Hemoglobin based oxygen carriers

# Overview
At the beginning of 20th century excellent work of Landsteiner and co-authors combine with significant progress in fields of heart and circulation physiology initiated development of modern transfusion medicine allowing that blood transfusion become a standard part of medical treatment.
Complexity of blood compatibility, lack of suitable anticoagulants, insufficient storage methods, and a disproportionate between demand and availability demonstrated in early the stages of transfusion medicine the need to find one universal blood substitute (red blood cells substitute or oxygen carrier substitute).
Perfect "blood substitute" was defined as one which mimics the oxygen-carrying capacity of haemoglobin, which requires no cross-matching or compatibility testing, with a long shelf life, which exhibits a long intravascular half life (over days and weeks), and is free of side effects and pathogens. Two main types of blood substitutes are in development, haemoglobin-based oxygen carriers and perfluorocarbon emulsions.
The search for a haemoglobin-based oxygen carrier began during World War II, and stroma-free haemoglobin has been investigated as an oxygen carrier since the 1940s. Early attempts and optimism in developing haemoglobin-based oxygen carrier were very quickly confronted with significant side effects which at the level of knowledge and technology of the time, could not be promptly eliminated. To overcome this problem, several types of Hb modification methods (purification, cross-linkage, polymerisation)  were developed in a last few decades. The appearance of HIV and AIDS in the 1980s, HCV in 1990s and Creutzfeld-Jakob disease with increasing sensationalism was the impulse necessary to improve blood safety but also renewed impetus for development of infection safe blood substitutes. Actually several haemoglobin-based oxygen carriers were studied in a clinical trials. In general, the new generation of haemoglobin based oxygen carriers have been shown to successfully reduce or eliminate the need for allogeneic blood transfusions in patients under various clinical conditions. Taking into consideration that the major limitation of haemoglobin solutions is still the short intravascular half-life, haemoglobin solutions could serve mainly as a bridge to transfusion, rather than as a definitive replacement for blood. Accordingly, significant progress in development as well as in registration affairs, commercialization, and routine applications could be expected in next few years. Possibility do decrease need of packed red blood cells through application of haemoglobin-based oxygen carriers in a situation of continuos decline of blood donation combine with the increased demand for blood transfusion (increasing aging of population, increasing incidence of invasive diagnostic, etc) have to initiate strategy how to incorporate HBOCs in Blood Bank logistic and transfusion practice.

# Introduction
The development of a perfect blood substitute has been ongoing for many years . Such a product would have certain advantages over human red cells,  including rapid and widespread availability, fewer requirements with regard to storage, transport, and compatibility testing, a longer shelf life, and a more consistent supply. An ideal substitute would be less antigenic than allogenic red cells, and would have less risk of disease transmission. Two main types of blood substitutes are in development; haemoglobin-based oxygen carrier (HBOCs) and perfluorocarbon emulsions . This review gives one short historical overview and summarised development and current clinical implementation of haemoglobin-based oxygen carriers. In addition we are attempting to initiate a development of strategy in regards to incorporation of  products in blood bank logistics and clinical practice.

# Historical review and actual statement
The idea to use a blood substitute is old as well as human intention to resuscitate a life with transfusion of real blood. In the past, many but often obscure trials were conducted. One recommendation from Sir Christopher Wren (17th century) who suggested wine and opium as blood substitute . At the beginning of the 20th century, the development of modern transfusion medicine initiated through the excellent work of Landsteiner and co-authors opened the possibility to understanding the general principle of blood group serology . Simultaneously, significant progress was made in the fields of heart and circulation physiology as well as in the understanding of the mechanism of oxygen transport and tissue oxygenation . These two points paved the way for blood transfusion to become a standard part of medical treatment. Complexity of blood compatibility, lack of suitable anticoagulants and insufficient storage methods combined with a disproportion between demand and availability implicated just in early phase of transfusion medicine needs to find one universal blood substitute. The term "blood substitute" is a misnomer. Under the term blood substitute, we understand in first line the substitution of a) facility of red blood cells e.g. haemoglobin as oxygen carriers and b) volume substitution. More accurately the term means "red-cell substitutes" or solutions of haemoglobin or non-haemoglobin based oxygen carriers.
Restrictions in applied transfusion medicine especially in disaster situations such as World War Two lay the grounds for an accelerated research in the field of blood substitutes . Early attempts and optimism in developing blood substitutes were very quickly confronted with significant side effects which according to for that time actual level of knowledge and technology could not be promptly eliminated. Appearance of HIV and infection in the 1980s with consecutive public sensibilisation was the impulse necessary to improve blood safety but also renewed impetus for development of infection-safe blood substitutes . This situation was more intense with the advent of HCV and Creutzfeld-Jakob Disease which showed the absence of absolutely safe blood . The continuous decline of blood donation combined with the increased demand for blood transfusion (increased ageing of population, increased incidence of invasive diagnostic, chemotherapy and extensive surgical interventions, terror attacks, international military conflicts) and positive estimation of investors in biotechnology branch make for a very positive environment for further development of blood substitutes . At the end of 2003 and March 2004  news that a selected patients in the Stockholms Karolinska Hospital and in two trauma centres in San Diego received red blood cell substitute without serious adverse events effects was just one positive reflection of  the described constellation.
Taking into consideration that blood was used as resuscitation fluid with the main goal of improving oxygenation  research interest was in first line to develop substitute which  mimic oxygen-carrying capacity of haemoglobin. In addition,  an ideal blood substitute was define as one 1.) that required no cross-matching or compatibility testing; 2.) with a long shelf life over a wide range of ambient temperatures; 3.) which exhibit a long intravascular half life (over days and weeks) 4.) free of side effects and pathogens.
Up until now, two types of oxygen carriers have been established: Perfluorocarbon emulsion and haemoglobin-based oxygen carrier (HBOCs)  . According to literature a significant increase of published case reports in which HOBCs were under different conditions applied was observed in the last few year .

# Haemoglobin-based oxygen carriers
The general task of blood within the frame of classic transfusion medicine is oxygen tissue supply (oxygen transport from lung to tissue, oxygen release and picking up carbon dioxide). All of this is accomplished with haemoglobin (Hb), the oxygen carrier protein contained within red cells. According to this simplified postulation, early attempts to develop blood substitutes was focused on simple cell-free solution of haemoglobin . 
Haemoglobin is a tetramer of two a and two b polypeptide chains, each of which is bound to an iron-containing heme group which each bind one oxygen molecule. This oxygen heme bond results in a conformational change in haemoglobin molecule, which progressively increases the affinity of haemoglobin for additional oxygen molecules. The main consequence is that the small change in oxygen partial pressure results in a large change in the amount of oxygen bound or released by the haemoglobin. This is widely known as oxygen-haemoglobin dissociation curves . Under conditions of increased pH or decreased temperature or 2,3 diphosphoglycerate (2,3-DPG, product of RBC glycolytic pathway) oxygen-haemoglobin dissociation curve is shifted to the left resulting in an increased affinity of haemoglobin for oxygen. In contrast, by decreased pH increased of temperature or an increase of 2,3-DPG-concentration curve is shifted to the right allowing the release of oxygen to tissue at higher than normal oxygen partial pressure . According to modern trends this ability today could be termed the intelligent natural nanotechnique product. However, it is of great importance that cell free haemoglobin maintains its ability to transport oxygen outside of the RBC. Stroma-free haemoglobin has been investigated as an oxygen carrier since the 1940s, when researchers realized that native haemoglobin is not antigenic. The ability to transport oxygen outside of the RBC and that application of haemoglobin solution did not require compatibility testing and allowed sterilisation promote isolated Hb as a substitute for red cells .
Further investigation and evaluation showed that unmodified cell-free haemoglobin had limitations, such as: an oxygen affinity that was too high for effective tissue oxygenation; a half-life within the intravascular space that was too short to be clinically useful; and a tendency to undergo dissociation in dimers with resultant renal tubular damage and toxicity . Early studies conducted in experimental animals showed that infusion of free haemoglobin caused also substantial increase in oncotic pressure because of its hyperosmolarity, coagulopathy, and hypertension . The general problem was that solutions of acellular haemoglobin were not as effective at oxygenation as packed red blood cells because of their high affinity for oxygen. Red blood cells have adapted to release oxygen at an oxygen half-saturation pressure of haemoglobin (P-50) of approximately 26.5 mm Hg, as a result of the allosteric effects of red bloods cell 2,3-disphosphoglycerate (2,3-DPG), which shifts the oxyhaemoglobin curve to the right . Without 2,3-DPG, stroma-free haemoglobin has a P-50 of 12-14 mm Hg, not allowing for the adequate release of oxygen to the tissues. This sides effects have been attributed to dissociation of a2b2 tetramer to ab dimers (short intravascular half life, high oxygen affinity, nephrotoxicity), contamination with RBC stroma and affinity of Hb for nitric oxide (abdominal pain, vasoconstrictive crises) . To overcome this problems several types of Hb modification methods (purification, cross-linkage, polymerisation)  were developed in the last few decades .
Haemoglobin can be cross-linked (a covalent bond between 2 globin chains is made through chemical modification), and then polymerised using reagents such as glutaraldehyde. These modifications result in a product that has a higher P50 than that of normal haemoglobin, and an increase in the plasma half life of up to 30 hours .
Prevention of rapid breakdown of tetramer into dimer could improve half life and consecutive also eliminate nephrotoxicity. In a first generation of modified HBOCs specific chemical cross-links are established between haemoglobin polypeptide chain to prevent the dissociation of the Hb tetramer  into dimers. Haemoglobin treatment with 3,5-dibromosalycil fumarate established strong covalent bond between a subunits (aa-cross-linked-Hb) and successfully prevent rapid tetramere dissociation (half life 12 hours compare to 6 hours of unmodified Hb). Efficiency of cross-linked Hb to transport and unload O2 was confirmed in a variety of shock animal models and there is no doubt that modified Hb solutions improve tissue oxygenation in similar rate as infusion of autologes or allogenes blood . In such form stabilised haemoglobin (diaspirin cross-linked haemoglobin, DCLHb; trade name HemAssist; Baxter Healthcare Corp) reached Phase III clinical trials. This is the most widely studied of the haemoglobin-based blood substitutes, used in more than a dozen animal and clinical studies . It has the advantages of a shelf life of approximately 9 months frozen and 24 hours refrigerated. Its intravascular half-life is limited to 2-12 hours and is dose-dependent. In phase II clinical studies, HemAssist increased perfusion and oxygen consumption in patients with septic shock and in other critically ill patients. This product underwent phase III clinical trials for coronary artery bypass grafting procedures and was determined to decrease the need for transfused packed red blood cells. The adverse effects include hypertension and gastrointestinal distress. Observed vasoconstriction as serious side effects manifested as an increase in systemic and pulmonary artery pressure without normalizing cardiac output or restoring intravascular volume. Decreases in the cardiac index may impair optimum oxygen delivery and outweigh the advantage of an oxygen-carrying solution. Severe vasoconstriction complications was reason for terminating this clinical trial  . The possible vasoconstriction mechanism involved penetration of modified (but unpolymerised) Hb molecules into interstitial space of the subendothelial layers of vessel walls with consecutive nitric oxide scavenging and a sensitisation of peripheral a-adrenergic receptors. NO produced by endothelial cells affect smooth muscle cells of the vessel wall  and modulate the vascular tone toward vasodilatation. Extravased Hb scavenges NO and shift vasomotor tone toward vasoconstriction . This problem was relative successfully solved with polymerisation (o-rafinose, glutaraldehyde) of the haemoglobine molecule. For example glutaraldehyde target specific amino groups and polymerised haemoglobin (polyhaemoglobin). Polyhaemoglobin (Poly-Hb) composed from 4-5 Hb molecules shows variation in molecular size and configuration, has intrvascular dwell times up to 24 hours and does not penetrate (or reduced penetrate) to subendothelium. Alternative to polymerisation Hb can be conjugated to variety of larger molecules such as dextran, polyoxyethylene or could be genetic modified and residual tetramers are with additional methods removed. Although  these processes are designed to optimise cross-linking with consecutively reduction of  vasoconstrictive HBOCs effects and prolongation of intravascular half life .
Hemolink (Hemosol, Inc., Missiassauga, Canada) is a haemoglobin solution that contains cross-linked an o-rafinose polymerised human haemoglobin which is currently in Phase II trials in cardiothoracic surgery in USA. Previous conducted Phase III in Canada demonstrated the effectiveness of Hemolink as substitute to conventional transfusion in cardio surgery patients [15,16,17). The intravascular half-life is 18 to 20 hours. The mode of excretion is not entirely clear, but a small amount is renal. Phase I clinical trials in healthy volunteers showed that the drug is fairly well tolerated, with dose-dependent moderate or severe abdominal pain and increase in mean arterial pressure. 
Two additional cross-linked polymers of bovine (Hemopure, Biopure, Cambridge, MA) and human (PolyHeme, Northfield Laboratories, Inc.) origin are being successfully applied in cardiac and abdominal surgery as well as in trauma patients . The intravascular half-life of Hemopure (polymerised form of bovine haemoglobin with a P-50 of 30 mm Hg)  is approximately 24 hours, and the excretion is non-renal . Administration of Hemopure leads to vasoconstrictive effects that may increase systemic and pulmonary vascular resistance with resultant decreases in cardiac index. The authors did emphasize that the product served as a bridge over days, until blood became available, or the patients own red cells were regenerated . Hemopure is undergoing phase III clinical trials as a perioperative alternative to red blood cell transfusion in orthopedic surgery in the United States, the European Union, Canada, and South Africa  .
PolyHeme is made from pyridoxylated polymerized outdated human blood with an intravascular half-life of 24 hours and a shelf life of longer than 12 months (refrigerated). Its P-50 is 28-30 mm Hg, thereby giving it favorable oxygen-unloading characteristics. In a phase II randomised trial in patients with acute trauma, this product reduced the required number of allogeneic red blood cell transfusions. No adverse clinical events including vasoactive properties were observed in this trial . Currently, this product is undergoing phase III studies for the treatment of patients with significant acute blood loss.
However, intensive studies conducted on animals as well as clinical observation showed the present generation of blood substitutes to successfully reduce or eliminate the demand for allogeneic blood transfusion . Even significant improved they are only oxygen carriers. The absence of enzymes which are integral to red blood cells and functional oxidation of Fe (II) to Fe (III) with consecutive formation of free radicals in some clinical situation could have deleterious effects .
Lack of tissue oxygen supply (severe haemorrhagic shock, stroke, myocardial infarction, organ transplantation) leads to ischaemia with alterations in metabolic reactions producing hypoxanthine and activating the enzyme xanthine oxidase. When the tissue is reperfused with oxygen carrying fluid, xanthine oxidase converts oxygen and hypoxanthine into superoxide. By several mechanisms, superoxide results in the formation of oxygen radicals with consecutive tissue injury . Superoxide dismutase (SOD) and catalase (CAT) in red blood cell converts superoxide into hydrogen peroxide that is in turn converted into water and oxygen . Considering that the described oxygen carriers do not contain these enzymes, this application could cause increased ischaemiareperfusion injury in certain conditions. Studies with polyhaemoglobin cross-linked with trace amounts of CAT and SOD showed that PolyHbSODCAT removes significantly more oxygen radicals and peroxides, stabilizes the cross-linked haemoglobin and decreased oxidative iron and hem release and generally reduces ischemia-reperfusion injury . Cross-linking these enzymes to PolyHb is important because otherwise, free SOD and CAT are removed rapidly from the circulation. In the form of PolyHbSODCAT, these enzymes circulate with a half-time more comparable with PolyHb which is about 24 h in human . A glutaraldehyde cross-linked bovine Hb was covalently attached with CAT and SOD in an attempt to prophylactically reduce ischemia-reperfusion injury (McGill University, Montreal Canada) is still in pre-clinical evaluation . In a study with the reperfusion of ischaemic rat intestine, PolyHbSODCAT significantly reduced the increase in oxygen radicals caused by PolyHb. HemoZyme (SynZyme Technologies USA) is polynitroxylated human Hb designed to reduce oxidative potential of Hb is in pre-clinical evaluation .
Alternatively to modified human or bovine Hb, the continuous development of recombinant techniques opened the possibility to the production of Hb in micro-organisms. So produced Hb is free from mammalian infectious agens and allowed possibility to be designed and constructed with specific conformational and functional characteristics that render them suitable for application in different clinical situations. In contrast to HBOCs solutions in which chemically modified Hb was present in heterogeneous mixture of different size polymers recombinant Hb represents homogeneous and stable polymer. Transfusion experiments performed on mice showed that recombinant Hb maintains physiologically relevant oxygen and heme affinity, stability toward denaturation and oxidation, and effective oxygen delivery as indicated by reduced cerebral ischemic damage .
One other form of HBOCs is encapsulated haemoglobin. In the 1950s, the first form of encapsulated haemoglobin was developed but limited technical possibility and absence of public interest slowed further development until the HIV crisis . Liposome-encapsulated haemoglobin (LEH) has been found to be an effective oxygen carrier, without the adverse effects of vasoconstriction . The liposome encapsulation appears to increase plasma retention time; however, adverse immune interactions occur with the liposome. Microcapsulation of Hb opens the possibility to constructing  real artificial red cells which contain some enzymes (SOD, CAT, reducing agents, 2,3 DPG) which are involved in reduction of ischemia reperfusion injury and solve problem of  methaemoglobin formation. This product is still in early experimental phase and large-scale production is considered difficult due to costs and technical constraints .
According to different property of HBOCs (or different stages of development), side effects and intention of investors for a prompt commercialisation, some of these products are in different phase of clinical trial and some are still registered for human or animal application.

# Conclusion and Perspectives
In general, haemoglobin-based oxygen carriers have been shown to reduce or eliminate the need for allogeneic blood transfusions in patients undergoing orthopaedic surgery, elective abdominal surgery, and coronary artery bypass graft surgery. Trauma victims can benefit from a rapidly available universal oxygen-carrying volume expander that does not require cross matching. HBOCs may also serve as a bridge to transfusion in patients for whom blood is temporarily difficult to find. They may also serve as a bridge in the temporary support of a patient who will not accept blood and who has a reasonable chance of recovering an adequate haemoglobin/ hematocrit within a few days. Blood substitutes  can also be used to prime cardioplegia machines during cardiac procedures in order to decrease the amount of packed red cells used. Given their low viscosity, substitutes can be used for reperfusion of ischemic organs during strokes or myocardial infarctions. These products potentially can serve as an organ preservative prior to organ transplantation in order to decrease reperfusion injury. Significant improvements in last few year have been made and some of the HOBCs have become available for routine use. The major limitation of haemoglobin solutions is still the short intravascular half-life. A transfused red cell can persist in the circulation for several weeks in a patient with no active bleeding or hemolysis. Taking into consideration that application of a haemoglobin solution requires frequent replacement significant costs could be considered. Because of this limitation, haemoglobin solutions could serve mainly as a bridge to transfusion, rather than as a definitive replacement for blood.
According significant progress in development as well as in registration affairs commercialisation and routine apply could be expect in next few years. One other side clear regulatory guidelines for application of HBOCs is still not established. What we now need is one strategy when and under which condition HBOCs could and have to be transfused. It is of great importance to construct a framework for application of HBOCs based on one broad consensus of different clinical specialists under co-ordination of blood banks or blood institutes.

# Military Use
In the 1980s, an HBOC was developed by the US Army at the Letterman Army Institute of Research (LAIR) which did not need typing. However, in clinical trials the HBOC were proven to be problematic, with more deaths using the HBOC than in the control group. Yet, their use would be of value to sustain the wounded in military conflicts. PolyHeme is currently in field trials with the US Army. Experiments continue (Blood volume and cardiac index in rats after exchange transfusion with haemoglobin-based oxygen carriers) with requirements include long shelf-life, no need for refrigeration, and minimal side-effects. (Oxygen Carriers Coursing Through Clinical Trials)