CAROLINE THYES, ROMAN KOCIAN AND DONAT R. SPAHN Department of Anesthesiology University Hospital Lausanne Lausanne, Switzerland

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1 CURRENT STATUS OF ARTIFICIAL O2 CARRIERS EUROANESTHESIA 2005 Vienna, Austria May RC4 CAROLINE THYES, ROMAN KOCIAN AND DONAT R. SPAHN Department of Anesthesiology University Hospital Lausanne Lausanne, Switzerland Saturday May 28, :00-16:45 Room I INTRODUCTION Although allogeneic red blood cell (RBC) transfusions are beneficial in specific situations, they are associated with many disadvantages: limited disposability, high costs, multiple risks and side effects, major adverse outcomes and a higher mortality (table 1) [1-6]. Therefore, alternatives such as artificial oxygen (O 2 ) carriers are being sought. The latter are intended to improve O 2 delivery as well as tissue oxygenation and function of organs with marginal O 2 supply. The aim of the present article is to describe the currently evaluated artificial O 2 carriers, to summarize their efficacy and to discuss their side effects. TABLE 1: RISK AND SIDE EFFECTS OF ALLOGENEIC RBC TRANSFUSIONS IN THE WESTERN WORLD [1,3] Infections Viruses Type of risk Incidence (per unit transfused) Human Immunodeficiency Virus (HIV) 1:1,468,000-1:4,700,000 Hepatitis B Virus (HBV) 1:31,000-1:205,000 Hepatitis C Virus (HCV) 1:1,935,000-1:3,100,000 Bacteria All 1:28,000-1:143,'000 Parasites Malaria 1:4,000,000 Prions New variant Creutzfeld-Jacob disease possible Immunologic Reactions Hemolytic Transfusion Reactions Acute hemolytic TR 1:13,000 Delayed hemolytic TR 1:9,000 Alloimmunization 1:1,600 Immunosuppression 1:1 Transfusion-related acute lung injury 1:70,000 Mistransfusion All RBC mistransfusions 1:14,000 1:18,000 Artificial O 2 carriers can be grouped into two general classes: modified hemoglobin (Hb) solutions and perfluorocarbon (PFC) emulsions. In addition, hemoglobin containing liposomes [7] are noteworthy as potential future O 2 carriers (table 2). 189

2 TABLE 2: ARTIFICIAL O2 CARRIERS Hemoglobin based O2 carriers (grouped by the source of the Hb) Outdated human blood - Diaspirin cross-linked hemoglobin - Human polymerized hemoglobin (PolyHeme) - Hemoglobin raffimer (Hemolink) - Maleimide-activated polyethylene glycol-modified hemoglobin (MP4) Bovine Hb - Polymerized bovine hemoglobin (HBOC-201) Human recombinant Hb - Human recombinant hemoglobin version 1.1 (rhb1.1) - Human recombinant hemoglobin version 2.0 (rhb2.0) Perfluorocarbon emulsions Perflubron emulsion (Oxygent) Nano-dimension artificial red blood cells HEMOGLOBIN SOLUTIONS The efficacy of modified Hb solutions to transport and unload O 2 has been shown in a variety of animal models. Hemoglobin solutions exhibit a sigmoidal O 2 dissociation curve and most of them provide O2 transport and unloading capacity similar to blood [7,8]. The native human hemoglobin molecule in hemoglobin based artificial O 2 carriers undergoes virus inactivation and removal [7]. Furthermore, modification of the human hemoglobin molecule is necessary to prevent rapid dissociation of the native α2β2-tetramer into αβ-dimers in order to prolong intravascular retention and to eliminate nephrotoxicity [9]. With the exception of surface-modified hemoglobins [7] these modifications also serve to decrease O 2 affinity, in order to improve O 2 off-loading to tissues [7-9]. In addition, polymerization is used to increase the hemoglobin concentration at physiological colloid oncotic pressure. Steps for protection against prion contamination are performed for hemoglobin based O 2 carriers based on bovine hemoglobin since the transmission of prion diseases via blood transfusion [2] appears possible. Both potential advantages and disadvantages associated with administration of Hb solutions have been observed (table 3). Vasoconstriction, resulting in an increase in systemic and pulmonary artery pressures, still remains problematic [7,8]. The underlying mechanism includes nitric oxide (NO) scavenging, endothelin release and a sensitization of peripheral α-adrenergic receptors [8]. Recently, a quite different degree of vasoconstriction for miscellaneous hemoglobin based O 2 carriers with very similar NO binding has been described. This had led to the assumption of an additional mechanism, based on the presumption that the P50 is another important factor in determining the vasoconstrictive properties of hemoglobin solutions [9]. The assumption is that cell-free hemoglobin increases the delivery of O 2 to arteriolar vascular walls and thereby engages autoregulatory vasoconstriction [9]. However, this hypothesis is not universally accepted and there are physiological observations which are not completely in keeping with this hypothesis [10-12]. 190

3 TABLE 3: ADVANTAGES AND DISADVANTAGES OF HB SOLUTIONS Advantages: Transport and unloading capacity of O2 similar to blood Sigmoidal O2 dissociation curve 100% FiO2 for maximal potency is not necessary Storage stability Sterilization, virus inactivation and protection against prion contamination possible Disadvantages: Vasoconstriction Gastrointestinal side effects: - esophageal dysmotility - abdominal pain Pancreatitis like syndrome with increases in lipase and amylase Interference with colorimetric laboratory methods TABLE 4: PHYSICO-CHEMICAL CHARACTERISTICS OF MODIFIED HEMOGLOBIN SOLUTIONS Source of Hb Conc. (g/dl) Met.Hb (%) Viscosity (cp) COP (mmhg) P50 (mmhg) T1/2 (h) Non polymerized (%) Clinical Develop. Phase DCLHb ODB 10 < na stopped rhb1.1 Hum. rec. 5-Oct na stopped rhb2.0 Hum. rec na stopped HBOC-201 bovine 13±1 < III PolyHeme ODB 10 < < 1 III Hemolink ODB 10 < ± III MP4 ODB 4.2±0.2 < ±1.0 55±20 6±2 24 na I / II Hb = hemoglobin, Met Hb = methemoglobin, COP = colloid oncotic pressure, P50 = O2 partial pressure associated with a 50% hemoglobin saturation, T1/2 = intravascular half life (maximal values listed), ODB = outdated donor blood, hum. rec. = human recombinant, PEG = polyethylene glycol, na = not applicable. DCLHb = diaspirin cross-linked hemoglobin (Baxter Healthcare Corp., Deerfield, IL), rhb1.1 = human recombinant hemoglobin version 1.1 (Somatogen Inc., Boulder, CO, later Baxter Healthcare Corp.), rhb2.0 = human recombinant hemoglobin version 2.0 (Baxter Healthcare Corp.), HBOC-201 = polymerized bovine hemoglobin (Biopure Corp., Cambridge MA), PolyHeme = human polymerized hemoglobin (Northfield Laboratories Inc., Evanston IL), Hemolink = hemoglobin raffimer (Hemosol Inc., Toronto, Ontario, Canada), MP4 = Maleimide-activated polyethylene glycol-modified hemoglobin (Sangart Inc., San Diego, CA). In the following sections, all hemoglobin based artificial O 2 carriers will be described including their principal modifications, their efficacy, side effect profile and their current development status as far as has been published in the public domain 1. DIASPIRIN CROSS-LINKED HEMOGLOBIN (HEMASSIST/DCLHB): α-subunits of the Hb molecule are internally cross-linked with diaspirin (bis(3,5-dibromosalicyl)fumarate) between lysine residues (Lysα 1 99 and Lysα 2 99) preventing rapid breakdown of the Hb molecule and resulting in a decrease of O 2 affinity and thereby in an increase of P50 (table 4) [9,13]. Based upon favorable findings in pre-clinical studies [9,13], DCLHb entered clinical trials in the 1990s. Two randomized, controlled trials in cardiac (n=209) and major non-cardiac surgery (n=178) showed significant reductions of RBC transfusions in DCLHb treated patients [13]. There was also an observed higher incidence of hypertension, jaundice, urinary side effects and laboratory evidence of pancreatitis in the DCLHb group. Furthermore, DCLHb has been used in trauma victims in the US and Europe but due to an increased mortality in DCLHb treated patients, both studies were terminated prematurely and the development of DCLHb has been stopped [14]. 191

4 2. HUMAN RECOMBINANT HEMOGLOBIN (RHB1.1 AND RHB2.0): rhb1.1, a hemoglobin molecule genetically modified by fusion of the α-chains and mutation of the β-chain, was first presented in 1992 (table 4). Although rhb 1.1 entered phase I/II clinical trials, its development was abandoned because of pronounced adverse side effects [13]. By alteration of the distal heme pocket rhb2.0 with reduced NO scavenging capacity (k NOD of 2-81 mm -1 s -1 ) and O 2 affinity (P50 of mmhg) has been developed (table 4). Large animal testing shows short lasting pulmonary hypertension, better preservation of pulmonary vasodilatation due to NO donors and the absence of an increase of total peripheral resistance. In addition, pancreatic microcirculation has been better restored after hemorrhagic shock [13]. Despite the above favorable biologic effects the pharmaceutical industry driven development of this recombinant hemoglobin was stopped in 2003 (press release of July 17, 2003 by Baxter Healthcare Corporation). 3. POLYMERIZED BOVINE HEMOGLOBIN-BASED O 2 CARRIER (HBOC-201): The intermolecular link for HBOC-201 is provided by glutaraldehyde reacting with surface lysines of the hemoglobin molecule [7]. Final filtration eliminates the majority of non-polymerized hemoglobin tetramers (table 4) to minimize NO scavenging within arterial walls due to presumed extravasation of hemoglobin molecules. HBOC-201 was the first artificial O 2 carrier to enter clinical trials. Several phase III studies, randomizing patients at the first perioperative transfusion decision, have recently shown its efficacy in reducing the amount of RBC transfusions: The percentage of patients avoiding RBC transfusions increased from 0% to 59% (693 patients undergoing major orthopedic surgery) and from 0% to 34% (98 patients undergoing cardiac surgery) respectively [15]. The side effect profile of HBOC-201 includes hypertension, elevated postoperative amylase and lipase levels, late methemoglobinemia, detection of IgG-antiHBOC-201 antibody, abdominal pain, dysphagia, nausea and vomiting and oliguria. In contrast, renal function, platelet count, blood coagulation parameters and general clinical laboratory values were similar in both groups [13]. Based on these and other non-published studies Biopure Corporation (Cambridge, MA), the manufacturer of HBOC-201, filed a biologics license application at the US Food and Drug Administration (FDA) in Although no definitive decision has been taken, the FDA is likely to ask for additional safety and efficacy information. 4. HUMAN POLYMERIZED HEMOGLOBIN (POLYHEME): The hemoglobin molecule is first pyridoxylated to alter the oxygen affinity (P50), then polymerized with glutaraldehyde and purified (table 4) [7]. Based on favorable findings in prior animal studies [13], PolyHeme underwent clinical testing in acute trauma and emergency surgery. In the largest study published so far, enrolling 171 patients, up to 20 units of PolyHeme (each containing 50g) were administered [16]. Forty patients had a nadir RBC hemoglobin of < 3 g/dl (mean: 1.5±0.7 g/dl) but total hemoglobin was maintained at 6.8±1.2 g/dl due to a plasma hemoglobin (PolyHeme) of up to 5.9±1.1 g/dl. Only 25% of patients with both a nadir RBC hemoglobin < 3.0 g/dl and < 1.0 g/dl died. However, the comparison with a historic control group of 300 patients originating from already published data is inadequate. In addition, the historic cohort refused any RBC transfusions for religious reasons and neither type of injuries, operations or co-morbidities were clearly described. Even more importantly, and quite surprisingly, no data on RBC transfusions was reported [16]. This is of great importance since extremely low RBC hemoglobin levels such as 1.1 g/dl may be well tolerated for a certain period of time but most often require subsequent RBC transfusions. Therefore, it is no surprise that mortality in patients with a nadir RBC hemoglobin of < 3.0 g/dl is higher in those refusing any RBC transfusion than in those treated with PolyHeme and eventually subsequent RBC transfusions. Nevertheless it is impressive to note that 75% of patients with a nadir RBC hemoglobin of < 3.0 g/dl, including patients with a nadir RBC hemoglobin of as low as 0.2 g/dl survive and that no major safety concerns were reported [16]. 192

5 Besides elevated bilirubin levels, which probably reflects the natural clearance of PolyHeme, no significant adverse events and no obvious hypertension were observed [13]. Enrollment is currently underway in a pivotal Phase III trial of PolyHeme in the pre-hospital setting. It is likely that Northfield Laboratories may subsequently file a biologics license application at the US Food and Drug Administration (FDA) for PolyHeme [13]. 5. HEMOGLOBIN RAFFIMER (HEMOLINK): Hemolink is an o-raffinose cross linked polymerized hemoglobin. O-raffinose covalently cross-links intramolecularly the β-chains to form a stable polymer. The final product is a mixture of ~40% stabilized 64 kda tetramers and ~55% polymers (table 4) [17]. Hemoglobin raffimer has been evaluated in several studies in patients undergoing coronary artery bypass surgery [(17,18]. These phase II studies, both enrolling 60 patients, have shown the product to be well tolerated for use in conjunction with intraoperative autologous blood donation. A higher percentage of patients completely avoiding RBC transfusions was observed [17]. In a phase III trial, 299 patients were investigated (Canada and UK). Exposure to RBC transfusions was reduced from 76% to 56% [18]. Adverse side effects like hypertension, jaundice, urine discoloration, and postoperative increase in certain liver and pancreatic enzymes were reported in Hemolink treated patients [17, 18]. 6. MALEIMIDE-ACTIVATED POLYETHYLENE GLYCOL-MODIFIED HEMOGLOBIN (MP4): MP4, a hemoglobin molecule modified by surface conjugation with maleimide-activated polyethylene glycol, was constructed to reduce NO binding through site-directed mutagenesis. MP4 has a high O 2 affinity (low P50), a high viscosity and a high oncotic pressure to enhance its effectiveness as plasma expander (table 4) [13,19,20]. MP4 has been tested in a variety of small animal models, observing no significant effect on blood pressure and vascular resistance as well as significantly better restoration of functional capillary density and tissue oxygenation [13,19,20]. Furthermore, the results of hemorrhagic shock models in animals have been reported demonstrating that hemodynamic and metabolic resuscitation after hemorrhage were more completely restored by MP4 than by stoma-free hemoglobin [13,19]. The development of a non-vasoconstrictive hemoglobin based O 2 carrier such as MP4 is certainly welcomed [9]. However, the MP4 concept may be viewed also as somewhat counterintuitive, mainly for two reasons: First, the high O 2 affinity as evidenced by the low P50 (6±2 mmhg) and second, the low MP4 concentration of 4.2±0.2 g/dl. A partial explanation, why MP4 was so well tolerated in the hemorrhagic shock model [19] is the relatively modest degree of hemodilution achieved (hematocrit of 15% (48% of baseline) in pigs), which is normally well tolerated even without any additional O 2 carrying capacity [8]. Recently, MP4 has been assessed in an extreme hemodilution model in rats assessing its O2 transport capacity [20]. In this study MP4 was compared with PEG-modified albumin, a solution with similar physicochemical properties. A continuous exchange transfusion of 2.5 times the blood volume was effected over 60 min to a hematocrit of < 5% followed by an observation period of another 70 min. Hemodynamics and overall homeostasis were better maintained in MP4 treated animals. In addition, the hematocrit at which the lactate started to rise was lower in MP4 (7%) than in PEG-modified albumin treated animals (15%). Last but not least, all MP4 treated animals survived, whereas none of the MPA treated animals did [20]. Currently, MP4 is formulated for human clinical development as 4,2 g/dl in lactated Ringer s solution [20]. Sangart Incorporated (San Diego, CA), the manufacturer of MP4, has announced the initiation of a phase II clinical trial, enrolling 90 patients undergoing elective orthopedic surgery in Sweden (press release, September 8, 2004 by Sangart Incorporated). 193

6 PERFLUOROCARBON (PFC) EMULSIONS The O 2 transport characteristics of PFC emulsions (PFCs) are fundamentally different from those of modified hemoglobin solutions. PFCs are carrying oxygen as dissolved gas and are characterized by a linear relationship between O 2 partial pressure and O 2 content. Relatively high arterial O 2 partial pressures are necessary to maximize their O 2 transport capacity. Therefore, PFCs are normally used in combination with pure O 2 breathing [11,12]. PFCs are virtually not miscible with water. Only manufacturing an emulsion with specific characteristics (size of droplets of approximately 0.16 µm diameter) rendered them biocompatible, with few side effects. Due to the development of Oxygent, a stable 60 % (58 % perfluorooctyl bromide and 2 % perfluorodecyl bromide) emulsion, PFCs are now clinically well tolerated (table 5). TABLE 5: ADVANTAGES AND DISADVANTAGES OF PFC EMULSIONS Advantages: High gas dissolving capacity Chemical and biological inertness Low viscosity Only few and mild side effects No known organ toxicity Disadvantages: Low O2 capacity at physiologic po2 100% FiO2 is necessary for maximal potency Additional colloid often necessary with potential side effects 1. PERFLUBRON EMULSION (OXYGENT): After intravenous application, the emulsion is being taken up by the reticulo-endothelial system (RES). This procedure determines intravascular half life, which is dose dependent and is approximately 10 hours after a 1.8 g/kg PFC dose [8,13]. Subsequently, the PFC droplets are slowly broken down; the PFC molecules are taken up by the blood again (bound to blood lipids), transported to the lungs and excreted unaltered via exhalation [8]. Oxygent was assessed in a variety of hemodilution studies in animals describing an increase in cardiac output, an immediate rise in mixed venous O 2 partial pressure and saturation as well as a preferential metabolisation of O 2 transported by Oxygent [13]. In addition, improvement of left ventricular contractility and an improved survival of cardiopulmonary bypass with extreme anemia was observed in PFC treated animals [13]. This might be explained by an augmented O 2 delivery through very narrow capillaries where PFC particles (<0.2 µm in diameter) may penetrate more easily than the relatively large red blood cells (7-8 µm in diameter) and thereby increase local tissue oxygenation including the myocardium [12,13]. Furthermore, PFCs have been assessed in several hemorrhagic shock models in animals indicating a rapid reverse of hemorrhagic hypotension as well as complete restoration of hepatic microcirculation and tissue oxygenation [13]. Oxygent has also been tested in several studies in humans [11-13]. In a prospective randomized multicenter study trial in orthopedic surgery, 147 patients were hemodiluted preoperatively to a hemoglobin level of 9 g/dl [11]. After reaching a predefined physiologic transfusion trigger, they were randomized into 4 groups: A, standard of care (retransfusion of 450 ml of autologous blood at an unchanged FiO 2 of 0.4); B and C, Oxygent (0.9 or 1.8 g/kg) with colloid to a total 450 ml and ventilation with an FiO 2 of 1.0 and D, infusion of 450 ml of colloid with ventilation with an FiO 2 of 1.0. Oxygent (1.8 g/kg) was most successful in reversing transfusion triggers in 97% of patients as compared to 60% in the control group. Transfusion trigger reversal lasted in the Oxygent 1.8 g/kg group longer (80 min) than in the control (55 min) and colloid groups (30 min) [11]. Thus, physiological transfusion triggers may be treated at least as successfully with Oxygent as with autologous blood. This illustrates the remarkable potency of Oxygent to deliver readily available O 2 to those areas in the body where the extra O 2 is most needed. 194

7 Another large European multi-center phase III study has shown that Oxygent in conjunction with acute normovolemic hemodilution (ANH) reduced the need for RBC transfusion in 492 patients undergoing major noncardiac surgery with an expected blood loss of more than 20 ml/kg [12]. PFC treated patients first underwent ANH to a hemoglobin of 8.0±0.5 g/dl, followed by dosing with Oxygent. The PFC group had significantly fewer RBC transfusions and the percentage of complete avoidance of RBC transfusions was significantly higher. The efficacy was particularly high in patients with major blood loss. In the PFC group a higher incidence of adverse events was reported, including mild flu-like symptoms, postoperative ileus (probably due to undertreatment with crystalloids and colloids) [12] and thrombocytopenia 3 days post-dosing with return to a normal platelet count by day 7 [11]. Traditional coagulation tests, bleeding time and platelet aggregation, however, were unaffected [12]. Finally, enrollment in a phase III study in cardiac surgery was voluntarily suspended in 2001 due to an apparent imbalance between the two groups with regards to adverse neurological outcome (press release, January 8, 2001 by Alliance Pharmaceutical Corporation). Experts, however, agree that these events were not directly related to the perflubron emulsion used but to the rapid blood harvesting procedure used early on cardiopulmonary bypass. HEMOGLOBIN CONTAINING LIPOSOMES The development of hemoglobin containing liposomes started more than 40 years ago but is still in the pre-clinical stage. Hemoglobin containing liposomes are prepared from a purified hemoglobin and a lipid mixture composed of phospholipids, cholesterol and α-tocopherol and may have a number of theoretical advantages (table 6) [7]. Hemoglobin vesicles have been assessed in animal models of acute normovolemic hemodilution and in hemorrhagic shock models, but currently they have not been used in humans. In these studies blood pressure was rapidly restored, microvascular blood flow, functional capillary density and tissue oxygenation were improved. In none of these studies was hypertension described [13]. TABLE 6: ADVANTAGES AND DISADVANTAGES OF HEMOGLOBIN CONTAINING LIPOSOMES Advantages: Long intravascular half-life Lesser pressor effect Co-encapsulating of other substances and enzymes possible (i.e. 2,3-DPG, catalase, superoxide dismutase, methemoglobin reductase) Disadvantages: Reproducibility of liposomes uniform in size technical difficult High costs OUTLOOK Great progress in the development of artificial O 2 carriers has been achieved in recent years but no artificial O 2 carrier has achieved market approval yet in the US, Canada or Europe. Obviously, this would be the next big goal. Therefore, we should now think of the necessary education for healthcare professionals, including physicians, to familiarize them with these new concepts, physiology and compounds. Only in the hands of the experienced can artificial O 2 carriers can be used to the benefit of patients. In addition, with the introduction of non-hemoglobin artificial O 2 carriers (i.e. PFCs), or low concentrated ( anemic ) hemoglobin solutions (i.e. MP4), physiologic transfusion triggers become more important since the hemoglobin concentration will decrease due to the treatment with these products. Therefore, objective measurement of the oxygenation status of the patient becomes highly desirable, but as of today no such generally applicable monitoring technology exists. 195

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