BLUKO82-Seeber March 19, 2007 10:7 Artificial Blood Components 113 Some of them are common to all or a group of HBOCs, while others are specific for one product. As nowadays, all tested hemoglobin solutions use highly purified hemoglobin; theoretically, stroma, viruses, and endotoxins are not a concern. oxygen affinity Most natural free hemoglobin molecules have a high oxy- gen affinity.To changetheiraffinitytomore normal values, hemoglobin has been modified. Pyridoxylation (adding pyridoxal-5-phosphate) [13] or cross-linking (e.g., by 3,5- dibromosalicylate) [14] increases the oxygen affinity of hemoglobin. The cross-linker, o-raffinose, modifies the 2,3-DPG pocket of the hemoglobin, resulting in a higher P50. However, it is not clear whether it is really benefi- cial to have free hemoglobin with an oxygen affinity near to normal. It is thinkable that the more rapid reaction of oxygen with free hemoglobin as opposed to hemoglobin in the red cell may offset the effects of an increased oxy- gen affinity. A higher oxygen affinity may ensure that the microcirculation is not unduly affected by high oxygen levels. oncotic effects Red cell transfusions, per se, do not exert relevant on- cotic pressure. In contrast, free hemoglobin does. This is a two-edged sword. To draw fluids from tissues into the cir- culation (plasma expansion) may be beneficial in hemor- rhaging patients or in patients with ischemia due to tissue swelling. However, in cases where additional intravascular fluids are detrimental, the oncotic effects of HBOCs may be harmful. The extent of the oncotic effects encountered depends on the number, not on the size of the particles. There- fore, many small particles (as in intramolecularly cross- linkedhemoglobins)exert a muchgreateroncotic pressure than fewer, yet bigger particles with the same amount of hemoglobin (as in LEH). rheological properties, viscosity A circulating viscous fluid in the vessels exerts a shear stress on the vessel wall. This results in the release of NO, a vasodilator. By this mechanism, whole human blood with a viscosity of about 4 cPoise (cP) is able to regulate the perfusion of the microvasculature. Artificial infusion solutions, including HBOCs, also exert certain effects on the microvascular system. However, since some HBOCs have a lower viscosity than blood, they may impair the microvascular blood flow. This effect is especially pro- nounced when a patient is so anemic that an increase in cardiac output cannot compensate for the decreased red cell level and viscosity. Since exactly that situation is the classical indication for HBOC therapy, the low viscosity of HBOCs may be detrimental. The rheological properties have to be taken into account when designing a solution [15]. Newer HBOCs do just that [16]. vasopressor effects Right from the early experiments with hemoglobin solutions, it was obvious that there is a strong vasopres- sor effect of free hemoglobin. There are two theories to explain this phenomenon [10]. The first relates the va- soactivity to the interaction of hemoglobin with NO (and endothelin, a strong vasoconstrictor). Hemoglobin in red cells can scavenge NO, but the extent is limited due to the red cell membrane acting as a barrier. Free hemoglobin can scavenge NO as well. This capability being stronger, when the red cell membrane—which formerly worked as barrier to NO diffusion—is missing [17]. Besides, small hemoglobin molecules can leave the vessels and interact directly with NO in the perivascular space. Modifications in the production of HBOCs can reduce the vasocon- strictive effect of hemoglobin. These modifications ad- dress the binding capacity of hemoglobin and NO, as well as the size of the hemoglobin molecules. Encapsulated hemoglobin may have a limited vasoactivity,sincetheneo- hemocytes mimic some effects of the red cells. This way, the hemoglobin cannot easily diffuse into the perivascu- lar space anymore and cannot react that easily with NO [18]. The interaction of hemoglobin and NO has also been altered by chemical modification of the hemoglobin sites where NO is typically bound. Besides, recombinant hemoglobin can be designed in a way that reduces its NO- scavenging properties. The NO-binding site of the recom- binant hemoglobin molecule can be modified, making it difficult for NO to bind to hemoglobin [19]. In addi- tion, tetrameric hemoglobin molecules, which are small and therefore diffuse outside the vasculature to scavenge NO, can be eliminated from the final HBOC. Vasoactive side effects of hemoglobin solution have been reduced in some products when tetrameric hemoglobin was re- moved [12]. The less tetrameric hemoglobin, the less vasoconstrictive side effects and the more HBOC can be infused. The second theory used to explain the vasoconstric- tive effects of HBOCs is the inference of regulative mech- anisms of the microvasculature. Precapillary arterioles sense the oxygen level in the blood coming toward the capillaries and, depending on the oxygen content, con- strict or dilate. Since free hemoglobin can react much BLUKO82-Seeber March 19, 2007 10:7 114 Chapter 9 faster than hemoglobin bound in red cells, oxygen may be released from the hemoglobin to the arterioles easily and prematurely, giving the arterioles the illusion there is plenty of oxygen available. As a result, arterioles may not dilate as they would if red cells would arrive. This may impair the microcirculation. In this context, hemoglobin with a higher oxygen affinity than that of red cells may be beneficial [20]. renal effects Normal human hemoglobin is a tetramer. In the red cell it is stable, but it easily disassembles into dimers, once it leaves the cell. The dimers pass through the glomeruli in the kidney and cause renal damage. intravascular half-life Compared with red cells, the intravascular half-life of HBOCs is very short. Therefore, repeated infusions are needed to provide sufficient oxygen-carrying capacity. In order to prolong the stability and intravascular half-life of acellular hemoglobin, intramolecular cross-linking, poly- merization, encapsulation, or conjugation with macro- molecules has been employed in the current generation of HBOCs. Besides, recombinant hemoglobin does not disintegrate easily, prolonging its half-life [13]. By these means, plasma half-life of HBOCs is about 5–40 hours. Further work on HBOCs attempts to increase the intravas- cular half-life even more. infectious risks and effects on the immune system It is believed that the production process of HBOCs ex- cludes every possibility of viral contamination coming from the original hemoglobin source. Moreover, since the cross-linking process of hemoglobin stabilizes the pro- tein, heat-sterilization is possible as well [14]. HBOCs are therefore thought to be free from infectious agents. Of greater concern is the effect of HBOCs on the im- mune system of the recipient. HBOCs may overload the reticuloendothelial system, impair bactericidal activities, and may promote bacterial growth by the iron content of HBOCs. Besides, these may potentiate the detrimental effects of endotoxin in the patients. Antigenicity from red cell membranes does not oc- cur with purified hemoglobins. Therefore, HBOCs can be administered independent of the blood group of the recipient. Another form of antigenicity may evolve with the use of HBOCs though. Reactions to polymerized hemoglobin molecules or to molecules from other species are of (theoretical) concern. During trials with the only bovine product, IgE production was not observed and only moderate levels of IgG were seen [21]. oxidative damage Superfluous delivery of oxygen may come with side ef- fects. Oxygen may cause oxidative damage. Reperfusion injury may occur after therapy of conditions with pro- longed ischemia, e.g., stroke, myocardial infarction, and severeprolonged hemorrhage. Also,bloodcells are notim- mune to the damaging effects of oxygen. The iron of free hemoglobin is prone to oxidation and so met-hemoglobin builds up easily. Besides, peroxides and other oxidative products may accumulate and result in the damage of hemoglobin and in endothelial damage. Red cells have enzymes that protect them from the influence of oxygen. SOD and CAT found in red cells may ameliorate the dam- aging effects of oxygen. The early HBOCs did not con- tain these enzymes. To control the oxygen-related dam- age to hemoglobin, the mentioned enzymes were added to the molecules, resulting in polyHb-SOD-CAT. These compounds were shown to remove free oxygen radicals and stabilize the hemoglobin molecule. Other approaches to add an antioxidant principle to hemoglobin are pos- sible (adding Tempol to form polynitroxylated ␣-␣-Hb) as well. Such protecting enzymes can also be included in the liposomes of LEH [22]. Simultaneous application of antioxidant agents (ascorbic acid, riboflavin) can protect from oxidative damage to the HBOCs [21]. hemostaseological alterations Both thrombocytopenia and thrombocytopathy have been observed after infusion of certain HBOCs. These ef- fects have been thought to be due to the NO-scavenging effects of hemoglobin. When scavenged, not enough NO is available to exert its antiplatelet effects. Besides, ac- tivation of the complementary system may lead to the thrombocytopenia—sometimes observed after HBOC in- fusion (especially LEH). effects on erythropoiesis IthasbeensuggestedthatHBOCs stimulateerythropoiesis [23, 24]. shelf life While red cells can be stored for a maximum of 49 days under standardconditions(about4 ◦ C), HBOCs are stored much longer. Some HBOCs can be refrigerated; others are stabile even under room temperature. Also, storage after lyophilization of the HBOC is possible [12]. This prolongs their shelf life to 1–2 years or longer. BLUKO82-Seeber March 19, 2007 10:7 Artificial Blood Components 115 other side effects In healthy volunteers, infusion of HBOCs resulted in gastrointestinal upset (with increased motility and sphincter spasms) and flu-like symptoms (fever, chills, headaches, and backaches) [25]. As a physiological re- sponse to infusion of hemoglobin, jaundice occurs. After administration of some HBOCs, liver enzyme levels increase. Current products Several types of HBOCs have been developed and are in different phases of clinical trials [12, 20, 21, 26, 27]. Table 9.1 summarizes the HBOCs developed and their properties. A comparison of the products demonstrates that they differ tremendously in one feature or the other. Obviously, HBOCs are a heterogenic group of drugs rather than the sole and perfect blood substitute. Potential indications Apart from use in blood management, HBOCs have been proposed and experimentally tested in a variety of settings. With that, HBOCs have the potential to become a drug rather than a blood substitute. In animal experiments, HBOCs were used to improve the outcome of animals subjected to brain ischemia [21]. HBOCs may be clinically useful in focal or global is- chemia of the brain, cardiopulmonary-bypass-induced brain lesions, and in brain trauma. These notions, how- ever, are only supported by animal studies [28]. In studies involving humans, the potential increase in strokes in pa- tients treated with HBOCs has stopped one trial using HBOCs. The vasoconstrictive and NO-scavenging “side effects” of certain HBOCs can be used in septic patients who are hypotensive. It is hoped that the microvascular alterations developing during sepsis may be influenced beneficially by HBOCs. In studies on septic animals, HBOCs did not alter the regional perfusion profoundly, as would be the case with other antihypotensive agents, such as cate- cholamines [29]. Studies are expected to demonstrate the benefits of HBOCs in humans with severe sepsis or septic shock. HBOCs have also been used to provide oxygen to ischemic tissues. Tissue beyond a peripheral or coro- nary stenosis may benefit from oxygenation provided by HBOCs. Since the HBOC particles are much smaller than red cells, a residual blood flow may suffice to provide the poststenotic tissue with oxygen [30]. HBOCs have also been proposed in acute pancreatitis to improve microcir- culation and to avoid tissue damage [31]. An improved oxygenation of organs by HBOCs is also beneficial to preserve organs reserved for transplantation. Oxygenation of tumors by HBOC infusion may improve the efficacy of anticancer treatment, which depends on the presence of oxygen in the tumor. Further developments After overcoming the first problems with stroma and dis- sociation of hemoglobin tetramers, a few first-generation HBOCs have been developed and refined and are more or less ready for use. However, while tested in thousands of patients, apart from a single exception, none of the prod- ucts is approved for clinical use. HBOCs are now developed, but mostly not in the area for which they were initially intended to be used, namely, to replace blood transfusions. Most of the first-generation HBOCs have been tested for indications that came up during development (“We have the solution, where is the problem?”). Likely, HBOCs can be used for the preserva- tion of microcircular perfusion in ischemic areas (stroke, heart attack, sickle cell crises), as antihypotensive therapy (sepsis), for organ preservation (transplantation), and as an ultrasound contrast medium [32]. Some of the problems of HBOCs have obviously turned into a useful property. Nevertheless, we seem not to have found what we were looking for in the first-generation products, namely, a red cell substitute for acute (let alone chronic) anemia. Second-generation HBOCs are about to address the problems that prevent HBOCs’ use in those settings. The hypertensiveeffects,short intravasal half-life, and antioxidative challenges of HBOCs must be addressed before we can use them the way we originally intended. However, during the years of research, much has been learned about hemoglobin as well as about anemia tol- erance. Maybe we really do not need to have any more of what we originally wanted. Perhaps we just need an agent with a higher viscosity than the traditionally used plasma expanders. A lower hemoglobin level may suf- fice, given other conditions as viscosity and blood volume are taken into account. New products are in the pipeline, and time will show how much they will change the blood management. albumin–heme conjugates as artificial oxygen carriers Recombinant human albumin can be engineered so that it includes up to eight heme molecules. These conjugates BLUKO82-Seeber March 19, 2007 10:7 116 Chapter 9 Table 9.1 HBOCs and their properties. Intravascular Oncotic Class t 1/2 pressure Viscosity Vasoactive Source of Hgb MP4, Hemospan (Sangart) Malemide-PEG- conjugated = MalPEG-Hb ca. 43 h High (55 ± 20) High (2.5 ± 1 cP) Low Human VTR-PHP (Apex Bioscience) Surface-modified polyoxyethylene- conjugated, pyridoxylated ca. 40 h Moderate to high Moderate to high Mild Human PEG Hgb (Enzon) Surface-modified 44 h Moderate to high Moderate to high Mild Bovine Liposome-encapsulated (Terumo) 16 h (rodent) 0.7 Bovine Polyheme (Northfield) GA-polymerized, pyridoxylated 24 h Low (20–25) Low Moderate to low Human Hemolink (Hemosol) o-Raffinose polymerized 14–24 h Low (COP 26 mm Hg) Low (1.15 cP) Moderate Human Hemopure (Biopure) similar to HBOC-201, HBOC-301, HbGlutoamer250bovine (Oxypure for veterinarian use approved) GA-polymerized 9–24 h Low (17) Low (1.3) Moderate Bovine Optro (Baxter) (rHb 1.1) ␣-␣-cross-linked recombinant hemoglobin from E. coli Low Low Marked Recombinant human, Presbyterian mutant of Hgb HemAssist (Baxter) Intramolecular cross-linked with diaspirin (DCLHb) 2–14 h (dose- dependent) (COP 42 mm Hg) Low (1.0) Marked Human PEG, polyethylene glycol; GA, glutaralydehyde; Hgb, Hemoglobin; GI, gastrointestinal; COP, colloid osmotic pressure; AST, aspartate transaminase. have been shown to transport and release oxygen [33, 34] and have an intravascular half-life of about 36 hours. Rats were able to survive a substantial exchange of their blood volume with albumin–heme conjugates, while the control group did not [35]. These early trials with albumin–heme conjugates show promise for a future generation of AOCs. hemoglobin aquasomes Hemoglobin aquasomes consisting of hemoglobin molecules attached to a hydroxylapatite core have recently been synthesized [36]. These have been successfully tested in animals to transport oxygen and may be another at- tempt to prepare a useful oxygen carrier. Perfluorocarbons The second group of AOCs is PFCs. They are greenhouse gases that develop during the production of aluminum. Chemically, they are related to Teflon r  . PFCs consist of carbohydrate chains with 8–10 carbon atoms. Most of the atoms are substituted with fluorine. Some of the carbon atoms may also be substituted with bromine. BLUKO82-Seeber March 19, 2007 10:7 Artificial Blood Components 117 % Hgb P50 Met-Hgb% tetramer Hgb g/dL Use Stable Side effects Status 6 ± 2 <0.5 4.2 g/dl Stable at −20 ◦ C GI and vasoactive effectsverylowto absent Phase I clinical trials 20 3 10 NO-induced shock (sepsis) 15 <5 6 Cancer (radiosensitizer) Trials suspended 18 12 10 28–30 <3 <1 10 Trauma, perioperative At room temperature >1yr No GI, no vasoconstrictive 39 ± 12 <10–25 30–36 10 Cardiac, general surgery, anemia Marked GI, purification improved this, rise in blood pressure 38–43 <10 <5 13 Perioperative, hemodilution, anemia At room temperature stable Chest pain (problem resolved), jaundice, mild increase in blood pressure, AST and lipase increased Approved in South Africa in 2001 30–33 <5 5 Surgery, trauma, hemodilution Fever, headaches, rising blood pressure, purification has improved the situation, amylase, lipase increased Pulled off market in 1998 30–32 4–18 10 Surgery, organ failure, trauma, stroke Pulled off market in 1998 PFCs can dissolve and thus carry gases. About 40–50 mL of oxygen can be dissolved in 100 mL of a PFC. The oxygen-carrying characteristics of PFCs are like the ones of saline or other liquids. They follow the physical prin- ciples as mentioned in Chapter 2 on oxygen physiology. The higher the partial pressure of a gas above the PFC, the more gas is dissolved in it. This characteristic is unlike hemoglobin which dissolves oxygen in a pH-dependent manner and cooperatively, as is depicted in hemoglobin’s S-shaped oxygen-binding curve (see Fig. 2.1). In contrast, the oxygen-binding curve of PFCs is a linear function. In clinical practice, this means that oxygen can be trans- ported in relevant amounts in PFC only when the inspira- tions oxygen fraction is high enough. That is why patients on PFCs always need supplemental oxygen. PFCs are insoluble in water. Therefore, they come emulsified. The first-generation PFCs were dissolved in pluronic F-68. Since complement activation was associ- ated with this compound, second-generation PFCs are now dissolved in a lecithin (and cholesterol) solution. The intravasal half-life of PFCs is very short, some hours only. However, the biological half-life is much longer. PFC particles are chemically and physically inert. They are taken up by phagocytosis by components of the BLUKO82-Seeber March 19, 2007 10:7 118 Chapter 9 reticuloendothelial system (spleen, liver, and lung). There, the lecithin emulator is metabolized and the PFCs remain inert and are slowly given back into the bloodstream to be excreted by the lung. There is concern regarding the PFC blocking the reticuloendothelial system, which may immunocompromise the patient transiently. Further- more, the emulgator of the PFC may be toxic to elements of the immune system. Therefore, there is a maximum dose of PFCs that should not be overstepped. Especially in patients with sepsis or an active infection, care must be taken. However, some beneficial effects of PFCs on the immune system have been postulated, since these may diminish the reperfusion damage in myocardial infarction or apoplexia. Overall, the clinical relevance of the immunomodulative effects is not yet clear. Apart from immunosuppression, PFC administration may lead to fever 4–6 hours after infusion, shaking, nausea, and transient leukopenia. A transient thrombo- cytopenia is observed 2–3 days after infusion. It is usually not severe (<20% from baseline) and seems to be self- limiting after about 7 days. The thrombocytopenia occurs without proof of platelet function problems or prolon- gation of prothrombin time and partial thromboplastin time in healthy volunteers [37]. The second-generation PFCs do not show relevant immunogenic reactions or complement activation (Table 9.2) [21, 26, 38, 39]. Oxygen delivery by perfluorocarbons Normal blood with a hematocrit of about 45% and at a PO 2 of 100 mm Hg carries about 20 mL of oxygen/100 mL blood. About 25%, that is, 5 mL is released during the blood’s trip through a normal, resting organism. The ve- nous blood that returns to the heart has about 75% of oxygen left in it. When PFC is given, the body first takes oxygen from the PFC, since this is easier than taking oxy- gen from the “neatly packed” oxygen in the red cells. Be- fore red cells can unload the oxygen and transfer it through their membrane, PFC already filled the needs of the tis- sue. Therefore, if enough PFCs are in the blood, the blood may not need to unload any oxygen and may return with nearly 100% of the oxygen it had when it left the lungs. Table 9.2 Characteristics of perfluorocarbons. Fluosol-DA (Green Cross Corp., Osaka, Japan) Oxygent (Alliance) Generation First Second Active constituents 14% perfluorodecalin and 6% perfluorotripropyl amine 58% perflubron (perfluorooctyl bromide, PFOB; C8F17 Br) and 2% perfluorodecyl bromide (PFDB, C10 F21 Br) Emulgator Pluronic F-68, egg yolk phospholipid Lecithin (in phosphate-buffered aqueous electrolyte solution) Size of particles 0.16–0.18 mcm Intravasal half-life 12–18 h 4–15 h Biological half-life 7 days 4 days Dose 30 mL/kg 2.7 g PFC/kg (1.4 mL PFC/kg) Oxygen dissolved in the presence of pure oxygen and STPD 6 mL/dL 17 mL/dL Viscosity ca. 4 cP Hemoglobin equivalency 4.0 ±/ 2.7 g/dL in a dose of 2.7 g/kg (recommended clinical dose) Storage Frozen, mix before use About 2 yr with standard refrigeration or room temperature More PFCs have been developed, among them are products based on perfluorodecalin (Synthetic Blood International and Sanguine Corp.; available for use in Russia since 1996 as Perftoran TM ) and emulsified perfluorodichloro octane (Oxyfluor TM , HemaGEN). STPD, standard temperature and pressure, dry. BLUKO82-Seeber March 19, 2007 10:7 Artificial Blood Components 119 In contrast, the PFCs deliver most of their oxygen. In fact, about 91% (under PaO 2 = 500 mm Hg) of the oxygen bound to PFCs is unloaded during one circulation [38]. The different characteristics of oxygen unloading make it difficult to compare hemoglobin and PFCs. The clini- cians typically want to know how much oxygen is theoreti- cally availableto the patient.Since most clinicians arecom- fortable with knowing that the patient has a hemoglobin level sufficient to meet the oxygen needs, they have a prob- lem when there is not enough hemoglobin, yet there is an agent that may mimic the hemoglobin’s function. To make matters easier, the concept of “hemoglobin equiv- alency” has been developed. It tells how much of a PFC is equivalent to a certain amount of hemoglobin as re- gards to oxygen transportation ability. It relates the per- centage of whole body oxygen consumption (VO 2 )from PFC to that from hemoglobin. For example: A patient with a hemoglobin level of 8 g/dL uses 50% of oxygen from hemoglobin and 25% from the PFC. This means that the PFC contributes half the amount of oxygen of that of hemoglobin to the tissue (25% is half of 50%.). Therefore, the PFC is equivalent to 4 g/dL of hemoglobin (since half of 8 g/dL is 4 g/dL) [38]. Another term used in this connection is the “effective hemoglobin,” which is the sum of the hemoglobin of the patient plus the hemoglobin equivalent of the PFC. PFCs also have effects on oxygen delivery that are be- yond that of increased intravascular oxygen transport. PFCs increase the availability of oxygen in the tissue, a phenomenon called “diffusion facilitation”. It is not clear how this occurs. It is thought that PFCs can travel into very small vessels in the tissue which the red cells cannot reach. Besides, PFCs may help red cells to deliver their oxygen. Since the PFCs flow near the vessel wall, and the red cells are near the center of the vessel, PFCs may serve as a bridge between red cells and the tissue (“near wall phenomenon”) [40]. Tested and potential indications PFCs are promising in blood management (see below). Additionally, PFCs can be of potential benefit in other areas. PFC particles are many times smaller than red cells. This enables them to travel to tissues that are is- chemic and where red cells can no longer travel, e.g., in myocardial infarction and ischemic limbs due to a tourniquet. PFCs are also used to avoid tissue ischemia during the repair of cerebral aneurysms and to improve the outcome of cardiopulmonary bypass (microbubbles, thought to cause problems, can be readily absorbed by PFC). PFCs augment tumor oxygenation and can preserve transplant organs [39]. PFCs were also proposed to be used in liquid ventilation in acute respiratory distress syn- drome or acute lung injury, or as an ultrasound-imaging medium [21]. Artificial oxygen carriers in blood management The original impetus to develop AOCs has been to pro- vide a solution, which substitutes for red cell transfusions. To a certain, limited extent, AOCs are able to do so. The following paragraphs outline how AOCs can be used in blood management—instead of red cell transfusions or in other areas related to blood management. Perfluorocarbons severe anemia Fluosol-DA, the first-generation PFC, has been used in severely anemic patients as an AOC. It was able to reverse the clinical signs of oxygen deficiency [40, 41]. However, the intravascular half-life was too short and, as a result, it was not able toimprovethesurvival of the anemic patients. Therefore, it has not been developed further into an agent suitable for blood management [21, 30]. A second-generation PFC was shown in animal stud- ies to be useful in resuscitation from hemorrhagic shock. Oxygen delivery can be maintained or reestablished with PFCs, so that tissues submitted to severe anemia can con- tinue with aerobic metabolism and the organ function is preserved [42, 43]. After a PFC became available in Rus- sia, extensive use of this PFC has been made in humans with severe anemia and it seems that the PFC can improve tissue oxygenation under such circumstances [44]. augmented acute normovolemic hemodilution TM Oxygent TM , a second-generation PFC, is not an approved drug. However, several trials have been performed, many of which directly relate to blood management. As learnt from the early trials with the first-generation PFCs, in se- vere anemia, current PFC products are not able to remain in the circulation long enough to work until the patient’s own erythropoiesis has provided the missing red cell mass. Many publications, therefore, discuss PFCs mainly in set- tings where short times of decreased oxygen delivery are to be bridged. The most prominent indication in this con- nection is augmented acute normovolemic hemodilution (A-ANH TM ) [45]. Intraoperatively,PFCshavebeenshown to reverse signs of anemia (“the transfusion trigger”) more effectively than the infusion of autologous blood or BLUKO82-Seeber March 19, 2007 10:7 120 Chapter 9 colloids. Besides, when used in conjunction with A- ANH TM , the transfusion rate of noncardiac surgical pa- tients, with a high intraoperative blood loss (defined as > 20 mL/kg), can be reduced [46, 47]. Despite the beneficial effects in reducing the transfusion of red cells, clinical trials with Oxygent TM were stopped in 2001. The reason was that in a trial with Oxygent TM , the treatment group had a higher incidence of stroke, com- pared to the control group. sickle cell anemia Patients with sickle cell anemia may benefit from PFCs when severe sickle cell-induced vasoocclusion occurs. Since vasoocclusion is often only partial and allows for a residual flow, small PFC molecules can still travel to the ischemic areas and ameliorate the effects of sickle cell crises. It was suggested that oxygenated PFC could even unsickle and dislodge red cells and thereby reduce the va- soocclusion [48]. Hemoglobin-based oxygen carriers trauma and hemorrhagic shock In theory, HBOCs are the ideal solutions for resuscitation of patients with trauma and in hemorrhagic shock [49]. In fact, in the emergency setting and in prehospital care, HBOCs can be used to treat catastrophic blood loss [14] without resorting to allogeneic blood transfusions. Early trauma trials with an HBOC consisting of diaspirin-cross-linked hemoglobin demonstrated an in- creased mortality in severely hemorrhaging trauma pa- tients treated with this agent. This was, likely, the re- sult of the strong vasoactive properties of the product. In contrast, Polyheme TM (see Table 9.1), which is much less vasoactive, has been used successfully to resuscitate trauma patients who do not receive red cells. Up to 20 units of the HBOC was given, and the 30-day mortal- ity was clearly reduced to 25%, compared with 64% in a control group [50]. Despite these and other encourag- ing results of animal trials, HBOCs are not licensed in most countries. More experience needs to be gained re- garding the use of HBOCs in trauma patients [51]. Until then, patients in only a few countries can benefit from HBOCs. when red cells are not an option HBOCs have also been used to treat patients with severe anemia who cannot take donor blood transfusions [52– 54]. The use of the HBOCs is often done in a “compas- sionate use protocol.” The inherent problem with this use of HBOCs in severe anemia is the short half-life of the HBOCs. Due to this, serial infusions over several days were necessary, until autologous erythropoiesis provided enough red cells. Initially, HBOCs were used for short periods only. Several case reports suggest that long-term survival is also possible by exclusively using HBOCs in- stead of red cells, as shown in reports of patients with sickle cell crisis [55], autoimmune hemolytic anemia [56], and leukemia [57]. acute normovolemic hemodilution In analogy to A-ANH TM using PFCs, HBOCs have been used to perform A-ANH TM . HBOCs can extend the tolerance of anemia during acute normovolemic hemodilution. surgical blood loss As a bridge until blood is available, different HBOCs have been used to substitute surgical blood loss in the setting of general, cardiac, and vascular surgery [58–62]. As a result, red blood cell transfusions have been reduced [62, 63]. Hemopure TM (see Table 9.1), which has been approved in South Africa for the therapy of patients with major surgical blood loss, avoided red cell transfusions in 34, 27, 43, and 60% of patients undergoing cardiopulmonary bypass, abdominal aortic reconstruction, general surgery, and orthopedic surgery, respectively [64]. sickle cell anemia Intheeventof ischemic complicationsofsicklecell disease, such as acute chest syndrome, exchange transfusions are recommended. As an alternative approach, HBOCs have been used in selected cases to treat anemia and sickle cell complications [55, 65]. Artificial platelet substitutes It has not only been tried to re-create red cells, but also attempts to re-create artificial platelets have been done. Platelets are a very complex entity. Although already much is known about the importance of platelets, their modes of action are only marginally understood. It is obvious how difficult it is to produce platelet substitutes artificially. In analogy to human HBOCs, outdated platelets have been used as source material for platelet substitutes. Nonviable platelets, platelet microvesicles (membrane fragments), and even membrane phospholipids have been tested as to their ability to support clotting [66]. Even such tiny microvesicles, as those used, have been shown to carry BLUKO82-Seeber March 19, 2007 10:7 Artificial Blood Components 121 platelet receptors and to be able to enhance endogenous clotting or clotting processes induced by recombinant clotting factors [67]. Attempts have been made to use known elements of plateletstocreateaproduct that at least mimics some ofthe platelet’s function. A plateletsome consisting of a liposome with a lipid bilayer has been employed as a carrier. Inte- grated in the lipid layer,platelet-derived receptors(e.g., for von Willebrand factor, fibrinogen, and thrombospondin) have been added. Infusion and topical application of the plateletsome solutions have been shown to decrease clin- ical bleeding in animals [68]. Such basic experiments aim at defining which components of platelets are needed to trigger a favorable response in bleeding. With the prospect of being able to produce the needed receptors in a recom- binant fashion, the research is promising. Another “artificial platelet substitute” called Syntho- cytes (Andaris Group Ltd., Quadrant Healthcare plc, Not- tingham, UK) consists of albumin microcapsules with fib- rinogen [69]. Since fibrinogen on the surface of Syntho- cytes can interact with receptors on platelets and activates platelets, Synthocytes, together with residual platelets in thrombocytopenia, may contribute to hemostasis [69]. It was hoped they would selectively target sites of hemor- rhage. And indeed, in animal experiments, Synthocytes were shown to reduce bleeding. Many other platelet substitutes, e.g., rehydrated, lyophilized platelets or thromboerythrocytes [70], are in the developing phase and are currently undergoing trials as to their usefulness as a clinically effective platelet substi- tute [71, 72]. It was considered that in the “far future, pro- coagulant cell surface transformation may be influenced by topicalapplicationofinhaledthrombomodulin-loaded liposomes or by sense or antisense oligonucleotides in- ducing thrombomodulin expression or suppressing tissue factor expression, respectively” [73]. However, since research on platelet substitutes is in a very early stage, basic parameters have to be established to evaluate the final products as to their ability to reduce bleeding. To this end, the Food and Drug Administration of the United States has compiled recommendations as for the testing of such products in humans [74]. Key points r It is impossible for human beings to re-create blood. r First-generation AOCs have a very short half-life and come with unwanted side effects that make them unsuit- able forgeneraluseasabloodsubstitute. AOCs arepromis- ing drugs with well-described indications and contraindi- cations. Questions for review r What are AOCs and what are their indications in blood management? r What are the sources of hemoglobin used for the pro- duction of HBOCs? r What are the differences between (a) hemoglobin in the viable red cell, (b) hemoglobin free after lysis of red cells, and (c) hemoglobin attached to polyethylene glycol? r What are common side effects of HBOCs and how have they been engineered to reduce them? r What is the source of PFCs? r How do PFCs transport oxygen? r What different platelet substitutes have been described? Suggestions for further research What is the current status of HBOCs and PFCs? Check the Internet and medical literature for more information. Exercises and practice cases Read the case report of Cothren and colleagues [75]. Dis- cuss the management of the described patient. What in- dicators have there been to support the claim that (a) the patient benefited from HBOC and (b) the patient did not benefit from HBOC. What lessons do you learn from this report about the oxygen transport of HBOCs? Homework 1 Find out whether there is somebody in your vicinity who uses AOCs. Record his/her contact information in the address book in the Appendix E. 2 Is it possible in your country to get AOCs? If so, record the contact information of the provider(s) in the address book in the Appendix E. References 1 Tunnicliffe, F.W. and G.F. Stebbing. The intravenous injec- tion of oxygen gas as a therapeutic measure. Lancet, 1916. (August 19): p. 321–323. BLUKO82-Seeber March 19, 2007 10:7 122 Chapter 9 2 Amberson, W., et al. On the use of Ringer–Locke solutions containing hemoglobin as a substitute for normal blood in mammals. 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Hematologic effects of a novel hemoglobin-based oxygen carrier in normal male and female subjects. J Lab Clin Med, 1995. 126(5): p. 444–451. 25 Viele, M.K., R.B. Weiskopf, and D. Fisher. Recombinant hu- man hemoglobin does not affect renal function in humans: analysis of safety and pharmacokinetics. Anesthesiology, 1997. 86(4): p. 848–858. 26 Mazer, C.D. Review of clinical trials of oxygen therapeutics. TATM, 2000. S1: p. 27–31. 27 Bjorkholm, M., et al. A phase I single blind clinical trial of a new oxygen transport agent (MP4), human hemoglobin modified with maleimide-activated polyethy- lene glycol. Haematologica, 2005. 90(4): p. 505–515. 28 Lenz, C. and F. Waschke. Artificial oxygen carriers and the cerebral circulation. Anasthesiol Intensivmed Notfallmed Schmerzther, 2001. 36(Suppl 2): p. S110–S113. 29 Bone, H.G. Hemoglobin-based oxygen carriers in sep- sis. Anasthesiol Intensivmed Notfallmed Schmerzther, 2001. 36(Suppl 2): p. S114–S116. 30 Kale, P.B., et al. Fluosol: therapeutic failure in severe anemia. Ann Pharmacother, 1993. 27(12): p. 1452–1454. 31 Strate, T., et al. The potential of HBOC in acute pancre- atitis. Anasthesiol Intensivmed Notfallmed Schmerzther, 2001. 36(Suppl 2): p. S119–S120. 32 Creteur, J., W. Sibbald, and J.L. Vincent. Hemoglobin solutions—not just red blood cell substitutes. Crit Care Med, 2000. 28(8): p. 3025–3034. 33 Wang, R.M., et al. Human serum albumin bearing covalently attached iron(II) porphyrins as O 2 -coordination sites. Bio- conjug Chem, 2005. 16(1): p. 23–26. 34 Komatsu, T., et al. O 2 and CO binding properties of artificial hemoproteins formed by complexing iron protoporphyrin IX with human serum albumin mutants. J Am Chem Soc, 2005. 127(45): p. 15933–15942. 35 Komatsu, T., et al. Exchange transfusion with synthetic oxygen-carrying plasma protein “albumin-heme” into an acute anemia rat model after seventy-percent hemodilution. J Biomed Mater Res A, 2004. 71(4): p. 644–651. 36 Khopade, A.J., S. 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[...]... sickle red blood cell-induced obstruction in an ex vivo vasculature Blood, 2001 98(10): p 3128–3131 49 Moore, E.E Blood substitutes: the future is now J Am Coll Surg, 2003 196(1): p 1–17 50 Gould, S.A., et al The life-sustaining capacity of human polymerized hemoglobin when red cells might be unavailable J Am Coll Surg, 2002 195 (4) : p 44 5 45 2; discussion 45 2 45 5 51 Sloan, E.P., et al Diaspirin cross-linked... hemodilution (A-ANH(tm)) in cardiac and non-cardiac patients Anasthesiol Intensivmed Notfallmed Schmerzther, 2001 36(Suppl 2): p S107–S109 47 Spahn, D.R., et al Use of perflubron emulsion to decrease allogeneic blood transfusion in high -blood- loss non-cardiac surgery: results of a European phase 3 study Anesthesiology, 2002 97(6): p 1338–1 349 48 Kaul, D.K., X Liu, and R.L Nagel Ameliorating effects of fluorocarbon... and hyperoxia alone Shock, 2005 24( 3): p 245 –2 54 44 Maevsky, E., et al Clinical results of Perftoran application: present and future Artif Cells Blood Substit Immobil Biotechnol, 2005 33(1): p 37 46 45 Spahn, D.R., P.F Willimann, and N.S Faithfull The effectiveness of augmented acute normovolemic hemodilution (A-ANH) Anaesthesist, 2001 50(Suppl 1): p S49–S 54 46 Kemming, G., O Habler, and B Zwissler... 2002 96 (4) : p 871–877 44 McLoughlin, P.L., T.M Cope, and J.C Harrison Hyperbaric oxygen therapy in the management of severe acute anaemia in a Jehovah’s Witness Anaesthesia, 1999 54( 9): p 891–895 45 Hart, G.B., et al Hyperbaric oxygen in exceptional acute blood- loss anemia J Hyperb Med, 1987 2: p 205– 210 46 Hart, G.B Exceptional blood loss anemia Treatment with hyperbaric oxygen JAMA, 19 74 228(8):... hemoglobin of 10 mg/dL equals a hematocrit of about 30%.) Step 4 The plan of care After completing the first three steps of the algorithm, all the information needed is available It is now time to tailor an individual treatment concept for the patient Having a detailed plan of care is the basis for successful blood management [39, 40 ], the lack of a plan may result in disaster [41 ] Take a sheet of paper... Artif Cells Blood Substit Immobil Biotechnol, 2001 29(6): p 43 9 45 1 72 Levi, M., et al Fibrinogen-coated albumin microcapsules reduce bleeding in severely thrombocytopenic rabbits Nat Med, 1999 5(1): p 107–111 73 Scherer, R.U Haemostaseological aspects of perioperative blood management Zentralbl Chir, 2003 128(6): p 47 3 48 0 74 US Department of Health and Human Services, Food and Drug Administration,... judgement” [28] Whether this recommendation is of use to specialists in the field of blood management is debatable But it gives a concept of hyperbaric oxygen therapy in blood management Severe anemia is usually treated with pressures up to a maximum of 2–3 atm The time of pressurization lasts about 60–90 minutes Depending on the clinical condition 1 34 Chapter 10 of the patient, the treatment is initially... Jehovah’s Witness Br J Haematol, 2002 118 (4) : p 1183–1186 54 Shander, A., et al Use of a hemoglobin-based oxygen carrier in the treatment of severe anemia Obstet Gynecol, 20 04 103(5, Pt 2): p 1096–1099 123 55 Lanzkron, S., et al Polymerized human Hb use in acute chest syndrome: a case report Transfusion, 2002 42 (11): p 142 2– 142 7 56 Mullon, J., et al Transfusions of polymerized bovine hemoglobin in a patient... Stebbing The intravenous injection of oxygen gas as a therapeutic measure Lancet, 1916 (August 19): p 321–323 11 Howitt, H.O The subcutaneous injection of oxygen gas CMAJ, 19 14 4: p 983–985 137 12 Haldane, J.S The therapeutic administration of oxygen BMJ, 1917 (February 10): p 181–183 13 Boerema, I., et al Life without blood Ned Tijdschr Geneeskd, 1960 1 04: p 949 –9 54 14 Boerema, I., N.G Meyne, W.K Brummelkamp,... 46 2 46 8 41 Meier, J., et al Hyperoxic ventilation reduces 6-hour mortality at the critical hemoglobin concentration Anesthesiology, 20 04 100(1): p 70–76 42 Meier, J., et al Hyperoxic ventilation reduces six-hour mortality after partial fluid resuscitation from hemorrhagic shock Shock, 20 04 22(3): p 240 – 247 43 Weiskopf, R.B., et al Oxygen reverses deficits of cognitive function and memory and increased heart . Shock, 2005. 24( 3): p. 245 –2 54. 44 Maevsky, E., et al. Clinical results of Perftoran application: present and future. Artif Cells Blood Substit Immobil Biotech- nol, 2005. 33(1): p. 37 46 . 45 Spahn,. A, 20 04. 71 (4) : p. 644 –651. 36 Khopade, A.J., S. Khopade, and N.K. Jain. Develop- ment of hemoglobin aquasomes from spherical hydroxya- patite cores precipitated in the presence of half-generation poly(amidoamine). S.A., et al. The life-sustaining capacity of human poly- merized hemoglobin when red cells might be unavailable. J Am Coll Surg, 2002. 195 (4) : p. 44 5 45 2; discussion 45 2 45 5. 51 Sloan, E.P.,