By the late 1800s efforts were being made to find a parenteral solution for treating anemia [1-3]. Interest in such solutions intensified as a result of World War I, when hypovolemia and hemorrhage became more widely appreciated as causes of "circulatory shock" [4-5]. Although no oxygen-carrying, volume-expanding solution was identified, clinical experience suggested that shock was caused by hypovolemia and could be reversed by administration of various fluids intravascularly [6]. Subsequent clinical experience and laboratory investigations have confirmed the utility of crystalloids, colloids, and/or blood administration in treating hemorrhagic shock, although the optimal timing of such treatment remains controversial [6-9]. The term "blood substitutes" has been used to broadly describe oxygen-carrying, volume-expanding solutions. However, blood performs a host of functions beyond the transport of oxygen whereas the goal of blood substitutes is to transport oxygen and expand blood volume [10]. A more accurate definition for blood substitutes is "oxygen-carrying volume expanders." Oxygen-carrying volume expanders have long been attractive to military medical organizations faced with the logistic constraints of the battlefield. An oxygen-carrying volume expander that is easy to store, transport, and administer could be life-saving for injured soldiers [10,11]. Concern over fatal blood-borne pathogens, including hepatitis and human immunodeficiency virus (HIV), also make oxygencarrying volume expanders attractive for civilian use [10,12]. Blood substitutes may be particularly useful in the next several decades. The United States blood supply faces major challenges associated with an aging population and potentially inadequate rates of volunteer donation by healthy citizens. Approximately 14 million units of whole blood are collected in the United States each year, and there are roughly 12 million transfusions of red blood cells (RBCs) [13-16]. Half of these RBCs are transfused to patients 65 yr or older, although this age group represents only 12.5% of the population Figure 1. It is estimated that by the year 2030, approximately 22% of the population will be elderly (65 yr of age or older), and the absolute number of this group will more than double [17]. At present rates of RBC utilization, the elderly population alone will require 12-13 million units of red cells per year by the year 2030 [13]. If present donation patterns in the United States remain constant, a shortfall of 4 million units of RBCs is projected in 2030. In this context, a safe and effective blood substitute could be useful to meet this projected shortfall in the red blood cell supply.Figure 1: The potential effects of aging on the future demand for red blood cells (RBCs) in the United States. In 1990, approximately 12 million units of RBCs were transfused. Half of these units were transfused to the elderly (age >or=to65 yr). In the year 2030, it is estimated that the total demand for RBCs will exceed 19 million units annually, with over 12 million of these units administered to the elderly. [Data from Vamvakas and Taswell 1994 [13].]A variety of substances that transport oxygen and augment intravascular volume in the absence of red cells are emerging as possible blood substitutes for use in humans. These include hemoglobin solutions, liposome-encapsulated hemoglobin (LEH), and perfluorocarbons [10]. None of these compounds replace the coagulant or immunologic functions of blood products, and all have a limited circulatory half-life in comparison to RBC transfusion. Since 60%-70% of RBC transfusions occur in surgical patients during the perioperative period [18], it is important that basic issues related to the emerging field of oxygen-carrying volume expanders be understood by anesthesiologists. The purpose of this review is to discuss issues related to the various oxygen-carrying, volume-expanding solutions by addressing the following questions: 1. What is the need for blood substitutes in clinical transfusion practice? 2. What are the ideal properties of an oxygen-carrying volume expander? 3. What types of oxygen-carrying solutions are under development? 4. What are the potential clinical applications of these products? 5. What is the ultimate definition of their efficacy? Need for Blood Substitutes Blood substitutes may be used to address three basic issues in transfusion medicine. First, RBCs can be difficult to store and transport, and their safe administration requires laboratory screening for donorrecipient compatibility and infectious disease. A product with a long shelf-life that is universally compatible could solve many of these problems. Second, despite rigorous screening for infectious disease, there is modest (1:3000) risk of hepatitis C transmission and a smaller (1:100,000 to 1:1,000,000) risk of HIV transmission per unit transfused [19-24]. Blood transfusion can also result in bacterial, parasitic, and other viral disease transmission, but the risk of clinically significant disease from these is low [25]. The risk of infectious disease transmission could be further minimized or eliminated with a safe blood substitute. Third, inadequate donation of blood by healthy citizens in the context of an aging population is already causing significant shortages of RBCs in some locales [26]. These local shortages could potentially be eliminated by an oxygen-carrying, volume-expanding solution that has a long intravascular half-life. Even if an ideal oxygen-transporting, volume-expanding solution was developed, there will still be a need for blood components, such as platelets, fresh frozen plasma, and cryoprecipitate. This conceivably could lead to a situation where whole blood was collected to harvest these components and the RBCs were subsequently wasted. Increased blood donation by volunteers may be the simplest and most cost-effective solution to the supply problem [13]. Ideal Properties As a result of a variety of factors, RBC transfusions have never undergone a classic broad-based clinical trial designed to demonstrate their efficacy. Nonetheless, RBC transfusions are widely used and they are the standard to which oxygen-carrying volume-expanding solutions are compared. RBCs are usually administered in a "packed" form that consists of approximately 225 mL volume with a hematocrit of 70%-80% (i.e., 25 g/dL of hemoglobin) [27]. Seventy percent of the red cells in a unit of packed RBCs will survive in the circulation for more than 24 h after transfusion [27]. The surviving cells are then assumed to have a life-span similar to native blood (i.e., weeks to months). Important properties of RBCs include: 1) high oxygen-carrying capacity; 2) ability to transport oxygen when oxygen tension is in the normal physiologic range; 3) desirable elimination characteristics; and 4) low incidence of side effects when appropriately screened and administered. Undesirable properties of RBCs include: 1) relatively short shelf-life (i.e., weeks) in most forms; 2) antigenicity requiring pretransfusion testing of donors and recipients; 3) transfusion reactions; 4) dependence on a limited donor pool; 5) infectious disease transmission; and 6) suppression of the normal immune system [10,28,29]. Although the oxygen-carrying volume expanders under development have few of the undesirable characteristics associated with RBCs, none has all of the desirable properties. With most products currently being developed, there are concerns related to short intravascular half-lives, routes of elimination, physiologic side effects (e.g., hypertension), and interactions with coexisting diseases. As blood substitutes emerge it should be emphasized that the current blood supply is safe in comparison to many forms of medical or industrial technologies. For example, the risk of dying in a fatal automobile accident in the United States is approximately 0.002% annually [30]. The overall yearly risk of dying from an allogeneic blood transfusion is several orders of magnitude less at approximately 0.0001% or lower [19,22,27]. Therefore, the overall safety of any RBC substitute will have to be very high to be as safe as allogeneic blood. In addition, the cost of these solutions will also have to compare favorably with that of RBCs. It is currently estimated that the hospital acquisition cost of one unit of RBCs is approximately $52-$64 [31,32]. Types of Oxygen-Carrying Volume Expanders Several types of products are under development as potential oxygen-carrying volume expanders [12]. These products are classified in one of three categories: 1) hemoglobin solutions, containing some modification of the hemoglobin molecule; 2) liposome-encapsulated hemoglobin solutions, containing hemoglobin within a synthetic membrane; 3) perfluorocarbons, organic solutions with high oxygen solubility. Hemoglobin Solutions In the 1930s Amberson began the first systematic investigation of an intravenous bovine hemoglobin-insaline solution as an oxygen-carrying volume expander in several animal species [33]. Animals survived for up to 36 h after exchange transfusions, with death due to hemoglobin loss from the circulation. In the initial hours after the exchange transfusions, oxygen consumption remained normal and arterial hypertension was observed. Subsequently, Amberson et al. [34] used a human hemoglobin-insaline solution a total of 77 times in 14 patients with anemia. Results included restoration of blood volume, increased oxygen-carrying capacity, and stimulation of hematopoiesis as evidenced by an increased reticulocyte count. When a modest volume was given to a patient suffering from acute hemorrhagic shock as a result of obstetrical bleeding, prompt restoration of arterial blood pressure was observed. Side effects reported in these patients included renal dysfunction and an acute increase in mean arterial pressure above normal values. When hemoglobin is liberated from human RBCs, its tetrameric structure of two alpha and two beta chains dissociates into dimers consisting of alpha and beta hemoglobin chains or hemoglobin monomers [35]. This dissociation decreases the molecular weight from approximately 64 kd per tetramer to 32 kd per dimer or 16 kd per monomer. With this smaller size, the dimer is filtered by the kidneys, reducing its intravascular retention time [36-38]. Additionally, the oxygen-binding cooperativity of hemoglobin in this dimeric form is lost, 2,3-diphosphoglycerate (2,3-DPG) is no longer a major regulator of the oxygen-hemoglobin dissociation curve, and the curve is shifted to the left [38]. This left shift causes P50 to decrease to a PO2 of 12-16 mm Hg and limits oxygen unloading at the tissues [38]. Renal damage occurs when filtered hemoglobin precipitates in the acidic ascending limb of the loop of Henle [36,39]. Red cell stromal debris remaining in the hemoglobin solution as a result of inadequate purification may also contribute to renal damage and systemic toxicity due to activation of the complement cascade [40,41]. Removal of red blood cell stroma from hemoglobin solutions reduces these side effects [41,42]. Four general classes of stroma-free hemoglobin solutions are under development at present. They are 1) intramolecularly cross-linked hemoglobin, 2) polymerized hemoglobin, 3) conjugated hemoglobin, and 4) the newly emerging hemoglobin microbubbles [43-47]. All four solutions contain a modified hemoglobin molecule. These modifications are used to increase molecular size and decrease renal filtration, prolong intravascular persistence, and to ensure a normal P50 of hemoglobin. Figure 2 is a schematic representation of some concepts related to hemoglobin solutions currently under development.Figure 2: Schematic representation of hemoglobin-based blood substitutes now under development. Animal, human, or genetically engineered (microorganism produced) sources of hemoglobin are used. The hemoglobin is then separated from either the red blood cells or microorganism. If the source of hemoglobin is animal or human, hemoglobin dimers and monomers result. These dimers and monomers are associated with a reduced P50, renal filtration, and a very short plasma half-life. To stabilize the smaller hemoglobin units obtained from animal or human red cells, these hemoglobin dimers and monomers are modified by either cross-linking, polymerization, or conjugation. The hemoglobin obtained from microorganisms is cross-linked during the synthetic process in the microorganism. The resultant cross-linked, polymerized, or conjugated hemoglobins have P50 values in the physiologic range, are subject to significantly less renal filtration, and also have a prolonged plasma half-life (h). Products based on these schemes represent the currently emerging hemoglobin-based blood substitutes. Such products are undergoing testing in animals and some are nearing trials in humans. In the future it is likely that various combinations of hemoglobin tetramers (known as oligomers) or liposome-encapsulated hemoglobin will be developed. One potentially attractive feature of such products would be a dramatically increased plasma half-life. The newly developed hemoglobin microbubbles (not represented) are an example of one of these products. Although not tested yet in animals or humans, the microbubbles theoretically will have an increased intravascular retention time due to their larger size (one-half that of an erythrocyte). Each of the basic forms of hemoglobin-based blood substitutes has been demonstrated to transport oxygen, sustain life during severe anemia, and be effective as a volume resuscitation fluid. However, it is unclear whether such products can be used in humans in a way that decreases the use of blood products, or significantly alters transfusion-related morbidity and mortality. Hgb = hemoglobin; RBC = red blood cells. (For discussion of issues related to the ultimate efficacy of blood substitutes, please see Figure 6).With cross-linked hemoglobin, the tetrameric structure of hemoglobin is maintained by an intramolecular cross-link between the alpha or beta hemoglobin chains. For example, when two alpha chains are cross-linked, the two beta chains remain associated with them on the basis of weak chemical forces [35,48]. Several cross-linking strategies are now under commercial development. These include creation of chemical bonds between hemoglobin chains [12,49-52] and use of genetic engineering to create one or more molecular bridges between two alpha chains that are synthesized in micro-organisms [53-55]. Polymerized hemoglobin solutions contain either oligomers of cross-linked hemoglobin or polymers of hemoglobin chains [45,56-58]. Conjugated hemoglobin is formed by linking free hemoglobin to soluble nonhemoglobin polymers [59-62]. In one strategy currently under development, bovine hemoglobin is conjugated with polyethylene glycol [46,63]. Solutions containing cross-linked hemoglobin, polymerized hemoglobin, and conjugated hemoglobin sustain life during exchange transfusions that eliminate almost all of the experimental animals native RBCs [44-46]. Several of these solutions have undergone extensive animal testing and are entering early safety trials in humans. Hemoglobin microspheres are a much more recent development that shows promise [47]. This technology uses high-intensity ultrasound to form microbubbles which have shells consisting of approximately one million hemoglobin molecules which are chemically cross-linked by superoxide formed during the sonication process. Characterization of these microspheres shows an oxygen-carrying capacity (0.32 mL of O2/mL solution) greater than that of blood, oxygen affinities similar to those of native hemoglobin, and minimal degradation in solution after storage for 6 mo at 4 degrees C. Development of hemoglobin microbubbles is in very early stages compared to the other types of hemoglobin solutions, and no animal or human studies have yet been conducted. Sources of Hemoglobin. If hemoglobin solutions were used to replace the oxygen-carrying capacity of 10% of the packed RBCs transfused annually in the United States, 60,000-70,000 kg of hemoglobin would be required each year [19,64]. Hemoglobin-based blood substitutes currently under development use human, animal, and biotechnologic sources of hemoglobin. There are potential logistic and safety problems associated with each of these sources. Several products under development use human hemoglobin derived from outdated banked blood [52,65,66]. With a shrinking donor pool in the United States and better inventory control in most blood banks, fewer units are becoming outdated. Therefore, it seems unlikely that outdated banked blood could provide enough hemoglobin to replace 10% of that volume now provided by RBCs unless there was substantial recruitment of new donors. A greater pool of hemoglobin might be available if it was liberated from donor RBCs as a processing step in blood component harvest from whole blood. In this concept, the hemoglobin in red cells would be viewed as a separate component of whole blood similar to platelets, fresh frozen plasma, cryoprecipitate, and albumin. This strategy would allow a shift of hemoglobin from relatively abundant types of red cells (i.e., A) to universally compatible forms. Unfortunately, since the half-life of solutions made from human-derived hemoglobin is short (i.e., hours), the need for red cell transfusion may merely be delayed and not eliminated by its use. Such solutions might be useful in an acute intraoperative hemorrhage, but RBCs would probably still be required in the postoperative period. If this occurred, the patient would be exposed to the risks and cost of both blood substitute administration and RBC transfusion. There would also be loss of some hemoglobin in the harvest, processing, and production steps in this strategy. Therefore, several questions remain about the utility and practicality of this approach. Just as the first hemoglobin solutions used bovine hemoglobin, so do several solutions currently under development [45,46,67]. The oxygen affinity (P50) of bovine hemoglobin is similar to human hemoglobin and is not controlled by 2,3-DPG but instead by chloride ion which is present in large concentrations of the plasma [68-70]. When human hemoglobin is removed from red cells, the effect of 2,3-DPG on the oxygen/hemoglobin dissociation is lost. However, since the oxygen/dissociation curve of bovine hemoglobin is regulated primarily by chloride ions, its dissociation curve can shift normally outside of red cells. There is also a more pronounced Bohr effect in bovine hemoglobin, resulting in enhanced delivery of oxygen to tissues at a lower pH [71]. Another attractive feature of bovine hemoglobin is its abundance. A 500-kg steer has approximately 35 L of blood containing approximately 12 g/dL of hemoglobin for an approximate total body hemoglobin content of 4.2 kg. A herd of 20,000 cattle could be routinely phlebotomized to provide the hemoglobin needed for a substantial amount of hemoglobin-containing solutions [64]. However, governmental regulatory agencies in several countries are concerned about the possibility of interspecies transmission of infectious disease, such as bovine spongiform encephalitis. The potential for interspecies disease transmission is poorly understood. In this context, it should be noted that hemoglobin can be successfully purified from human RBC units containing the HIV virus [72]. Recombinant DNA technology has been used to produce modified human hemoglobin molecules in Escherichia coli and Sarcomyces cerevisiae [53-55,73,74]. Unfortunately, it is unclear whether the yield of hemoglobin per unit of microorganism is sufficient to make large scale commercial production of hemoglobin possible. There are also concerns about complete separation of bacterial components from the hemoglobin and waste management of the byproducts of its production [55]. Another biotechnologic approach to producing large amounts of hemoglobin involves transgenic manipulation of animals to produce RBCs that contain a substantial proportion of human hemoglobin [75,76]. This approach has been attempted in pigs, and approximately 50% human hemoglobin has been produced in these animals. This approach is noteworthy because it could provide an abundant source of human hemoglobin. Physiologic Effects. When animals are given hemoglobin solutions in exchange transfusions, oxygen consumption and carbon dioxide production do not change, cardiac output remains the same, and increases in mean arterial pressure, pulmonary artery pressure, and systemic vascular resistance are seen Figure 3[33,44,45,77-79]. Long-term survival (7-30 days) is possible after partial and complete exchange transfusion with hemoglobin solutions [79-82]. After complete exchange transfusion, there is regeneration of native red blood cells. In these cases the iron made available by the hemoglobin solution may aid hematopoiesis and speed the return of RBCs.Figure 3: Hemodynamic impact of exchange transfusion of alpha-alpha cross-linked hemoglobin in a conscious swine model. In this experiment chronically instrumented swine underwent a total exchange transfusion during which their native blood volume was replaced with a cross-linked hemoglobin solution. The top panel demonstrates the time course of red blood cell (RBC) hemoglobin loss from the circulation along with the increase of free hemoglobin associated with the exchange transfusion. The second panel demonstrates that the exchange transfusion was associated with an increase in both systolic and diastolic blood pressures. This increase was highly significant (P < 0.05) and observed in the 28 animals studied. The bottom two panels demonstrate that the hypertension observed during the exchange transfusion was not associated with marked changes in cardiac output or heart rate. [Figure adaptedfrom Hess et al. [84].]In animal models of hypovolemic, hypotensive hemorrhagic shock, hemoglobin solutions restore circulating blood volume and provide adequate tissue oxygenation [46,83,84]. The use of hemoglobin-containing solutions compared to crystalloid or colloid solutions has been associated with improved survival when animals have been subjected to acute blood loss and resuscitated [65,85]. Several studies have suggested that oxygen delivery is similar with transfusion of hemoglobin solutions and RBCs [45,78,83,86]. However, these experimental models used venous oxygen saturation as an indicator of end-organ oxygenation. Recent studies by Winslow et al. [87] that directly measured tissue oxygen content with indwelling electrodes, suggest that tissue oxygenation can be significantly lower after transfusion of hemoglobin solutions compared to RBCs despite similar mixed-venous oxygen saturations. These studies high-light the need for better physiologic indicators of actual oxygen delivery to tissues. Increases in mean arterial and pulmonary artery pressures and systemic and pulmonary vascular resistances have been reported with the infusion of most hemoglobin solutions [45,83-86,88]. Figure 4 demonstrates the time sequence of these changes in an anesthetized dog being transfused with alpha-alpha cross-linked hemoglobin. The increases in these pressures appear to be greater than expected from the restoration of normovolemia. This additional pressor effect may be related to the binding of nitric oxide (NO) by the free hemoglobin molecule [89-92]. NO is a potent vasodilator that is synthesized in and released by the vascular endothelium and nonadrenergic, noncholinergic vasodilator nerves [93,94]. There is continuous release of NO by vascular endothelium, and NO contributes to the maintenance of normal systemic and pulmonary arterial blood pressures [95]. Additionally, the hemoglobin molecule itself may possess vasoconstricting properties [96,97].Figure 4: Individual record of a barbiturate-anesthetized dog demonstrating the pressor effects of hemorrhage and transfusion with a cross-linked hemoglobin solution. Prior to baseline measurements the animals received systemic alpha and beta blockade so that the impact of changes in arterial pressure on reflex control in the circulation would be minimized. The dog's blood volume then was reduced by approximately one-third. This magnitude of hemorrhage caused marked reductions in both systemic and pulmonary artery pressures. Immediately after hemorrhage the animal was transfused over 1-2 min with an equal volume of cross-linked hemoglobin solution. This transfusion caused an immediate increase in systemic and pulmonary artery pressures. Five minutes after the replacement transfusion there was a further increase in systemic arterial pressure. Post XL Hgb = post cross-linked hemoglobin. (From Dietz et al. Unpublished observation.)The mild-to-moderate vasopressor effect associated with transfusion of hemoglobin solutions may be beneficial in the setting of hypovolemic shock and other clinical situations that usually warrant RBC transfusion. An increase in mean arterial pressure, in conjunction with decreased viscosity of hemoglobin solutions, may improve oxygen delivery by hemoglobin solutions in various vascular beds [98]. For example, in a rat model of brain ischemia, infarct size is reduced in the presence of a hemoglobin solution [99-101]. However, much more needs to be known about how this pressor effect interacts with common coexisting diseases frequently seen in older surgical patients (e.g., renal insufficiency, coronary artery disease, hypertension, etc.) [102] before this "side effect" can be considered beneficial. In animal models, transfusion with highly purified solutions of modified hemoglobin can be associated with mild transient increases in blood urea nitrogen and creatine [103]. Precipitation of trace amounts of free hemoglobin can be found in the kidneys [103]. The vasopressor effect seen with transfusion of hemoglobin solutions may affect renal blood flow and its distribution. Although no long-term renal damage has been seen in young, healthy animal models or healthy human volunteers, it is unclear whether these renal effects will also be mild and transient in older patients who have reduced baseline renal function [104]. Unmodified, purified human hemoglobin that is free from RBC cellular components has no apparent immunogenicity [65]. Hemoglobin that is modified by nonspecific cross-linking agents can, however, be antigenic under some circumstances [105,106]. After repeated exposure of dogs and rabbits to heterologous hemoglobin solutions, in conjunction with maneuvers designed to maximize their immune responses, intravenous administration of heterologous polyhemoglobin or stroma-free hemoglobin solutions resulted in antibody responses significantly greater than in control animals [105]. In some forms (e.g., conjugated), heterologous hemoglobin is much less immunogenic [107]. Feola et al. [67] reported that single large infusions of modified or unmodified bovine hemoglobin in rabbits produced no detectable antibodies. Other hemoglobins which have been tested for possible antigenicity include those of sheep, rabbits, and dogs [108]. These animal hemoglobins were found only weakly antigenic in the various species. Further studies are required to determine whether either single or repeated infusions of heterologous hemoglobin will evoke a clinically significant immune response in humans [109]. Like saline, hemoglobin and polyhemoglobin solutions have minimal direct effect on coagulation. In an animal model, replacement of 10% of the blood volume with hemoglobin solution (7 g/dL) did not significantly alter prothrombin time, partial thromboplastin time, factor chi, fibrinogen, antithrombin III, antiplasmin, or plasminogen when compared to saline control [110]. Hemoglobin solutions do not affect adenosine diphosphate-induced platelet aggregation or platelet activation [110,111]. Surprisingly, the effects of massive transfusions of hemoglobin solutions and concurrent administration of other blood products on coagulation profiles has not been studied extensively. Purified hemoglobin solutions have little effect on complement activation. There are no differences in C3 or C3a levels between plasma incubated with hemoglobin solutions and saline controls [112]. However, endotoxin and membrane stroma found in unpurified hemoglobin solutions significantly increase C3 and lower C3a[112]. Although purified stroma-free hemoglobin and polyhemoglobin do not activate complement, contaminants such as membrane stroma or endotoxin do activate the complement system. Biodistribution. Biodistribution studies of isotope-labeled modified hemoglobin show that modified hemoglobin is scavenged mainly by the reticuloendothelial system (RES) [66,104,113,114]. The long-term effects of increased whole body uptake of free hemoglobin have not been established. Due to the efficiency of the RES, intravascular retention time averages 6-8 h with most hemoglobin solutions [43,115]. The iron liberated from the breakdown of hemoglobin solutions appears to be recycled and may facilitate hematopoiesis [116]. Status of Current Trials and Applications. A variety of hemoglobin-based, oxygen-carrying volume expanders have been developed by the pharmaceutical industry and several are in Phase I and II clinical trials. Several solutions have been used in healthy anesthetized patients who are undergoing surgical procedures. Companies currently are developing variables for Phase III trials. In addition to the obvious uses for intraoperative blood restoration and volume resuscitation for hemorrhage, proposed uses of hemoglobin solutions include cardioplegia, bypass pump prime during extracorporeal circulation, perfusion of organs awaiting transplantation, perfusion of ischemic microvascular beds, and enhancement of tumor susceptibility to radio