Blood Substitutes What They Are and How They Might Be Used

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1 Pathology Patterns Reviews Blood Substitutes What They Are and How They Might Be Used Qun Dong, MD, PhD, 1 and Christopher P. Stowell, MD, PhD 1,2 Key Words: Blood substitute; Hemoglobin-based oxygen carrier; Perfluorocarbon DOI: /6TKP72PU3NYH4VM5 Abstract Three classes of materials have been studied as potential blood substitutes: modified hemoglobin solutions, perfluorocarbon emulsions, and liposomeencapsulated hemoglobin. The first two have reached phase III clinical trials, while the third remains in the preclinical stage of testing. Hemoglobin is a highly active molecule; hence, modification has been required to avoid potential deleterious effects. Although there has been considerable progress toward bringing such a product to the clinical setting, its development has challenged our understanding of oxygen delivery and use. The perfluorocarbon emulsions have been studied primarily for roles other than as equivalents of conventional banked units of RBCs for transfusion. The study of these molecules has added to our understanding of basic physiologic processes. During the 1980s, concerns about the adequacy of the blood supply and the public focus on the HIV epidemic spurred substantial interest in the development of a safe and effective blood substitute. 1,2 Blood substitutes are products that generally are intended to serve the functions of oxygen delivery and volume expansion. Studies have shown that these artificial products also can serve additional functions (eg, a nitric oxide [NO] scavenger during septic shock). Considerable progress has been made in the development of blood substitutes during the last 15 years, and several products are in, or have completed, phase III clinical trials. It is hoped that the development of blood substitutes will provide an alternative to banked blood and help us better understand oxygen delivery to tissues. This article summarizes the advantages and limitations of current blood substitutes (modified hemoglobin solutions, perfluorocarbon emulsions, and liposome-encapsulated hemoglobin) and their applications in various clinical settings. This topic has been the subject of several recent reviews. 3-5 Hemoglobin Solutions Hemoglobin as an Oxygen Carrier Hemoglobin is a tetrameric protein that contains 2 alpha subunits and 2 beta subunits. One alpha and 1 beta chain combine to form a stable alpha/beta dimer, and the 2 dimers are loosely associated to form a tetramer. Each subunit contains a single iron-containing heme group, which binds and releases oxygen. A cell-free hemoglobin solution would seem to be an obvious candidate for a blood substitute, because hemoglobin S71 S71

2 Dong and Stowell / BLOOD SUBSTITUTES is the natural oxygen carrier in RBCs. The advantages of hemoglobin as an oxygen carrier are high capacity for oxygen and carbon dioxide, efficient function at physiologic PO 2 levels, high oncotic pressure, and stability Table 1. 3 The absence of the RBC membrane and its antigens obviates the need for compatibility testing. The purification and modification procedures also virtually eliminate the risk of transmission of infectious disease. Despite these appealing features, the use of cell-free hemoglobin has a number of potential drawbacks, including short plasma half-life, renal toxic effects, high affinity for oxygen, and hypertensive effects. When cell-free hemoglobin is present in solution, it rapidly dissociates to alpha/beta dimers that are cleared by the kidney, resulting in a very short plasma half-life. In addition, cell-free hemoglobin is toxic to the kidney. 6 To overcome the problems of short circulation time and renal toxic effects, chemical modification of cell-free hemoglobin is necessary. The hemoglobin-based substitutes that have been created can be classified according to the modification strategy: surface-modified hemoglobins, polymerized hemoglobins, and cross-linked hemoglobins Table 2. All of these hemoglobin solutions have a stabilized hemoglobin tetramer (or oligomers thereof) and an increase in the apparent molecular weight relative to unmodified hemoglobin, thereby preventing rapid clearance by the kidneys. Cell-free hemoglobin also may affect vascular tone, which is manifested by the mild to moderate hypertensive effects seen in recipients of some of these products. 7,8 The mechanisms that have been proposed include NO scavenging by the heme iron producing vasoconstriction. 9,10 Unlike RBC-encapsulated hemoglobin, cell-free hemoglobin can diffuse much closer to endothelial cells and bind NO molecules, which are potent vasodilators, thereby producing vessel constriction. In the intact RBC, some NO also is present in a membrane-associated compartment as nitrosothiol adducts, which can dissociate to release NO and perhaps function to regulate vascular tone. 11 Hemoglobin also may exert a direct effect on peripheral nerves, stimulating noradrenaline Table 1 Potential Advantages and Disadvantages of Unmodified Hemoglobin Advantages Disadvantages High capacity for O 2 and CO 2 Rapid clearance Functions at physiological PO 2 level Renal toxicity Low viscosity (?) Vasoactivity High oncotic pressure Increased oxygen affinity (?) Absence of RBC antigens Autoxidation Prolonged shelf-life Immunogenicity (modified or nonhuman) Purification/viral inactivation possible Potentiation of sepsis (?)?, effect equivocal. production and inducing vascular constriction. 12 Other factors that have a role in the vasoactivity of hemoglobin are its effects on endothelin-mediated regulation of vascular tone 13,14 and the influence of reduced viscosity, which reduces shear stress and output of NO by endothelial cells. 15 Delivery of oxygen by the substitute also may exert local vasoconstrictive effects by triggering an oxygen-sensitive autoregulatory response It has been suggested that without the RBC membrane as a barrier, oxygen can more easily diffuse into the tissue and activate the autoregulatory vasoconstriction reflex. 19 The hypertensive effect has been exploited by Baxter Healthcare (Deerfield, IL) 20,21 and Apex Bioscience (Research Triangle Park, NC) 22 in the treatment of patients with septic and hemorrhagic shock, in which NO levels usually are elevated. Another potential difficulty with cell-free hemoglobin is that it loses its complement of 2,3-bisphosphoglycerate (BPG). Thus, its affinity for oxygen is higher than that of red cell associated hemoglobin and might require a greater degree of hypoxia to release oxygen to the tissues where it is needed. 23 In some of the hemoglobin-based oxygen carriers (HBOCs), additional chemical modifications have been used to decrease the affinity of modified hemoglobin for oxygen, so that it more closely replicates the behavior of intraerythrocytic hemoglobin in vivo. The beta/beta cross-linking into the 2,3-BPG pocket of Hemolink (Hemosol, Mississauga, Ontario) has the effect of shifting the oxyhemoglobin dissociation curve to the right, 24 as does the diaspirin linkage modification used in HemAssist (Baxter Healthcare). 25 Pyridoxylation of PolyHeme (Northfield Laboratories, Evanston, IL )26 and PHP (Apex Bioscience) 27 achieves the same effect. Bovine hemoglobin, which is used in Hemopure (Biopure, Cambridge, MA), Oxypure (Biopure), and PEG-Hemoglobin (Enzon, Piscataway, NJ), is regulated by Cl ion rather than 2,3-BPG. Therefore, cell-free bovine hemoglobin does not have a marked leftward shift; at physiologic Cl concentrations, its dissociation curve resembles that of human hemoglobin with bound 2,3-BPG. Somatogen (Boulder, CO) designed its HBOC, Optro, around a recombinant version of a naturally occurring human hemoglobin variant with a high P 50 (hemoglobin Presbyterian). Although the early assumption guiding the development of the HBOCs was that a low P 50 was an undesirable characteristic, it may in fact be advantageous because the more parsimonious release of oxygen may blunt the autoregulatory response. 28 In addition, by retaining oxygen in the face of a greater degree of hypoxemia, a high-affinity hemoglobin may create a steeper oxygen gradient. Since oxygen delivery beyond the vascular space depends on diffusion, the steeper oxygen gradient may drive oxygen more deeply into the tissue. 29 Sangart s (San Diego, CA) Hemospan has been deliberately designed to have a low P 50 to minimize the autoregulatory effect. S72 S72

3 Pathology Patterns Reviews Table 2 Modifications to Prevent Rapid Clearance and Nephrotoxic Effects of Hemoglobin Class/Technique Product Name Company Product Description (kd) Intramolecular cross-linking Diaspirin HemAssist Baxter Healthcare, Deerfield, IL Hemoglobin tetramers (64) Recombinant di-alpha chain Optro Somatogen, Boulder, CO Hemoglobin tetramers (64) Intermolecular cross-linking Glutaraldehyde PolyHeme Northfield Laboratories, Evanston, IL Hemoglobin oligomers (150) Glutaraldehyde Hemopure/Oxyglobin Biopure, Cambridge, MA Hemoglobin oligomers (240) o-raffinose Hemolink Hemosol, Mississauga, Ontario Hemoglobin oligomers (156) Surface conjugation Polyethylene glycol PEG-Hemoglobin Enzon, Piscataway, NJ Hemoglobin tetramers (117) Polyoxyethylene PHP Apex Bioscience, Research Triangle Park, NC Hemoglobin tetramers (97) Polyethylene glycol Hemospan Sangart, San Diego, CA Hemoglobin tetramers (90) In some of the earliest studies infusing cell-free hemoglobin in human subjects, anaphylactic reactions were observed that have since been shown to be due mainly to contaminating phospholipids from the RBC membranes that had not been removed completely by the manufacturing process. The presence of these phospholipids activated the complement cascade. 6 Improvements in purification techniques have since resulted in essentially phospholipid-free hemoglobin solutions so that hypersensitivity reactions have not been observed. Hemoglobin Sources There are 3 major sources of hemoglobin for the products that have been studied in clinical trials to date: human hemoglobin from outdated units of banked blood, bovine hemoglobin, and recombinant human hemoglobin. These sources are compared in Table 3. The projected shortage in the blood supply may adversely affect the availability of outdated human RBC products for further processing into blood substitutes. Production of recombinant human hemoglobin may solve the problem. However, producing recombinant hemoglobin on a large scale at a low cost will be a technical challenge. Bovine hemoglobin is abundant; however, the use of nonhuman hemoglobin may create other problems. One potential problem is an immune response to the foreign hemoglobin molecules, which also is a concern when using chemically modified human hemoglobin. Fortunately, hemoglobins are in general ineffective immunogens, and an immune response to modified or nonhuman hemoglobin molecules has not emerged as a problem in clinical trials to date. 30 Still, the long-term effects, especially of multiple doses, have yet to be determined. With any naturally occurring source material, such as human or animal hemoglobin, there always is the possibility of contamination with microorganisms. The purification, modification, and viral inactivation techniques used on the various HBOCs under development are quite hostile to microorganisms and have been shown to be highly effective in eliminating them. Prion-mediated diseases are of particular concern with products based on bovine hemoglobin. Biopure uses hemoglobin from closed herds and has data showing that model prions are effectively inactivated by its preparation process. Hemoglobin-Based Oxygen Carriers Under Clinical Development To date, 8 HBOCs have been studied as possible blood substitutes for human use, 6 of which remain under active development Table 4. In addition, one product has been licensed for veterinary use (Oxyglobin, Biopure). Table 3 Comparison of Hemoglobin Sources Human Bovine Recombinant (Human) Quantity Limited * Abundant Moderate P 50 Immunogenicity Very low Low Very low Infectious risks Very low;? new pathogen Very low;? bovine pathogens Very low, decrease;, no change;?, possible. * Outdated RBCs. Compared with native hemoglobin. May be dependent on modification procedures. S73 S73

4 Dong and Stowell / BLOOD SUBSTITUTES Table 4 Modified Hemoglobin-Based RBC Substitutes * Product (Manufacturer) Hemoglobin Source Trial Phase Application PolyHeme (Northfield Laboratories) Human III Trauma; surgery Hemolink (Hemosol) Human II Cardiopulmonary bypass ANH, orthopedic surgery acute blood loss; dialysis III Cardiac surgery HemAssist (Baxter Healthcare) Human II Septic shock; hemodialysis; hemorrhagic shock; cardiopulmonary bypass III (all trials terminated) Acute blood loss surgery, trauma; stroke PHP (Apex Bioscience) Human III Nitric oxide induced shock Hemospan (Sangart) Human Preclinical PEG-Hemoglobin (Enzon) Bovine Ib Radiosensitizer for solid tumor treatment Hemopure (Biopure) Bovine Preclinical Erythropoiesis I Radiosensitizer for glioblastoma treatment II Sickle cell crisis; oncology; trauma; orthopedic, urologic, vascular, and cardiac surgery III Cardiac and orthopedic surgery Oxyglobin (Biopure) Bovine Approved Veterinary uses: anemia; acute blood loss Optro (Somatogen) Recombinant I Erythropoiesis in ESRD, refractory anemia II (all trials terminated) ANH (acute blood loss); surgery ANH, acute normovolemic hemodilution; ESRD, end-stage renal disease. * Information current as of July For company locations, see Table 2. Approved in South Africa. Stabilized Tetramer HBOCs HemAssist The HBOC developed by Baxter Healthcare was based on a stabilized hemoglobin tetramer cross-linked using the diaspirin linkage technique initially developed by the US Army. The product then was purified, heat inactivated, and stored frozen. 31 Although this product was one of the first to reach phase III clinical trials, the company halted its trials in trauma, surgery, and acute ischemic stroke in 1998 and discontinued development of the product altogether, citing safety concerns. 32 Mortality rates were higher in patients receiving HemAssist in trials in acute, hemorrhagic stroke 33 and trauma, 34 although not in a trial in cardiac surgery. 35 A post hoc, systematic analysis of the trauma trial did not find an explanation for the marked difference in mortality, although it confirmed that administration of HemAssist was an independent risk factor for mortality. 36 Optro Optro is the only product to date based on a recombinant human hemoglobin. The tetramer was cross-linked by combining a single polypeptide consisting of 2 alpha subunits joined by a short linker peptide with 2 conventional beta chains to produce a stabilized hemoglobin tetramer. 37 The parent company, Baxter Healthcare, discontinued the development of Optro in 1998 when it closed down its trials with HemAssist. Optro had been in phase I 38 and II clinical trials. Polymerized HBOCs PolyHeme Northfield Laboratories has been developing a product consisting of glutaraldehyde polymerized human hemoglobin, which has been pyridoxylated and extensively purified to remove residual hemoglobin tetramers. 39 It is being studied principally as an alternative to use of RBCs in surgery and trauma Preliminary studies in trauma have shown that patients receiving PolyHeme required fewer transfusions of banked blood. 40 Northfield is the first company to have submitted data for approval of a human blood substitute product approval to the US Food and Drug Administration (FDA). Five units of PolyHeme (250 g of hemoglobin) were transfused on a compassionate use basis to a victim of a motor vehicle collision who was a Jehovah s Witness. 43 The patient hemorrhaged to a nadir of 3.2 g/dl (32 g/l) of cellular hemoglobin. PolyHeme helped sustain the patient for a period of several days until hemorrhage was controlled and erythropoiesis (stimulated by exogenous erythropoietin) could compensate for blood loss. Hemopure Biopure uses glutaraldehyde polymerization of bovine hemoglobin to produce both Hemopure and Oxyglobin. The product undergoes extensive purification and viral inactivation steps to produce a substitute with less than 3% hemoglobin tetramers. The primary indication for Hemopure is in the perioperative period as a bridge delaying the need for S74 S74

5 Pathology Patterns Reviews banked RBCs until the patient s condition has been stabilized and blood loss curtailed, although it has also been studied in a phase II trial in sickle cell crisis in which it improved exercise tolerance. 48 Hemopure has a slight pressor effect, which correlates with increased systemic vascular resistance and decreased cardiac index. 44 In a phase II study in patients undergoing infrarenal aortic aneurysm resection, 27% of patients receiving Hemopure intraoperatively avoided allogeneic transfusion compared with none of the patients receiving banked RBCs; however, the median number of allogeneic units infused was not different. 45 Biopure also has completed enrollment in several phase III trials in noncardiac surgery in the United States and Europe. Biopure is the first to have obtained licensure for a blood substitute product. In 1997, Oxyglobin, the veterinary formulation of Biopure s HBOC, was licensed by the FDA, and in April 2001, Biopure announced that Hemopure had been approved for clinical use in South Africa, although it is available in very limited quantities only. Hemopure was infused on a compassionate use basis into a patient with warm autoimmune hemolytic anemia who was refractory to treatment and required RBC transfusion support. 49 This patient received 11 U (330 g of hemoglobin) of Hemopure during a period of several days, which improved hemodynamics and relieved ischemic symptoms. Hemolink Hemosol has used an oxidized form of the trisaccharide raffinose to polymerize human hemoglobin followed by a reduction step and extensive purification and viral inactivation procedures. 50 Hemolink also has been found to have a mild pressor effect. 51 It has been studied in phase II clinical trials in dialysis and as an oxygen-carrying replacement fluid in acute normovolemic hemodilution. Patients receiving up to 4 U of Hemolink in a phase II study in cardiac surgery required fewer transfusions of banked RBCs up to 5 days after surgery compared with control subjects receiving pentastarch. 52 Phase III trials in cardiac surgery have been completed in Canada, the United States, and Europe. Surface-Conjugated HBOCs PEG-Hemoglobin Enzon has prepared PEG-Hemoglobin by conjugating polyethylene glycol (PEG) to bovine hemoglobin tetramer followed by purification. PEG-Hemoglobin has a much larger molecular weight and radius than native hemoglobin, as well as increased viscosity and oncotic pressure. 53,54 It also has a slightly longer plasma half-life than most of the other HBOCs (48 hours). This product is being developed for use as a sensitizer for radiation treatment of solid tumors. 55 Adequate oxygenation of tumor tissue is required for optimal effectiveness of radiation therapy; however, the anomalous vasculature of many tumors impedes the flow of RBCs. PEG-Hemoglobin, which is much smaller, may navigate through the vasculature of the tumor more readily than RBCs, thereby improving oxygen delivery and enhancing the effect of radiation. PHP Apex Bioscience prepares its product by pyridoxalation of human hemoglobin followed by conjugation with polyoxyethylene. 56 This preparation has a hypertensive effect and is being studied as an NO scavenger in patients with septic or hemorrhagic shock. Hemospan The most recent product to undergo development is Hemospan. This HBOC is prepared by conjugating PEG to hemoglobin tetramer obtained from outdated units of human blood. It is available in 2 formulations, one with a high concentration of the PEG-conjugated hemoglobin (Hemospan) and the other with a lower concentration but with pentastarch (Hemospan PS). The product has been designed by Sangart to have a large molecular diameter, high viscosity, and high oxygen affinity to minimize autoregulatory and vasoconstrictive effects. 19 The product is still in the preclinical phase of testing, but phase I trials are being planned. Perfluorocarbon Emulsions Perfluorocarbon Emulsions as Oxygen Carriers Perfluorocarbons (PFCs) are chemically and biologically inert, water-insoluble compounds that are capable of dissolving large volumes of gases, including oxygen. Some PFCs can dissolve 100 times more oxygen per volume than plasma. The oxygen capacity of PFCs was first illustrated in 1966 when mice were fully submerged in oxygenated PFCs, where they survived for several hours. 57 Unlike the cooperative, saturable binding of oxygen to hemoglobin within RBCs, however, the capacity of PFCs for oxygen is directly proportional to the ambient oxygen tension. Since PFCs are immiscible in water, they must be prepared as emulsions for most clinical applications. At the dosing levels currently being tested in clinical trials, the oxygen content of the PFC emulsions is significantly less than that of whole blood at ambient PO 2. To increase the oxygen content of PFC emulsions to levels closer to that found in blood, patients need to receive supplemental oxygen. PFCs are cleared from the circulation by the reticuloendothelial system and ultimately are exhaled through the lungs. In general, they have a short lifetime in the circulation (ie, hours). Several PFC S75 S75

6 Dong and Stowell / BLOOD SUBSTITUTES emulsions have been reported to produce a flu-like syndrome, with back pain, malaise, and flushing 58 that has been attributed to the release of cytokines and arachidonic acid metabolites by cells of the reticuloendothelial system as they ingest the PFC droplets. 59 Despite these disadvantages, PFCs have a number of attractive features, which have been responsible for their advancement into clinical trials Table 5. These include the ability to control the composition of emulsions, large scale production at relatively low cost, minimal infectious risk, and minimal immunogenicity. Perfluorocarbon Emulsions Under Clinical Development Fluosol-DA The first blood substitute to reach clinical trials was Fluosol-DA Table 6, a PFC emulsion prepared by Green Cross (Osaka, Japan) and developed in the United States by Alpha Therapeutic (Los Angeles, CA). Fluosol-DA consisted of 2 PFCs, a surfactant, and an emulsion stabilizer. Its performance in a phase II clinical trial in acutely hemorrhaging patients was disappointing. 60 In retrospect, the lack of efficacy was attributed to the low oxygen-carrying capacity of the product, the small amounts that were infused, and the severe illness of the patients in the trial. Although the development of Fluosol-DA as an RBC substitute was halted, it was eventually licensed for use in percutaneous transluminal coronary angioplasty, where it was used to perfuse and oxygenate the capillary beds distal to the balloon. 61,62 It eventually was removed from the market because it was inconvenient to use and improvements in catheter technology obviated the need. The experiences with Fluosol-DA provided proof of concept and informed the development of a second generation of PFCs represented by perflubron (Alliance Pharmaceutical, San Diego, CA) and Oxyfluor (HemaGen/PFC, Waltham, MA). Table 5 Potential Advantages and Disadvantages of Perfluorocarbon RBC Substitutes Advantages Disadvantages Control of composition Requirement for emulsification/ stabilization Possibility of specific Heterogeneous particle size modification Large-scale production Variable (long) reticuloendothelial system clearance Low production costs High FIO 2 required Prolonged shelf life Low O 2 capacity at physiologic PO 2 Minimal infectious risk Rapid plasma clearance Minimal immunogenicity Low viscosity (?)?, effect equivocal. Perflubron Alliance Pharmaceutical has developed a substitute based on the new PFC, perfluorooctylbromide (perflubron), which is emulsified with egg yolk phospholipid (lecithin). 63 The perflubron emulsion, named Oxygent, has a much higher capacity for oxygen than did Fluosol-DA. In safety trials it was shown to be well tolerated; the principal adverse effects were a mild, reversible thrombocytopenia and transient flu-like symptoms. 64 Oxygent s primary application is as an extender in acute normovolemic hemodilution in general and cardiac surgery, 65,66 although it also has been studied as a sensitizing agent for radiation therapy of solid tumors. 67 Phase III clinical trials had been discontinued but were cleared to resume in 2002 (Table 6). Alliance also had pursued other applications for perflubron. Perflubron has 1 bromine substituent, hence it is radiopaque. It has been studied as a contrast agent for several imaging applications and has been licensed, as Imagent GI, for use in gastrointestinal radiography. 68 Another formulation, Imagent US, was licensed in 2002 for use in cardiac ultrasound, capitalizing on its propensity to form stable microbubbles that are highly echogenic. 69 One particularly novel application that Alliance studied was partial liquid ventilation in which oxygenated perflubron was instilled, neat (ie, not as an emulsion), into the lungs of patients with acute respiratory distress syndrome (ARDS). The PFC was intended not only to deliver oxygen, but also to act as a surfactant to improve gas diffusion. Early results in infants with ARDS 70 were promising, but subsequent phase III trials in adults and children with ARDS were discontinued because of poor outcomes among patients receiving the PFC. Oxyfluor HemaGen/PFC produced an emulsion based on the PFC perfluorodichlorooctane that was stabilized with lecithin and safflower oil (triglycerides) and could be stored for 1 year at room temperature. 64 In safety trials, the adverse effect profile was similar to that seen with Oxygent. This product was studied for use as an RBC substitute in surgery, although its principal application was as a scavenger of the microbubbles that form in blood exposed to the oxygenation systems of cardiopulmonary bypass circuits. 71,72 These microemboli are thought to be responsible for some of the characteristic neuropsychiatric changes seen in many patients who have undergone cardiopulmonary bypass. 73,74 In model systems, Oxyfluor was found to protect animals from central nervous system damage when air was introduced into the carotid artery. 75 However, this product has not been studied actively for several years. S76 S76

7 Pathology Patterns Reviews Table 6 Perfluorocarbon RBC Substitutes Studied * Product (Manufacturer) Perfluorocarbon Trial Phase Application Fluosol-DA (Green Cross, Osaka, Japan/` Perfluorodecalin II (discontinued) Acute blood loss Alpha Therapeutic, Los Angeles, CA) Perfluoropropylamine Approved (withdrawn) Percutaneous transluminal coronary angioplasty Oxygent (Alliance Pharmaceutical, Perflubron II ANH during CABG; acute blood loss during San Diego, CA) orthopedic surgery III Cardiac surgery; ANH during surgery Imagent GI (Alliance Pharmaceutical) Perflubron Approved Gastrointestinal imaging Imagent US (Alliance Pharmaceutical) Perflubron Approved Cardiac ultrasound Liquivent (Alliance Pharmaceutical) Perflubron (neat) Ib/II Liquid ventilation for treatment of IRDS II/III (discontinued) Liquid ventilation for treatment of pediatric and adult ARDS Oxyfluor (HemaGen/PFC, Waltham, MA) Perfluorodichlorooctane II (discontinued) Surgery; neuroprotectant during bypass ANH, acute normovolemic hemodilution; ARDS, acute respiratory distress syndrome; CABG, coronary artery bypass graft; IRDS, infant respiratory distress syndrome. * Information current as of July Liposome-Encapsulated Hemoglobin Liposome-encapsulated hemoglobin (LEH) is the least well-developed class of blood substitutes, and these products have remained in the preclinical stage for a number of years. Theoretically, LEH could act like a miniature erythrocyte to transport and deliver oxygen, presumably in a manner similar to that of an RBC. LEHs eventually are cleared by the reticuloendothelial system. Currently, phosphatidylcholine is the phospholipid most commonly used to prepare the liposome bilayers that encapsulate the hemoglobin. 76 The potential advantages of LEH include a somewhat longer half-life and lower toxicity. In addition, the ability to include or omit 2,3-BPG from the encapsulated hemoglobin solution offers the opportunity to modify the P 50 to suit the application. The major limitation of LEH is that it is technically challenging to prepare liposomes that are uniform in size on a large scale. Another matter of concern is that the clearance of large amounts of phospholipids such as phosphatidylcholine may induce adverse effects. 77 Potential Applications of Blood Substitutes Clinical Applications: A Summary Although these oxygen carriers originally were conceived of as alternatives to banked blood, their unique characteristics suit them for applications beyond the scope of the RBC. The variety of these potential applications is indicated in Table 7. Blood substitutes are most likely to be useful in clinical settings in which a long-term need for volume and oxygen supplementation is not expected, such as in trauma or during the perioperative period (Table 7). However, they also may be useful when it may be difficult to find compatible blood as, for example, in patients with rare blood types, multiple alloantibodies, or RBC autoantibodies. Some substitutes may be acceptable to patients with religious objections to receiving banked human blood. Although the blood supply in most developed countries is quite safe, the same is not necessarily true in many developing nations that lack the resources to support the infrastructure required to maintain a safe supply of donated blood, particularly those with endemic transfusion-transmitted diseases (eg, malaria, Chagas, hepatitis B, HIV). In the perioperative setting, a blood substitute may be superior to crystalloid or colloid solutions for volume replacement or for intraoperative acute normovolemic hemodilution because of its greater capacity for oxygen. In cardiothoracic surgery, blood substitutes may be used for priming the bypass pump, replacing the use of other solutions with low oxygen solubility. Table 7 Applications for RBC Substitutes Acute blood loss due to trauma or surgery Acute blood loss in Jehovah s Witnesses or in cases of multiple RBC alloantibodies, rare blood type, endemic infection in donor blood supply Extender in acute normovolemic hemodilution Nitric oxide scavenger in septic shock Anti-ischemic therapy in sickle cell crisis, percutaneous transluminal coronary angioplasty, myocardial infarction, cardiopulmonary bypass, vaso-occlusive stroke Ex vivo organ or tissue preservation Neuroprotectant in cardiopulmonary bypass Sensitizer for chemotherapy and radiotherapy Imaging Partial liquid ventilation for acute respiratory distress syndrome, near drowning, smoke inhalation, infection Erythropoiesis S77 S77

8 Dong and Stowell / BLOOD SUBSTITUTES Because HBOC and PFC emulsion particles are much smaller than RBCs, they are able to diffuse into and deliver oxygen to poorly vascularized hypoxic tissues. As mentioned earlier, the FDA approved the use of the PFC emulsion Fluosol-DA in percutaneous transluminal coronary angioplasty. Other potential uses include situations in which enhanced oxygen perfusion of ischemic tissues is needed, such as in sickle cell crisis, in conjunction with thrombolytic therapy for myocardial infarction and stroke, and in peripheral vascular disease. Some oxygen carriers are undergoing clinical trials as sensitizers for radiotherapy and chemotherapy in patients with solid tumors. Other applications capitalize on the fact that some cellfree hemoglobin preparations are efficient NO scavengers. Therefore, clinical trials have been conducted to test the vasopressor effects of several hemoglobin-based solutions in patients with shock. The ability of PFC emulsions to dissolve gases was exploited to prevent the neuropsychiatric complications of cardiopulmonary bypass attributed to gaseous microemboli. Effects on Blood Transfusion Practices With an increasing number of elderly people and a decreasing number of potential blood donors, it has been estimated that there could be a shortfall of as many as 4 million U of RBCs per year by the year Substitutes based on PFCs, bovine hemoglobin, and, conceivably, recombinant hemoglobin may mitigate this projected shortage; substitutes based on outdated units of human RBCs obviously will have a limited effect. A key factor affecting the implementation of blood substitutes will be their safety; specifically, the adverse effects of the substitutes will have to be less than the risks associated with the transfusion of banked blood, which, at this point, are extremely low. 79 Another important factor will be their cost relative to that of conventional blood products. As recent experience with solvent-detergent treated plasma has shown, the market for blood components is not completely insensitive to costs. A very expensive product that offers only a small increase in safety may not be readily adopted, as happened with solvent-detergent treated plasma. Finally, the blood substitutes will have to compete against other technologies for improving the safety of the blood supply, most notably pathogen inactivation. Blood substitutes are not just like blood. Appropriate clinical use of blood substitutes will require substantial knowledge of the oxygen transport and delivery characteristics of the products, as well as an understanding of the limitations and potential toxic effects of each product. In addition, implementation of blood substitutes will impose new analytic challenges for the laboratory medicine community, because transfusion recipients will have the equivalent of hemolyzed or lipemic plasma, which may interfere with some clinical laboratory assays. From the 1 Department of Pathology and 2 Blood Transfusion Service, Massachusetts General Hospital, Harvard Medical School, Boston. Address reprint requests to Dr Stowell: MGH Blood Transfusion Service, 55 Fruit St, GRJ 212, Boston, MA References 1. Winslow RM. Blood substitutes, a moving target. Nat Med. 1995;1: Scott MG, Kucik DF, Goodnough LT, et al. Blood substitutes: evolution and future applications. Clin Chem. 1997;43: Stowell CP, Levin J, Spiess BD, et al. Progress in the development of red blood cell substitutes. Transfusion. 2001;41: Winslow RM. Blood substitutes. Curr Opin Hematol. 2002;9: Stowell CP. Hemoglobin based oxygen carriers. Curr Opin Hematol. In press. 6. Savitsky JP, Doczi J, Black J, et al. A clinical safety trial of stroma-free hemoglobin. Clin Pharmacol Ther. 1978;23: Swan SK, Halstenson CE, Collins AJ, et al. Pharmacologic profile of diaspirin cross-linked hemoglobin in hemodialysis patients. Am J Kidney Dis. 1995;26: Przybelski RJ, Dailey EK, Kisicki JC, et al. Phase I study of the safety and pharmacologic effects of diaspirin cross-linked hemoglobin solution. Crit Care Med. 1996;24: Rioux F, Drapeau G, Marceau F. Recombinant human hemoglobin (rhb1.1) selectively inhibits vasorelaxation elicited by nitric oxide donors in rabbit isolated aortic rings. J Cardiovasc Pharmacol. 1995;25: Ritchie AJ, Hartshorn S, Corsbie AE, et al. The action of diaspirin cross-linked haemoglobin blood substitutes on human arterial bypass conduits. Eur J Cardiothorac Surg. 2000;18: Pawloski JR, Hess DT, Stamler JS. Export by red blood cells of nitric acid bioactivity. Nature. 2001;409: Gulati A, Rebello S. Role of adrenergic mechanisms in the pressor effect of diaspirin cross-linked hemoglobin. J Lab Clin Med. 1994;124: Gulati A, Singh S, Rebello S, et al. Effect of diaspirin crosslinked and stroma-reduced hemoglobin on mean arterial pressure and endothelin-1 concentration in rats. Life Sci. 1995;56: Saxena R, Wijnhoud AD, Man in t Veld AJ, et al. Effect of diaspirin cross-linked hemoglobin on endothelin-1 and blood pressure in acute ischemic stroke in man. J Hypertens. 1998;16: Karmaker N, Dhar P. Effect of steady shear stress on fluid filtration through the rabbit arterial wall in the presence of macromolecules. Clin Exp Pharmacol Physiol. 1996;23: Gulati A, Sharma AC, Burhop KE. Effect of stroma-free hemoglobin and diaspirin cross-linked hemoglobin on the regional circulation and systemic hemodynamics. Life Sci. 1994;55: S78 S78

9 Pathology Patterns Reviews 17. Ulatowski JA, Nishikawa T, Matheson-Urbaitis B, et al. Regional blood flow alterations after bovine fumaryl betabetacrosslinked hemoglobin transfusion and nitric oxide synthase inhibition. Crit Care Med. 1996;24: Caron A, Malfatti E, Aguejouf O, et al. Vasoconstrictive response of rat mesenteric arterioles following infusion of cross-linked, polymerized, and conjugated hemoglobin solutions. Artif Cells Blood Substit Immobil Biotechnol. 2001;29: McCarthy MR, Vandegriff KD, Winslow RM. The role of facilitated diffusion in oxygen transport by cell-free hemoglobins: implications for the design of hemoglobin-based oxygen carriers. Biophys Chem. 2001;92: Garrioch MA, McClure JH, Wildsmith JA. Haemodynamic effects of diaspirin crosslinked haemoglobin (DCLHb) given before abdominal aortic aneurysm surgery. Br J Anaesth. 1999;83: Reah G, Bodenham AR, Mallick A, et al. Initial evaluation of diaspirin cross-linked hemoglobin (DCLHb) as a vasopressor in critically ill patients. Crit Care Med. 1997;25: Yabuki A, Matsushita S, Malchesky PS, et al. In vitro evaluation of a pyridoxalated hemoglobin polyoxyethylene conjugate in reversing cell sickling. ASAIO Trans. 1988;34: Marik PE, Sibbald WJ. Effect of stored-blood transfusion on oxygen delivery in patients with sepsis. JAMA. 1993;269: Rohlfs RJ, Bruner E, Chiu A, et al. Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide. J Biol Chem. 1998;273: Vandegriff KD, Medina F, Marini MA, et al. Equilibrium oxygen binding to human hemoglobin cross-linked between the alpha chains by bis(3,5-dibromosalicyl) fumarate. J Biol Chem. 1989;264: Seghal LR, Rosen AL, Noud G, et al. Large volume preparation of pyridoxylated hemoglobin with high P 50. J Surg Res. 1981;30: Iwashita Y, Yabuki A, Yamaji K, et al. A new resuscitation fluid stabilized hemoglobin preparation and characteristics. Biomater Artif Cells Artif Organs. 1988;16: Winslow RM, Vandegriff KD. Hemoglobin oxygen affinity and the design of red cell substitutes. In: Winslow RM, Vandegriff KD, Intaglietta M, eds. Advances in Blood Substitutes: Industrial Opportunities and Medical Challenges. Boston, MA: Birkhäuser; 1997: Intaglietta M. Microcirculatory basis for the design of artificial blood. Microcirculation. 1999;6: Estep TN, Gonder J, Bornstein I, et al. Immunogenicity of diasprin cross-linked human hemoglobin solutions. Biomater Artif Cells Immobilization Biotechnol. 1992;20: Chatterjee R, Welty EV, Walder TY, et al. Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99alpha1-99alpha2). J Biol Chem. 1986;26: Burton TM. Baxter suspends trial of blood substitute. Wall Street Journal. June 3, 1998:B Saxena R, Wijnhoud AD, Carton H, et al. Controlled safety study of a hemoglobin-based oxygen carrier, DCLHb, in acute ischemic stroke. Stroke. 1999;30: Sloan EP, Koenigsberg M, Gens D, et al. Diaspirin crosslinked hemoglobin (DCHLb) in the treatment of severe traumatic hemorrhagic shock: a randomized controlled efficacy trial. JAMA. 1999;282: Lamy ML, Dailey EK, Brichant JF, et al. Randomized trial of diaspirin cross-linked hemoglobin solution as an alternative to blood transfusion after cardiac surgery. Anesthesiology. 2000;92: Sloan EP, Koenigsberg M, Brunett PH, et al. Post hoc mortality analysis of the efficacy trail of diaspirin cross-linked hemoglobin in the treatment of severe traumatic hemorrhagic shock. J Trauma. 2002;52: Looker D, Abbott-Brown D, Cozart P, et al. A human recombinant hemoglobin designed for use as a blood substitute. Nature. 1992;356: Murray JA, Ledlow A, Launspach J, et al. The effects of recombinant human hemoglobin on esophageal motor functions in humans. Gastroenterology. 1995;109: Sehgal SA, Gould LR, Rowen AL, et al. Polymerized pyridoxylated hemoglobin: a red cell substitute with normal O 2 capacity. Surgery. 1984;95: Gould SA, Moore EE, Hoyt DB, et al. The first randomized trial of human polymerized hemoglobin as a blood substitute in acute trauma and emergent surgery. J Am Coll Surg. 1998;187: Gould SA, Moore EE, Moore FA, et al. Clinical utility of human polymerized hemoglobin as a blood substitute following acute trauma and urgent surgery. J Trauma. 1997;43: Moore EE, Gould SA, Hoyt DB, et al. Clinical utility of human polymerized hemoglobin as a blood substitute following trauma and emergent surgery [abstract]. Shock. 1997;7(S1): Cothren C, Moore EE, Offner PJ, et al. Blood substitute and erythropoietin therapy in a severely injured Jehovah s Witness. N Engl J Med. 2002;346: Sprung J, Kindscher JD, Wahr JA, et al. The use of bovine hemoglobin glutamer-250 (Hemopure) in surgical patients: results of a multicenter, randomized single-blinded trial. Anesth Analg. 2002;94: LaMuraglia GM, O Hara PJ, Baker WH, et al. The reduction of allogeneic transfusion requirement in aortic surgery with hemoglobin-based solution. J Vasc Surg. 2000;31: Kasper SM, Grune F, Walter M, et al. Effects of increased doses of bovine hemoglobin on hemodynamics and oxygen transport in patients undergoing preoperative hemodilution for elective abdominal aortic surgery. Anesth Analg. 1998;87: Standl T, Wilhelm S, Horn EP, et al. Präoperative Hämodilution mit bovinem Hämoglobin: Akute hämodynamische Auswirkungen bei Patienten in der Leberchirurgie. Anaesthesist. 1997;46: Hughes GS, Yancey EP, Albrecht R, et al. Hemoglobin-based oxygen carrier preserves submaximal exercise capacity in humans. Clin Pharmacol Ther. 1995;58: Mullon J, Giacoppe G, Clagett C, et al. Transfusions of polymerized bovine hemoglobin in a patient with severe autoimmune hemolytic anemia. N Engl J Med. 2000;342: Adamson JG, Bonaventura BJ, Song SE, et al. Production, characterization, and clinical evaluation of Hemolink, an oxidized raffinose cross-linked hemoglobin-based blood substitute. In: Rudolph AS, Rabinovici R, Feuerstein GZ, eds. Red Blood Cell Substitutes. New York, NY: Marcel Dekker; 1998: Carmichael FJL, Ali ACY, Campbell JA, et al. A phase I study of oxidized raffinose cross-linked human hemoglobin. Crit Care Med. 2000;28: S79 S79

10 Dong and Stowell / BLOOD SUBSTITUTES 52. Cheng DCH. Safety and efficacy of o-raffinose cross-linked human hemoglobin (Hemolink) in cardiac surgery. Can J Anaesth. 2001;48(4 suppl):s41-s Conover CD, Malatesta P, Lejeune L, et al. The effects of hemodilution with polyethylene glycol bovine hemoglobin (PEG-Hb) in a conscious porcine model. J Investig Med. 1996;44: Nho K, Linberg R, Johnson M, et al. PEG-hemoglobin: an efficient oxygen delivery system in the rat exchange transfusion and hypovolemic shock models. Artif Cells Blood Substit Immobil Biotechnol. 1994;22: Robinson MF, Dupuis NP, Kusumoto T, et al. Increased tumor oxygenation and radiation sensitivity in two rat tumors by a hemoglobin-based, oxygen-carrying preparation. Artif Cells Blood Substit Immobil Biotechnol. 1995;23: de Figueiredo LF, Cruz RJ, Neto AC, et al. Initial management of severe hemorrhage with an oxygen-carrying hypertonic saline solution. Artif Organs. 2001;25: Clark LC, Gollan F. Survival of mammals breathing liquids equilibrated with oxygen at atmospheric pressure. Science. 1966;152: Spence RK. Perfluorocarbons in the twenty first century: clinical applications as transfusion alternatives. Artif Cells Blood Substit Immobil Biotechnol. 1995;23: Keipert PE, Otto S, Flaim SF, et al. Influence of perflubron emulsion particle size on blood half-life and febrile response in rats. Artif Cells Blood Substit Immobil Biotechnol. 1994;22: Gould SA, Rosen A, Sehgal L, et al. Fluosol-DA as a red cell substitute in acute anemia. N Engl J Med. 1986;314: Kerins DM. Role of perfluorocarbon Fluosol-DA in coronary angioplasty. Am J Med Sci. 1994;307: Williams GA, Kent SP. Specific identification of hemoglobin and myoglobin in renal tubular casts by the fluorescent antibody technic on fixed embedded tissues. Am J Clin Pathol. 1981;75: Flaim SF. Perflubron-based emulsion: efficacy as temporary oxygen carrier. In: Winslow R, Vandegriff K, Intaglietta M, eds. Advances in Blood Substitutes: Industrial Opportunities and Medical Challenges. Boston, MA: Birkhäuser; 1997: Kaufman RJ. Clinical development of perfluorocarbon-based emulsions as red cell substitutes. In: Winslow RM, Vandegriff KD, Intaglietta M, eds. Blood Substitutes: Physiological Basis of Efficacy. Boston, MA: Birkhäuser; 1995: Wahr JA, Trouwborst A, Spence RK, et al. A pilot study of the effects of perflubron emulsion, AF0104, on mixed venous oxygen tension in anesthetized surgical patients. Anesth Analg. 1996;82: Stern SA, Dronen SC, McGoron AJ. Effect of supplemental perfluorocarbon administration on hypotensive resuscitation of severe uncontrolled hemorrhage. Am J Emerg Med. 1995;13: Rockwell S, Kelley M, Irvin CG, et al. Preclinical evaluation of Oxygent as an adjunct to radiotherapy. Biomater Artif Cells Immobilization Biotechnol. 1992;20: Rubin DL, Muller HH, Nino-Murcia M, et al. Intraluminal contrast enhancement and MR visualization of the bowel wall: efficacy of PFOB. Radiology. 1991;22: Andre M, Nelson T, Mattrey R. Physical and acoustical properties of perfluorooctyl-bromide, an ultrasound contrast agent. Invest Radiol. 1990;25: Leach CL, Greenspan JS, Rubenstein DS, et al. Partial liquid ventilation with perflubron in premature infants with severe respiratory distress syndrome. N Engl J Med. 1996;335: Blauth CI, Arnold JV, Schulenberg WE, et al. Cerebral microembolism during cardiopulmonary bypass: retinal microvascular studies in vivo with fluorescein angiography. J Thorac Cardiovasc Surg. 1988;95: Blauth CI, Smith P, Newman S, et al. Retinal microembolism and neuropsychological deficit following clinical cardiopulmonary bypass: comparison of a membrane and a bubble oxygenator: a preliminary communication. Eur J Cardiothorac Surg. 1989;3: Shaw PJ, Bates D, Cartlidge EF, et al. Neurologic and neuropsychological morbidity following major surgery: comparison of coronary artery bypass and peripheral vascular surgery. Stroke. 1987;18: Roach GW, Kanchugar M, Mangano CM, et al. Adverse cerebral outcomes after coronary artery bypass surgery. N Engl J Med. 1996;335: Cochran RP, Kunzelman KS, Vocelka CR, et al. Perfluorocarbon emulsion in the cardiopulmonary bypass prime reduces neurologic injury. Ann Thorac Surg. 1997;63: Rabinovici R, Rudolph AS, Vernick J, et al. Lyophilized liposome encapsulated hemoglobin: evaluation of hemodynamic, biochemical and hematological responses. Crit Care Med. 1994;22: Rudolph AS, Cliff RO, Spargo BJ, et al. Transient changes in the mononuclear phagocyte system following administration of the blood substitute, liposome-encapsulated hemoglobin. Biomaterials. 1994;15: Marwick C. More than a trickle of interest in blood substitutes [editorial]. JAMA. 1994;271: Goodenough LT, Brecher ME, Kanter MH, et al. Transfusion medicine. N Engl J Med. 1999;340: S80 S80