through randomized controlled trials, the current focus is on finding an artificial oxygen carrier that may infer a morbidity or mortality benefit to patients for whom blood transfusion is not even an option. Artificial oxygen carriers offer several advantages over allogeneic erythrocytes, including eliminating the need for cross match, longer storage duration, and a decreased or eliminated risk of pathogen transmission. Furthermore, new research suggests they may have utility in other areas as well, including organ preservation for transplant surgery,^² sickle cell crisis,^³ and brain oxygenation during circulatory arrest.^⁴ This article aims to review the artificial oxygen carriers as an alternative to transfusion when blood is not an option. The history and development will be discussed, as well as clinical trials, potential future technology, and the current standing of artificial oxygen carriers.

HISTORY

The concept of blood substitutes has long been studied, including use of human and bovine hemoglobin (Hb) in the 1930s.^⁵ Although infusion of purified human Hb into maternal hemorrhage victims allowed for rapid resuscitation and feeling of well-being, patients subsequently developed jaundice, abdominal/back pain, chemical pancreatitis, and ultimately died from renal failure around 10 days later. It turns out that the removal of the red blood cell coat and the infusion of stroma-free Hb mimics extreme hemolysis, which has severe effects on some end-organ function. Hb-based oxygen carriers use purified human, animal, or recombinant Hb in a cell-free preparation, modified for stability. The adverse effects of Hb-based oxygen carriers noted by Amberson^⁵ have largely been attributed to the dissociation of the Hb tetramer into dimers, resulting in renal toxicity, NO scavenging leading to vasoconstriction, and oxidation leading to methemoglobinemia.^⁶^,^⁷ In the 90 years since Amberson^⁵ infused stroma-free Hb, Hb-based oxygen carrier development has matured. Modifications such as cross-linkage, polymerization, and encapsulation with polyethylene glycol (PEGylation) have allowed the development of Hb-based oxygen carriers that have a longer half-life and lessened degrees of vasoconstriction and the other earlier noted adverse effects of infused stroma-free Hb. Additionally, molecular cross-linkage of Hb-based oxygen carriers have led to a shift of the oxygen–Hb dissociation curve, which dictates the relationship between the partial pressure of oxygen in the blood and the oxygen saturation, or the “oxygen affinity.” Hb-based oxygen carriers have a sigmoid shaped curve, similar to native Hb, while perfluorocarbons carry dissolved oxygen in solution in a linear fashion, with a slope determined by the amount of oxygen that they carry.^⁶ The partial pressure at which 50% oxygen saturation occurs (p50), is different for Hb-based oxygen carriers from native Hb, some shifted to the right of normal adult Hb (27 torr), with the strategy to load and offload more oxygen that adult Hb, while others have been designed to be left-shifted, even more than fetal Hb (17 torr), with the rationale that the Hb-based oxygen carrier should only load and offload oxygen under hypoxic conditions.^⁶ There is no definitive study demonstrating which strategy is more beneficial in preventing oxygen toxicity and NO scavenging.^⁶ It may be that different clinical conditions warrant different oxygen delivery strategies and therefore the need for multiple different types of Hb-based oxygen carriers. A new focus on providing these products to patients who are unable to be transfused, and measuring outcomes, will hopefully be more successful than comparing these products directly to allogeneic erythrocytes.^⁶^,^⁸ Although numerous products have been developed and studied, only a few remain available today, due to continued concerns over earlier increased morbidity and mortality. The history of the development of Hb-based oxygen carriers is as follows.

A major step forward was the US Army research, headed by Robert Winslow, to develop α–α cross-linked Hb. This chemically modified Hb was further developed into the product 2,3 diaspirin cross-linked Hb (HemAssist, Baxter Healthcare, Deerfield, IL). This product underwent extensive clinical trials in Europe and the United States, specifically in trauma patients. After the first 100 patients were enrolled, the company halted the study in the United States due to an increased incidence of mortality in the HemAssist subjects compared with those receiving blood. This finding was judged to be significant by the data safety monitoring board.^⁹^,^¹⁰ These results may have been due to vasoconstrictive adverse events. Other explanations include that trauma patients are an exceedingly heterogeneous population and it is possible that an increased number of patients in the HemAssist group had head trauma. Alternatively, preclinical models may have been able to predict this clinical failure as they had demonstrated that the breakdown products of Hb should be filtered by the kidneys and cause renal failure.^¹⁰ Nevertheless, production of this product ceased in 1998.

To avoid these adverse effects, scientists and engineers postulated that larger Hb-based oxygen carrier molecules would not breakdown into small toxic dimers and should be less likely to be fraught with complications. Subsequently, other novel products emerged into clinical trials. Optro (Somatogen, Boulder, Colorado), Hemolink (Hemosol, Toronto, Canada), PolyHeme (Northfield, Evanston, IL), and Hemospan (Sangart, San Diego, CA) were all trialed but eventually abandoned. The Somatogen product was produced by recombinant DNA and had fewer renal risks, but suffered from endotoxin effects.^¹¹ The Hemosol product was evaluated for acute normovolemic hemodilution in cardiac surgery and had worse outcomes including higher mortality and myocardial ischemia, as did the Northfield product in trauma, although both may have been victim to poor study designs (such as severe anemia before bypass in the acute normovolemic hemodilution study and late randomization when survival was unlikely for the trauma study) rather than product issues.^¹²^,^¹³ Hemospan, developed by Robert Winslow and Marcos Intelietta, used Hb derived from outdated human blood, with an extreme leftward shift on the oxyhemoglobin dissociation curve, an increased viscosity and a PEGylated coat surrounding a small Hb-based oxygen carrier molecule, yet failed due to a similar side effect profile of earlier Hb-based oxygen carriers.^¹⁴ After extensive preclinical and human trials, all of these products had a worse outcome in at least 1 area, forcing cessation of clinical trials and lack of Food and Drug Administration approval.^¹⁵ This led to a negative meta-analysis, by Natanson et al,^¹⁵ that was widely criticized for nonuniform definitions and ambiguous control-group comparators. Nonuniform definitions included multiple products, each with different pharmacologic properties and each studied for different indications/applications, and ambiguous control-group comparators included that controls were different in each study, whether they were crystalloid, blood, colloid, etc.^¹⁶ Nonetheless, it prompted a serious review of the data by multiple authors and regulatory agencies.^¹⁷

Hb-BASED OXYGEN CARRIERS IN CURRENT USE AND DEVELOPMENT

The Hb-based oxygen carrier that remains in use and has been the most widely studied to date is HBOC-201 (Hemopure, Hemoglobin glutamer-250 [bovine] originally developed by Biopure, Cambridge, MA, then sold to OPK Biotech, Cambridge, MA, and then to Hemoglobin Oxygen Therapeutics, Souderton, PA). It is a purified bovine Hb (Hb concentration of 12–14 g/dL), chemically cross-linked (polymerized with glutaraldehyde) for stability and formulated in a modified lactated Ringer’s solution. Each unit contains 32.5 g of HBOC-201 in 250 mL. Its p50 is approximately 40 mm Hg.^¹⁸ OxyGlobin, a veterinary product structurally related to Hemopure, was US Food and Drug Administration approved for canine anemia in 1997 and in the European Union in 1998, and remains an approved product in veterinary clinical use.^¹⁹ A case series in 48 felines showed an increase in Hb with product administration, but adverse side effects related to circulatory overload in cats suffering from cardiac disease.^²⁰

There have been 22 clinical trials using HBOC-201, the majority of which compared the product to allogeneic blood transfusion for acute perioperative anemia. There were 2 phase III studies looking at HBOC-201 as a “blood substitute.” The first was in general surgery, and randomized 81 subjects to receive either a single dose of HBOC-201 (55 subjects) or an equivalent volume of lactated Ringer’s solution (26 subjects), and found that although HBOC-201 was generally well tolerated, there was no difference in transfusion requirements between the 2 groups.^²¹ The second and larger of these studies enrolled 688 patients undergoing elective orthopedic surgery (350 patients in the study group and 338 patients in the packed red blood cells group), with randomization to HBOC-201 or packed red blood cells at the time of first transfusion decision.^²² This study by Jahr et al^²² demonstrated a reduction in the need for transfusion using HBOC-201, and eliminated the need for transfusion in up to 50% of subjects. However, the investigators found that certain patients could not tolerate the volume associated with Hb-based oxygen carrier administration: patients over 80 years old or with preexisting cardiac disease. Adverse events were also higher in the patients receiving Hb-based oxygen carrier, with 8 cases of methemoglobinemia, 2 of which were at levels above 10% (clinically significant threshold).^²³ Although methemoglobinemia is treated with methylene blue, in a severely anemic patient, even modest increases in methemoglobin can reduce the oxygen-carrying capacity of the available Hb, and thus should be treated early.

A randomized trial in 98 cardiac surgery patients also found a reduction in erythrocyte transfusion as well as an increased oxygen extraction is those patients receiving HBOC-201.^²⁴ Common adverse effects included elevation in liver enzymes, jaundice, gastrointestinal side effects, a modest increase in arterial blood pressure, and an increase in methemoglobin level.^²⁵ A thorough review of the use and side effect profile of HBOC-201 has indicated that when used for the newer indications, it is relatively safe, and does increase the Hb concentrations.^²⁵

Although earlier clinical studies investigated HBOC-201 as a substitute to allogeneic erythrocytes, in a comparison of outcomes in patients having received HBOC-201 against historical patients for whom blood is not an option, there appears to be a mortality benefit of Hb-based oxygen carrier in patients with severe anemia.^¹ Although only available in the United States through the Food and Drug Administration’s Expanded Access program, HBOC-201 was approved for use in South Africa in 2001 to treat adult surgical patients with acute anemia, and to replace allogeneic erythrocyte transfusion when blood is unavailable or refused.^²⁶ Until alternative therapies are available in the United States, obtaining an Expanded Access investigational new drug remains the only option. Hemopure is currently being marketed by Hemoglobin Oxygen Therapeutics (Souderton, PA) in South Africa.^²⁷ Investigations continue to work for a niche application in the United States and European Union for the above applications, for organ preservation, and for use in acute coronary syndromes.^²²^,^²⁶

Sanguinate (Prolong, South Plainfield, NJ) is a PEGylated (covalent attachment of polyethylene glycol) Hb-based oxygen carrier with different qualities than Hemopure, which uses glutaraldehyde polymerization.^²⁸ In contrast to Hemopure, which has a normal oncotic pressure, has a Hb of 13 g/dL, and a p50 of 40 torr, Sanguinate is hyperoncotic, has a Hb of 4–5 g/dL, and a p50 of 7–16 torr. Although a product whose Hb is only 4–5 g/dL is unlikely to provide any substantial improvement in anemia, the theoretical benefit comes from the increased release of oxygen to ischemic tissue due to the oxygen–Hb dissociation curve shifted to the left. Sanguinate serves both as an oxygen and carbon monoxide (CO) transfer agent, to help vasodilate, reduce inflammation, and offload oxygen to ischemic tissues.^²⁹ The small size of the molecule allows it to travel even through stenotic regions to improve oxygen delivery to those tissues, while the use of polyethylene glycol increases the half-life and reduces immunogenicity while preventing NO scavenging. CO serves to vasodilate, decrease NO depletion, and reduce inflammation. Therefore, Sanguinate may offer benefits beyond oxygen delivery, especially in diseases where inflammation or ischemia reperfusion plays a role, such as sickle cell disease and organ failure.

Only results from phase I trials have been published using Sanguinate, with a phase II trial recently completed in sickle cell patients with vaso-occlusive crisis. Results from the phase I trial, performed in 3 cohorts of 8 healthy volunteers, demonstrated a dose-dependent half-life ranging from 7.9 to 13.8 hours and no serious adverse events.^²⁹ The most commonly reported adverse events were mild and included dizziness and lethargy. There was a trend toward increased systolic and diastolic blood pressure that self-resolved, with no clinically significant changes in cardiac function or biomarkers. Results from the phase II trial have not yet been published.

A phase Ib trial in stable sickle cell anemia patients demonstrated a transient increase in troponin in 3 of 15 patients treated with the study drug, thus, further independent work is necessary to define risk/benefit.^³⁰ In another phase Ib evaluation in end-stage renal disease, 2 of 5 patients developed an increase in troponin, 1 of whom was determined to have a non-ST elevation myocardial infarction.^³¹ Although both of these studies suggest possible risk of myocardial injury from Sanguinate, larger studies are needed to determine if this risk is greater than that from severe anemia alone.

A novel use for Hb-based oxygen carriers may be in the setting of cerebral ischemia. In a study in 12 patients with subarachnoid hemorrhage treated with Sanguinate, an improvement in regional cerebral blood flow was demonstrated, although the benefit did not persist to 24 hours.^³² The combination of increasing oxygen delivery plus vasodilation from release of CO may serve to reduce ischemic damage and has been shown to reduce infarct volume in an animal model.^³³ Additionally, CO in low doses acts both as an anti-inflammatory and antioxidant offering further beneficial effects.^³⁴

Hb-based oxygen carriers still in early phases of testing include HemO₂Life (Hemarina SA, Morlaix, Brittany, France), an extracellular Hb from the invertebrate Arenicola marina, a marine worm subjected to periodic hypoxia and hypothermia during high tide.^³⁵ A hexagonal-bilayer composed of 156 globin molecules linked together, it can carry up to 156 oxygen molecules and has a p50 of 7 mm Hg. HemO₂Life is marketed as an additive to preservation solutions and is being researched in organ preservation, and has recently been studied in renal transplant patients.^³⁵^,^³⁶ In the first trial in kidney preservation before human transplantation, HemO₂life was added to existing hypothermic organ preservation solutions to reduce ischemia reperfusion injuries due to hypoxia during preservation of the graft. Preliminary data from over 60 subjects in 6 French reference transplant centers demonstrated the following results 3 months after transplantation: no graft loss related to product, no deaths, no major adverse event related to HemO₂Life, and no immunological, allergic, or prothrombotic effects from the additive.^³⁶

OxyVita (OXYVITA, Windsor, NY), a stroma-free Hb-based oxygen carrier that was created using a novel zero-linked polymerization process that utilizes cross-linked bovine tetramers and no exogenous binding agent like raffinose or glutaraldehyde, was developed to avoid the NO scavenging effects of earlier Hb-based oxygen carriers. It is supplied as a lyophilized powder and can be prepared by mixing with IV fluid for infusion; it was studied in the past with indirect Defense Advanced Research Projects Agency blood farming funding,^³⁷ although it has not yet been administered to humans in clinical trials to date.

Table 1. - Synopsis of Artificial Oxygen Carriers That Have Been Tested in Clinical Investigations^6,24

Product	Company	Source	Modification	Half-Life	State of Development
PEG-Hb	Enzon, Piscataway, NJ	Bovine	Polyethylene glycol-conjugated (PEGylated)	Not available	Phase Ib, tumor radiosensitization, discontinued in 1996
HemAssist (DCLHb, diaspirin-crosslinked Hb	Baxter, Deerfield, IL	Human	Intramolecular diaspirin α–α cross-linked tetramer	Not available	Phase III cardiac surgery, acute normovolemic hemodilution, trauma/stroke discontinued in 1999
Optro	Somatogen, Boulder, CO	Recombinant	Intramolecular cross-linked β-chain mutation (108 Lys)	Not available	Phase II discontinued in 1999
PHP/Hemoximer	Curacyte/Apex Bioscience, Triangle Park, NC	Human	Surface-modified polyoxyethylene pyroxilated polymer	Not available	Phase III, distributed shock, discontinued in 1999
Oxygent	Alliance Pharmaceutical Corp, San Diego, CA	Chemical	Perfluorochemical emulsion	Not available	Phase III, discontinued in 2001
HemoLink (hemoglobin-raffimer)	Hemosol, Toronto, Canada	Human	Intra- and intermolecular cross-linking with O-raffinose	Not available	Phase II/III, surgery, acute normovolemic hemodilution/cardiac surgery discontinued in 2004
PolyHeme (polymerized human Hb)	Northfield, Evanston, IL	Human	Glutaraldehyde polymerization	Not available	Phase III, trauma, surgery, discontinued in 2009
Hemospan (MP4)	Sangart Inc, San Diego, CA	Human	Maleimide-polyethylene glycol-modified Hb	Not available	Phase II published, phase III completed, discontinued in 2015
Hemopure (hemoglobin glutamer-250 [bovine])	Acquired by Hemoglobin Oxygen Therapeutics in 2014, Souderton, PA	Bovine	Glutaraldehyde polymerization	19–24 h	Phase III, perioperative transfusion, acute normovolemic hemodilution cardiac surgery. Hb glutamer-250 (bovine) approved for perioperative treatment of anemia in adult elective surgical patients in South Africa and Russia. Available for expanded access in United States
Sanguinate	Prolong Pharmaceuticals, South Plainfield, NJ	Bovine	Polyethylene glycol-conjugated (PEGylated) carboxyhemoglobin	13–20 h	Phase II trials in process
HemO₂Life	Hemarina, Morlaix, Brittany, France	Marine Invertebrate	Hexagonal-bilayer linked globin molecules	2.5 d	Phase I in progress
OxyVita Hb	OXYVITA Inc, Windsor, NY	Bovine	Hb stabilized with sebacoly diaspirin	72 h	Preclinical trials in progress

Advances in the second- and third-generation Hb-based oxygen carriers have made the products safer than discontinued products, and the inherent toxicities are mild.^²⁹^,^³⁸^,^³⁹ Further study is ongoing with these and other products. See Table 1 for a summary of the clinical trials to date.

Perfluorocarbons

Perfluorocarbon emulsions are synthetic products composed of a hydrophobic oxygen perfluorocarbon with the supporting elements (surfactants and salts) to allow them to be miscible with water and carry dissolved oxygen. An ideal perfluorocarbon would have dissolved oxygen that is readily available with a high extraction ratio, have a linear oxygen versus Po₂ uptake without saturation, have passive oxygen delivery, allow for carbon dioxide dissolution and removal, not be metabolized, and have emulsion stability and a steady excretion rate. The perfluorocarbon molecule is a hydrocarbon molecule that has had the hydrogen atoms replaced with fluoride ions and is referred to as a fluorine-alkylated compound. These molecules are not able to bind gases chemically; however, they can dissolve them and function as a gas-like fluid when in an appropriate emulsion.

Examining volume percent oxygen solubility, the oxygen solubility is 20 times greater for perfluorocarbons than for water. The fundamentals of these compounds have been extensively reviewed.^⁴⁰

In 1966, perfluorocarbons were shown to keep a mouse alive when breathing an oxygen-saturated liquid perfluorocarbon.^⁴¹ After this discovery of perfluorocarbon utilization, the race began to convert this perfluorocarbon into a perfluorocarbon emulsion that could be utilized either intravascularly or as an inhaled fluid for liquid ventilation. The first-generation perfluorocarbons Fluosol, Oxypherol, and Perftoran all had significant limitations to include: low oxygen delivery, labor intensive preparation, long organ half-life, and short shelf lives among others.^⁴² The second generation products addressed some of these issues by having higher perfluorocarbon content in the emulsion, utilizing natural phospholipids as emulsifiers, and not requiring frozen storage. These compounds included Oxygent, Oxyfluor, and Oxycyte, which had studies initiated and then terminated due to adverse events. These included increased risk of stroke for Oxygent and Oxyfluor or futility for Oxycyte.^¹⁸^,^⁴²^,^⁴³ Specific data on the rate of adverse events have not been published in the literature and is currently Food and Drug Administration–only information. In addition, during the past decade, there have been multiple reports of thrombocytopenia related to perfluorocarbon administration with speculated causes including platelet-white cell conjugates or platelet aggregates.^⁴⁴ Out of all the perfluorocarbons, Perftoran has been used the most, reportedly more than 35,000 administrations, and appears to have the lowest adverse effects perhaps due to its small size and unique surfactant–Proxanol 268.^⁴⁵ Based on a Russian study, side effects ranged from 0% to 25% with an overall rate of 6.9% based on 912 patients receiving Perftoran (as this is a study from Russia, it is difficult to interpret a side effect range of 0%–25%).^⁴⁶ Of note, Perftoran has been rebranded as Vidaphor in North America in 2017 and has been administered extensively in Russia since 1996 as well as having approval for short periods of time in the Republic of Kazakhstan, Ukraine, Kirghiz Republic, and Mexico.^⁴² FluorO2 Therapeutics, Inc plans to manufacture Vidaphor in the United States and further study it internationally.

There has been steady interest in perfluorocarbons and their utilization in traumatic brain injuries. This is due to a common secondary sequela of traumatic brain injury being cerebral ischemia with the thought that perfluorocarbons may be able to increase oxygen delivery to the injured areas. In 2004, perfluorocarbon emulsions given after injury were shown to increase cerebral oxygenation significantly after traumatic brain injury in rats.^⁴⁷ Moon-Massat et al^⁴⁸ demonstrated that the perfluorocarbon emulsion NVX-108 did not cause vasoconstriction in the cerebral pial arterioles of healthy rats nor did it cause significant hemodynamic changes. Most recently, a study using Oxycyte given with 100% inspired oxygen fraction and postinjury, demonstrated neither cerebral vasoconstriction nor an increase in brain tissue oxygenation.^⁴⁹ This 1 study contradicts the hypothesis that perfluorocarbons increase brain tissue oxygenation following trauma as was shown previously. Nonetheless, perfluorocarbon compounds are still an area of continued research with potential large benefits as oxygen carriers, and with the recent rebranding of Vidaphor, there is likely more to come.

Current and Future Technologies

The quest for development of oxygen carriers has been a complex journey of conflicting results. The initial ineffectiveness and side effect profiles of the perfluorocarbons was followed by further development of Hb-based oxygen carriers. Though extensively modified, the cross-linked, polymerized, and conjugated Hb-based oxygen carriers have been characterized by short circulatory half-lives and adverse event profiles due to free Hb and NO scavenging. These challenges have led scientists to develop other forms of oxygen-carrying capacity such as protein-based oxygen carriers, bionanotechnology, and the use of erythrin-based products.

Protein-based artificial oxygen carriers have been predominantly based on modifications of the protein Hb to alter its redox state so that it functions optimally in oxygen transport and is minimally toxic to humans. Modifications of Hb have included intramolecular cross-linking, polymerization, PEGylation, genetic modification, encapsulation, and using derivatives of Hb to create many of the recently tested Hb-based oxygen carrier products.

The advent of Hb-loaded nanoparticles (particles between 1 and 100 nm in size) has eliminated many of the toxic side effects of protein-based Hb-based oxygen carriers. Nanoparticle-based Hb-based oxygen carriers can provide a larger oxygen-carrying capacity by incorporating a higher amount of Hb per particle, while controlling effects on viscosity and oncotic pressure.^⁵⁰^,^⁵¹ The nanoparticle encapsulation of erythrocytes, and the coencapsulation of enzymes and reducing agents further mitigate the formation of toxic metabolites, as the formation of methemoglobin is avoided. A typical limitation is that biodegradable polymer-based Hb-loaded nanoparticles are rapidly cleared from the circulation by phagocyte uptake and thus their circulatory half-life is short. However, their surface can be modified to not only eliminate immunogenicity but to prolong circulatory lifespan (1–3 days). The addition of polyethylene glycol to these Hb polymers has significantly prolonged their survival time in circulation by providing a hydrophilic neutral-charged surface that evades opsonization by the body’s mononuclear phagocytic system.^⁵² In fact, half-life in the circulation is proportional to the percentage polyethylene glycol coating.^⁵³ The interaction of Hb with various other nanoparticles has been investigated and the most promising substrate to date is silica nanoparticles. The adsorption of Hb to silica nanoparticle surfaces leads to a modification of the protein structure that preserves its quaternary structure, enhances oxygen affinity, and is completely reversible, under physiologic conditions, after dissociation from the nanoparticle. The silica surface provides a substrate for Hb adsorption in its tetrameric form and allows it to retain its oxygen affinity properties. The duration of circulatory effect in many nanoparticle formulations is 1–3 days, depending on the composition, and thus a temporary solution for anemia is offered.

One novel nanoparticle, ErythroMer, developed at Washington University in St Louis, is a product funded by the National Institutes of Health and US Department of Defense that can be reconstituted from a lyophilized state, and while still in preclinical testing, may be useful in combat situations. It is a Hb-based synthetic polymer that can sense pH changes through its shell properties, and using a 2,3-diphosphoglycerate shuttle, it permits it to load oxygen in places where the pH is high and offload when the pH is low, due to a proprietary process.^⁵⁴

Hemerythrin is a protein of oxygen transport in marine invertebrates that may prove useful in the creation of oxygen carriers.^⁵⁵ Hemerythrin is inert to hydrogen peroxide, NO, and nitrite, and has a higher molecular weight than Hb; thus, it will not be filtered as easily by the kidney. Polymerized and derivatized hemerthyrin has been successfully chemically modified and its potential to serve as an oxygen carrier is worthy of further study. In culture, hemerythrin has a proliferative effect on human lymphocytes and its chemical derivatives have shown to be less toxic than native Hb.

An exciting prospective source of human red blood cell production is through the use of stem cell technologies. Through induced hematopoietic differentiation of multiple different stem cell types, human erythrocyte growth and development can be mimicked. Through these techniques, a near limitless supply of erythrocytes that most closely approximate the biologic and physiochemical properties of native erythrocytes can be produced, although industrialization of stem cell technology is not feasible in the current legal and political climate. Our failure thus far to produce the ideal artificial oxygen carrier has led scientists to these new nano and genetic technologies; they hold great promise for providing increased oxygen-carrying capacity in severe anemia.

WHAT’S AVAILABLE TODAY AS EXPANDED ACCESS/COMPASSIONATE USE

Food and Drug Administration defines “expanded access” as the use of an investigational medical product outside of a clinical trial. Expanded access is available through 3 mechanisms: emergency use, compassionate use, and treatment use. All 3 mechanisms share the same requirements that the patient’s condition must be life-threatening or serious in need of immediate treatment and there is no approved acceptable alternative treatment for the condition. Under emergency use program, there should be an immediate need with no time to obtain Food and Drug Administration approval for the use, while under compassionate use a case-by-case Food and Drug Administration approval process is in place. Obtaining compassionate use requires going through both the institutional review board as well as the Food and Drug Administration. The process will vary slightly for each institution and the clinical urgency, but the manufacturer may assist with this process. Treatment use program may be used when the patient cannot be enrolled in ongoing trials, or in clinical trials have reached maximum enrollment.

According to ClinicalTrials.gov, currently there are 3 active compassionate use programs for artificial oxygen carriers in the United States (ClinicalTrials.gov Identifiers NCT02684474,^⁵⁶ NCT03633604,^⁵⁷ and NCT02934282^⁵⁸). They all offer HBOC-201 for critically ill patients with life-threatening anemia for whom blood is not an option. The life-threatening anemia is defined as Hb ≤6 g/dL in the first 2 and ≤5 g/dL in the third program (or Hb 7–8 g/dL in the first 2 or 6–7 g/dL in the third program in the presence of significant active bleeding and physiologic evidence of critical ischemia).

Clinical use of artificial oxygen carriers requires knowledge of their laboratory interference. All Hb-based oxygen carriers affect certain laboratory results, and the manufacturers developing these products have done extensive testing of equipment to determine each product’s specific issues. However, there are a number of commonalities that affect all of them.^⁵⁹ When measuring Hb, there are actually 3 measurements: erythrocyte Hb, plasma Hb (from the Hb-based oxygen carrier), and the sum of these, or total Hb.^⁶⁰ Hematocrit will only rely reflect the Hb concentration in the erythrocytes and measuring the Hb in the plasma will give the Hb-based oxygen carrier Hb concentration.^⁶¹ Clinical decisions during the phase III trials used total Hb, as accurately measured by the Coulter counter.^²² Other laboratory alterations are with creatine phosphokinase muscle/brain fraction, although use of troponin has been determined to be accurate.^²² Lactate level and other tests using colorimetric testing are less accurate than use of spectrophotometry.^⁶² Pulse oximetry may also be erroneous, and arterial blood gases and cooximetry are often necessary to determine accurately oxygen saturation and content.^⁶³ With the potential eventual approval of Hb-based oxygen carriers, extensive guidance will be necessary from the manufacturer in laboratory testing and accuracy and interference with specific tests for that each Hb-based oxygen carrier, as there are differences among products.

USE IN PATIENTS FOR WHOM BLOOD IS NOT AN OPTION

Numerous reports of patients treated with artificial oxygen carriers under compassionate use programs in various settings have been published (Table 2). In 1 report, 54 patients with median baseline Hb of 4 g/dL received HBOC-201 (6–300 g). About 40% of the patients survived and no serious adverse event was attributed to HBOC-201. The authors noted that the time taken from Hb dropping to ≤8 g/dL to initiation of infusion of Hemopure was significantly shorter in survivors versus nonsurvivors, underscoring the importance of earlier treatment with these agents in life-threatening anemia.^⁶⁴ Timeliness can be a challenge in the context of a compassionate use program, and thus many patients in this and other similar reports spend extended periods of time with severe deficits in oxygen-carrying capacity before the artificial oxygen carrier can be started, already putting them at a survival disadvantage.

Table 2 - . Summary of Compassionate Use of Hb-Based Oxygen Carriers in Patients for Whom Blood Is Not an Option

Author, Publication Year	Patients, N=	Population	Nadir Hb (g/dL)	Units of HBOC-201 Received	Outcome
Davis et al,³ 2018	3	Sickle cell (2 Jehovah’s Witnesses patients)	3.6–3.8	6–27 U	All survived to hospital discharge
Donahue et al,⁶⁵ 2010	1	Jehovah’s Witnesses with acute lymphoblastic leukemia	3.1	15	Completed chemotherapy and discharged
Fitzgerald et al,⁶⁶ 2011	1	Jehovah’s Witnesses trauma patient	2.9	5	Discharge to rehabilitation center
Jordan and Alexander,⁶⁸ 2013	1	Jehovah’s Witnesses with autoimmune hemolytic anemia	2.8	2	Discharge to home
Lundy et al,⁶⁹ 2014	1	Jehovah’s Witnesses burn patient	5	6	Death from multiorgan failure
Mackenzie et al,⁶⁴ 2010	54	Jehovah’s Witnesses patients with severe anemia	3.9^a	8^b	Survival of 41.8%, no adverse events attributed to Hb-based oxygen carrier
Posluszny and Napolitano,⁶⁷ 2016	1	Jehovah’s Witnesses trauma patient	3.9	7	Discharge to home

^aMedian Hb before Hb-based oxygen carrier administration.

^bMedian units received.

Another case report details the management of a Jehovah’s Witnesses patient diagnosed with acute lymphoblastic leukemia with a nadir Hb of 3.1 g/dL during the induction chemotherapy.^⁶⁵ The patient received a total of 15 units (U) of HBOC-201 over 12 days and was able to complete the chemotherapy and was eventually discharged in stable condition. Fitzgerald et al^⁶⁶ reported on their experience with treating a Jehovah’s Witnesses trauma patient with Hb dropping to as low as 2.9 g/dL with signs of ischemia. After slow infusion of 5 U of Hemopure, Hb level rose to 6.2 g/dL and signs of organ ischemia were resolved. The patient was eventually discharged with no evidence of lasting ischemic sequelae. Additionally, Posluszny and Napolitano^⁶⁷ reported on their successful management of a Jehovah’s Witnesses trauma patient with Hb as low as 3.9 g/dL who was treated with an Hb-based oxygen carrier as an “oxygen bridge” agent while waiting for the effects of erythropoiesis stimulating agents and iron on bone marrow and Hb to reach clinical level. Similar successful results were reported from a case of a Jehovah’s Witnesses patient with symptomatic autoimmune hemolytic anemia and nadir Hb of 2.8 g/dL who received 2 U of HBOC-201.^⁶⁸

Lundy et al^⁶⁹ reported on their experience with management of an elderly Jehovah’s Witnesses patient with extensive full-thickness burns, necessitating surgical excision and wound closure. The patient received a total of 6 U of HBOC-201 in the postoperative period with no initial signs of adverse events, but eventually developed progressively worsening neurologic, respiratory, cardiovascular, and renal dysfunction and passed away. In their report, the authors listed the steps taken to obtain the Hb-based oxygen carrier product, including the initial communication with the manufacturer, submission of an emergency investigational new drug request to Food and Drug Administration, and submission of a compassionate use protocol to the local institutional review board that eventually allowed them to approach the patient’s family to obtain an informed consent. While these steps are in place to protect patients, there is no denying that time required could be defining the line between significant morbidity and mortality for patients in such grave situations.

Davis et al^³ reported 3 critically ill patients with multiorgan failure during sickle cell crisis treated with Hemopure, including 1 patient who received a total of 27 U of the product, making this the largest volume of Hemopure infused to a single patient reported up to the time of that publication. All 3 patients survived.

Mackenzie et al^²⁵ have provided documentation of the lessons learned during treatment of more than 1700 patients with HBOC-201 under clinical trials and compassionate use programs. The authors listed the increased blood pressure, oliguria, gastrointestinal symptoms, yellow discoloration of sclera and skin, and transient increase in blood methemoglobin and liver/pancreatic enzymes as the main reported adverse events and indicated that most were transient, self-limiting, or easily manageable. The authors noted that issues related to improper consideration of the volume expanding effects of HBOC-201 and its half-life in the body that necessitates repeated infusions were most common clinical management errors. As before, early use of Hb-based oxygen carrier under compassionate use programs as soon as Hb drops below 5 g/dL was recommended to improve survival.

Recently, Weiskopf et al^¹ published a comparison of historical mortality data from severely anemic patients for whom transfusion was not an option (Jehovah’s Witnesses) versus mortality data from patients treated with an HBOC-201. Survival was significantly improved for the 102 patients treated with HBOC-201 through compassionate use programs (HR, 0.44; P < .0001) as well as for the 350 surgical patients treated in a HBOC-201 trial (HR, 0.42; P = .014) compared to the historical Jehovah’s Witnesses patients. Morbidity and mortality remains high in patients with severe anemia and unable to be transfused,^⁷⁰ and with no other treatment available, these results hold promise for improving survival in severely anemic patients who are unable to be transfused.

While multiple products were originally designed as “blood substitutes,” none have ever been able to replace erythrocytes. Instead, as oxygen therapeutics, multiple potential indications have emerged, such as augmenting acute normovolemic hemodilution (the perfluorocarbons may be particularly useful here, given their relatively short half-lives), use where blood is not an option or not available, organ preservation, improved graft survival, sepsis, and sickle cell crisis. To hypothesize that 1 product could conceivably perform all these indications is unreasonable, far more likely is that each product will find its niche and be valuable after considerable study for that particular area. With the passage of the “Right to Try” law in the United States in 2018, in addition to the multiple Food and Drug Administration mechanisms for cases where blood is unavailable or not an option, further collection of data is possible and should allow for increased indications and usage of these products.^⁷¹

SUMMARY/CONCLUSIONS

Despite decades of research looking for a substitute to allogeneic blood, transfusion of erythrocytes has been and remains the standard for increasing oxygen delivery to anemic patients. As a consequence of heightened concerns about enveloped viral transmission with allogeneic transfusion, the ongoing search for an acceptable oxygen carrier has accelerated in the past few decades. Chemically produced agents that deliver oxygen to tissues and their clinical use as artificial oxygen carriers are still fraught with significant challenges. Despite overcoming issues of toxicity, there is still reluctance to accept these agents because clinical validity has always been judged in comparison to erythrocyte transfusion.

After years of search, we now recognize that these agents are meant for patients suffering life-threatening anemia when erythrocyte transfusions are not an option. Although not currently Food and Drug Administration approved for clinical use in the United States, the future is showing more promise for possible approval and adaptation of artificial oxygen carriers into clinical practice for the proper patient population. With the recent passage of the “Right to Try” law in the United States, it may become easier for the US Food and Drug Administration to permit use of more of these products as a life-saving measure and give more exposure to the field and specific artificial oxygen carriers.

ACKNOWLEDGMENTS

This article has been prepared by members of and on behalf of the American Society of Anesthesiologists Committee on Patient Blood Management. The authors thank Dr Bruce Spiess for his valuable assistance on the section on perfluorocarbons.