Haemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers are one of two main types of oxygen-carrying blood substitutes in development, the other one being perfluorocarbon emulsions. As of June 2008 there are no haemoglobin-based oxygen carriers or perfluorocarbon emulsions available for commercial use in North America or Europe.^[1][2] The only countries where these products are available for general use is South Africa and Russia.^[1]

This is because they significantly increase the risk of death and myocardial infarction.^[3] It has been recommended that further phase III trails not be conducted until these products are as effective as the current standard of care.^[1]

Optimism about near term approval of oxygen carriers has decreased recently due to poor result from a number of clinical trials.^[1]

Classification

The development of a “perfect” blood substitute has been going on for many years.^[4] It is hoped that 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.^[5]

Haemoglobin-based oxygen carriers

Unmodified cell-free haemoglobin

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.^[6][7]

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.^[4][6][8][9] 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.^[4][9] 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.^[4][6]

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.^[4][10] 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.^[10] 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.^[6][8] 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).^[6][11] To overcome this problems several types of Hb modification methods (purification, cross-linkage, polymerisation) were developed in the last few decades.^[6][7][8]

Crosslinked haemoglobin

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.^[4][6][7]

Prevention of rapid breakdown of tetramere into dimere 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.^[8]

HemAssist

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.^[6][12][13] 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.^[6][13] 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.^[8][11]

Polymerised haemoglobin

The problem of vasoconstriction was relative successfully solved with polymerisation (o-raffinose, glutaraldehyde) of the haemoglobin 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.^[4][7]

Hemolink

Hemolink (Hemosol, Inc., Mississauga, 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.^[14][15][16] 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.

Hemopure and PolyHeme

Two additional cross-linked polymers of bovine (Hemopure, Biopure, Cambridge, MA) and human (PolyHeme, Northfield Laboratories, Inc.) origin have been used in trials during cardiac and abdominal surgery as well as in trauma patients.^{[17][18][19][20]} 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.^[21] 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 patient’s own red cells were regenerated.^[22] 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.^[22]

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.^[19][20] 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.^{[4][6][19][22][23]}

OxyVita

OxyVita Hb is a polymerised HBOC developed at the University of Maryland, Baltimore and undergoing pre-clinical studies in the United States. Polymerisation is via a novel synthetic process involving the linkage of activated carboxyl groups with lysyl residues, to form a so-called "zero-linked" polymerised haemoglobin lacking chemical residues. The zero-linked polymerisation process can be applied to a wide variety of mammalian haemoglobins, resulting in the formation of a very large superpolymer, with an average molecular weight of 17MDa.

OxyVita Hb has a low P50 and exhibits low cooperativity (n=1.2). The high molecular weight of OxyVita reduces extravasation from the circulatory system, potentially reducing the possibility of renal failure. An intravascular time greater than 10 hours has been observed in cats. Several preclinical studies in cats and various other mammals have shown no increase in normal blood pressure after transfusion with OxyVita Hb.^{[24][25][26][27][28][29][30][31]}

Complexes with superoxide dismutase and catalase

HBOCs are only oxygen carriers, and 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.^[32] 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.^[33] Superoxide dismutase (SOD) and catalase (CAT) in red blood cell converts superoxide into hydrogen peroxide that is in turn converted into water and oxygen.^[33] Considering that the described oxygen carriers do not contain these enzymes, this application could cause increased ischaemia–reperfusion injury in certain conditions. Studies with polyhaemoglobin cross-linked with trace amounts of CAT and SOD showed that PolyHb–SOD–CAT 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.^[7] Cross-linking these enzymes to PolyHb is important because otherwise, free SOD and CAT are removed rapidly from the circulation. In the form of PolyHb–SOD–CAT, these enzymes circulate with a half-time more comparable with PolyHb which is about 24 h in human.^[34] 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.^[7] In a study with the reperfusion of ischaemic rat intestine, PolyHb–SOD–CAT 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.^[6]

Recombinant haemoglobin

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 agents 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.^[35]

Encapsulated haemoglobin

One other form of HBOCs is encapsulated haemoglobin, where the haemoglobin is packed inside an artificial neohemocyte ("new blood cell"). 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.^[7] 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.^[7]

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.

Safety

HBOCs have not been found to be safe in human. They increase both the risk of death and risk of myocardial infarction.^[3]

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.^{[citation needed]} PolyHeme is currently in field trials with the US Army.^{[citation needed]} Experiments continue^[36] with requirements include long shelf-life, no need for refrigeration, and minimal side-effects.^[37]

History

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 .^[4] 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.^[38] 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.^[39][40] 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.^[6] 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.^[4] This situation was more intense with the advent of HCV and Creutzfeld-Jakob Disease which showed the absence of absolutely safe blood.^[4][41] 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.^[41] At the end of 2003 and March 2004 news that a selected patients in the Stockholm’s 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,^[8] 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).^[4][5][8] According to literature a significant increase of published case reports in which HOBC’s were under different conditions applied was observed in the last few years.^[6]

References

1. ^ ^{a b c d} "UpToDate Inc.". http://www.uptodate.com/online/content/topic.do?topicKey=transfus/11560&selectedTitle=1~8&source=search_result. 
2. ^ Spahn DR, Kocian R (2005). "Artificial O2 carriers: status in 2005". Curr. Pharm. Des. 11 (31): 4099–114. doi:10.2174/138161205774913354. PMID 16378514. http://www.bentham-direct.org/pages/content.php?CPD/2005/00000011/00000031/0011B.SGM. 
3. ^ ^{a b} Natanson C, Kern SJ, Lurie P, Banks SM, Wolfe SM (May 2008). "Cell-free hemoglobin-based blood substitutes and risk of myocardial infarction and death: a meta-analysis". JAMA 299 (19): 2304–12. doi:10.1001/jama.299.19.jrv80007. PMID 18443023. 
4. ^ ^{a b c d e f g h i j k l} Squires JE (2002). "Artificial blood". Science 295 (5557): 1002–5. doi:10.1126/science.1068443. PMID 11834811. http://www.sciencemag.org/cgi/pmidlookup?view=long&pmid=11834811. 
5. ^ ^{a b} Remy B, Deby-Dupont G, Lamy M (1999). "Red blood cell substitutes: fluorocarbon emulsions and haemoglobin solutions". Br. Med. Bull. 55 (1): 277–98. doi:10.1258/0007142991902259. PMID 10695091. http://bmb.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=10695091. 
6. ^ ^{a b c d e f g h i j k l m} Reid TJ (2003). "Hb-based oxygen carriers: are we there yet?". Transfusion 43 (2): 280–7. doi:10.1046/j.1537-2995.2003.00314.x. PMID 12559026. http://www.blackwell-synergy.com/openurl?genre=article&sid=nlm:pubmed&issn=0041-1132&date=2003&volume=43&issue=2&spage=280. 
7. ^ ^{a b c d e f g h} Chang TM (2003). "Future generations of red blood cell substitutes". J. Intern. Med. 253 (5): 527–35. doi:10.1046/j.1365-2796.2003.01151.x. PMID 12702030. http://www.blackwell-synergy.com/openurl?genre=article&sid=nlm:pubmed&issn=0954-6820&date=2003&volume=253&issue=5&spage=527. 
8. ^ ^{a b c d e f g} Spahn DR, Pasch T (2001). "Physiological properties of blood substitutes". News Physiol. Sci. 16: 38–41. PMID 11390946. http://nips.physiology.org/cgi/pmidlookup?view=long&pmid=11390946. 
9. ^ ^{a b} Hébert PC (1998). "Transfusion requirements in critical care (TRICC): a multicentre, randomized, controlled clinical study. Transfusion Requirements in Critical Care Investigators and the Canadian Critical care Trials Group" (– ^{Scholar search}). Br J Anaesth. 81 Suppl 1: 25–33. PMID 10318985. http://bja.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=10318985. ^{[dead link]}
10. ^ ^{a b} Rabiner SF, O'Brien K, Peskin GW, Friedman LH (1970). "Further studies with stroma-free hemoglobin solution". Ann. Surg. 171 (4): 615–22. doi:10.1097/00000658-197004000-00020. PMC 1396709. PMID 5436128. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1396709. 
11. ^ ^{a b} Schechter AN, Gladwin MT (2003). "Hemoglobin and the paracrine and endocrine functions of nitric oxide". N. Engl. J. Med. 348 (15): 1483–5. doi:10.1056/NEJMcibr023045. PMID 12686706. http://content.nejm.org/cgi/pmidlookup?view=short&pmid=12686706&promo=ONFLNS19. 
12. ^ Barve A, Sen AP, Saxena PR, Gulati A (1997). "Dose response effect of diaspirin crosslinked hemoglobin (DCLHb) on systemic hemodynamics and regional blood circulation in rats". Artif Cells Blood Substit Immobil Biotechnol 25 (1-2): 75–84. doi:10.3109/10731199709118899. PMID 9083628. 
13. ^ ^{a b} Saxena R, Wijnhoud AD, Carton H, et al. (1999). "Controlled safety study of a hemoglobin-based oxygen carrier, DCLHb, in acute ischemic stroke". Stroke 30 (5): 993–6. PMID 10229733. http://stroke.ahajournals.org/cgi/pmidlookup?view=long&pmid=10229733. 
14. ^ Cheng DC (2001). "Safety and efficacy of o-raffinose cross-linked human hemoglobin (Hemolink) in cardiac surgery". Can J Anaesth 48 (4 Suppl): S41–8. PMID 11336436. 
15. ^ Hill SE, Gottschalk LI, Grichnik K (2002). "Safety and preliminary efficacy of hemoglobin raffimer for patients undergoing coronary artery bypass surgery". J. Cardiothorac. Vasc. Anesth. 16 (6): 695–702. doi:10.1053/jcan.2002.128416. PMID 12486649. http://linkinghub.elsevier.com/retrieve/pii/S1053077002001258. 
16. ^ Cheng DC, Mazer CD, Martineau R, et al. (2004). "A phase II dose-response study of hemoglobin raffimer (Hemolink) in elective coronary artery bypass surgery". J. Thorac. Cardiovasc. Surg. 127 (1): 79–86. doi:10.1016/j.jtcvs.2003.08.024. PMID 14752416. http://linkinghub.elsevier.com/retrieve/pii/S0022522303015575. 
17. ^ Levy JH, Goodnough LT, Greilich PE, et al. (2002). "Polymerized bovine hemoglobin solution as a replacement for allogeneic red blood cell transfusion after cardiac surgery: results of a randomized, double-blind trial". J. Thorac. Cardiovasc. Surg. 124 (1): 35–42. doi:10.1067/mtc.2002.121505. PMID 12091806. http://linkinghub.elsevier.com/retrieve/pii/S0022522302000375. 
18. ^ Sprung J, Kindscher JD, Wahr JA, et al. (2002). "The use of bovine hemoglobin glutamer-250 (Hemopure) in surgical patients: results of a multicenter, randomized, single-blinded trial". Anesth. Analg. 94 (4): 799–808, table of contents. doi:10.1097/00000539-200204000-00006. PMID 11916776. http://www.anesthesia-analgesia.org/cgi/pmidlookup?view=long&pmid=11916776. 
19. ^ ^{a b c} Gould SA, Moore EE, Hoyt DB, et al. (1998). "The first randomized trial of human polymerized hemoglobin as a blood substitute in acute trauma and emergent surgery". J. Am. Coll. Surg. 187 (2): 113–20; discussion 120–2. doi:10.1016/S1072-7515(98)00095-7. PMID 9704955. http://linkinghub.elsevier.com/retrieve/pii/S1072-7515(98)00095-7. 
20. ^ ^{a b} Gould SA, Moore EE, Hoyt DB, et al. (2002). "The life-sustaining capacity of human polymerized hemoglobin when red cells might be unavailable". J. Am. Coll. Surg. 195 (4): 445–52; discussion 452–5. doi:10.1016/S1072-7515(02)01335-2. PMID 12375748. http://linkinghub.elsevier.com/retrieve/pii/S1072-7515(02)01335-2. 
21. ^ Pearce LB, Gawryl MS (2003). "The pharmacology of tissue oxygenation by biopure's hemoglobin-based oxygen carrier, Hemopure (HBOC-201)". Adv. Exp. Med. Biol. 530: 261–70. PMID 14562723. 
22. ^ ^{a b c} Levy JH (2003). "The use of haemoglobin glutamer-250 (HBOC-201) as an oxygen bridge in patients with acute anaemia associated with surgical blood loss". Expert Opin Biol Ther 3 (3): 509–17. doi:10.1517/14712598.3.3.509. PMID 12783619. http://www.expertopin.com/doi/abs/10.1517/14712598.3.3.509. 
23. ^ Greenburg AG, Kim HW (2004). "Hemoglobin-based oxygen carriers". Crit Care. 8 Suppl 2 (Suppl 2): S61–4. doi:10.1186/cc2455. PMID 15196328. http://ccforum.com/content/8%20Suppl%202//S61. 
24. ^ Bucci, E., Rasynaska, A., Kwansa, H., Matheson-Urbaitis, B., O’Hearne, M., Ulatowski, J.A. and Koehler, R.C. (1996) J. Lab Clin Med 128, 146-153.
25. ^ Estep, T., Bucci, E., Farmer, M., Greenburg, G., Harrington, J.P., Kim, H.W., Klein, H., Mitchell, P., Nemo, G., Olsen, K., Palmer, A., Valeri, C.R. and Winslow, R.M. (2008) Transfusion January 7 (Epub).
26. ^ Hirsch, R.E. and Harrington, J.P. (2000) Einstein Quart. J. Biol. Med. 27, 123-134.
27. ^ Jahr, J.S., Walker, V. and Manoochehri, K. (2007) Curr. Opin. Anaesthesiol. 20, 325-330.
28. ^ Matheson, B., Kwansa, H., Bucci, E., Rebel, A. and Koehler, R.C. (2002) J. Appl. Physiol. 93,
29. ^ Ness, P.N. and Cushing, M.M. (2007) Arch Pathol Lab Med 131, 734-741.
30. ^ Rohlfs, R.J., Bruner, E., Chiu, A., Gonzales, A., Gonzales, M.L., Magde, D., Magde, M.D., Vandergriff, K.D. and Winslow, R.M. (1998) J. Biol. Chem. 273, 12128-12134.
31. ^ Winslow, R.M. (2007) Semin. Hematol. 44, 51-59.
32. ^ Reddy BR, Kloner RA, Przyklenk K (1989). "Early treatment with deferoxamine limits myocardial ischemic/reperfusion injury". Free Radic. Biol. Med. 7 (1): 45–52. doi:10.1016/0891-5849(89)90099-3. PMID 2753395. 
33. ^ ^{a b} Fattman CL, Schaefer LM, Oury TD (2003). "Extracellular superoxide dismutase in biology and medicine". Free Radic. Biol. Med. 35 (3): 236–56. doi:10.1016/S0891-5849(03)00275-2. PMID 12885586. http://linkinghub.elsevier.com/retrieve/pii/S0891584903002752. 
34. ^ D'Agnillo F, Chang TM (1998). "Polyhemoglobin-superoxide dismutase-catalase as a blood substitute with antioxidant properties". Nat. Biotechnol. 16 (7): 667–71. doi:10.1038/nbt0798-667. PMID 9661202. 
35. ^ Bobofchak KM, Mito T, Texel SJ, et al. (2003). "A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2 transporter". Am. J. Physiol. Heart Circ. Physiol. 285 (2): H549–61. doi:10.1152/ajpheart.00037.2003. PMID 12689854. http://ajpheart.physiology.org/cgi/pmidlookup?view=long&pmid=12689854. 
36. ^ "Blood volume and cardiac index in rats after exchange transfusion with hemoglobin-based oxygen carriers -- Migita et al. 82 (6): 1995 -- Journal of Applied Physiology". http://jap.physiology.org/cgi/content/full/82/6/1995+. Retrieved 2008-02-13. ^{[dead link]}
37. ^ "DefenseLink News Article: Oxygen Carriers Coursing Through Clinical Trials". Archived from the original on December 17, 2007. http://web.archive.org/web/20071217140851/http://www.defenselink.mil/news/Nov2005/20051110_3316.html. Retrieved 2008-02-13. 
38. ^ Bendum J: Geschichte der Bluttransfusion; in Mueller-Eckhardt C (ed): Transfusionsmedizin. Berlin Heidelberg New York, Springer, 1996, pp 3-18.
39. ^ Feigl EO (1983). "Coronary physiology". Physiol. Rev. 63 (1): 1–205. PMID 6296890. http://physrev.physiology.org/cgi/pmidlookup?view=long&pmid=6296890. 
40. ^ Lahiri S (2000). "Historical perspectives of cellular oxygen sensing and responses to hypoxia". J. Appl. Physiol. 88 (4): 1467–73. PMID 10749843. http://jap.physiology.org/cgi/pmidlookup?view=long&pmid=10749843. 
41. ^ ^{a b} Goodnough LT, Brecher ME, Kanter MH, AuBuchon JP (1999). "Transfusion medicine. First of two parts--blood transfusion". N. Engl. J. Med. 340 (6): 438–47. doi:10.1056/NEJM199902113400606. PMID 9971869. http://content.nejm.org/cgi/pmidlookup?view=short&pmid=9971869&promo=ONFLNS19.