The need for an alternative to allogeneic red blood cells (RBCs) remains a challenge that man has attempted to solve for more than a century. Although the fundamental function and sacred nature of blood was known since the first time of our era by prehistoric civilizations (Baudin-Creuza et al, 2008), concerns about the infectious and immunosuppressive dangers of allogeneic blood products persist, and the increased discrepancy between blood donation and consumption continues to feed the impetus towards the realization of alternative erythrocyte transfusion strategies (Napolitano, 2009). But while it has been demonstrated that research aimed at the development of blood substitutes based on either haemoglobin (Hb) or perfluorocarbon (PFC) emulsions has been ongoing, and in spite of the fact that heavy investments have been made by the pharmaceutical industry, the military, public institutions and researchers toward the development of a viable artificial blood substitute, no such product has yet been approved for widespread clinical use in North America or Europe (Kluger, 2010; Winslow, 2000; Winslow, 2006b; Akira, 2009). This paper reviews underlying issues in artificial blood substitutes with a view to critically evaluate past and current research in blood substitutes.
Blood has fascinated mankind throughout history, with past and current research demonstrating that blood has distinctive functions in the body that are absolutely vital for survival (Kjellstrom, 2003). It is therefore understandable that studies aimed at finding a viable replacement for allogeneic blood have interested many researchers, public and private institutions, and the military (Kim & Greenburg, 2006). In simple terms, “blood is a special type of connective tissue that is composed of white cells, red cells, platelets, and plasma” (Sarker, 2008 p. 140). Plasma, an extracellular material composed of water, salts, and assorted proteins, functions with platelets to facilitate blood clotting. The white blood cells reinforce the immune defence of the body by seeking out invading organisms or agents and reducing their effect in the body. The red blood cells, on their part, function to transport oxygen and carbon dioxide throughout the body, and are also responsible for the ‘typing’ phenomena (Sarker, 2008).
Research in blood substitutes have basically revolved around developing alternative means by which oxygen can be transported to vital organs in the body (Hae & Greenburg, 2004).
Hirsch & Harrington (2000) argue that the term “blood substitute” is a misnomer by virtue of the fact that no single product will ever replace the compound and varied multi-functionality of human blood. Since the principal function of erythrocytes in the blood is to transport oxygen to tissues, the concept of “blood substitute,” as presently used, refers to a therapeutic oxygen carrier as opposed to full replacement of human blood with some form of artificial blood alternative (Tsuchinda, 1998; Nose, 2004).
According to Tappenden (2007), “artificial oxygen carrier infusions offer volume expansion and oxygen carriage” (p. 3). To date, the advancement of artificial blood substitutes have mainly followed two diverse pathways of development, which are synthetic blood substitutes grounded on perfluorocarbons and haemoglobin-based oxygen carriers employing human or bovine haemoglobin as the main product (Chang, 2002). As postulated by Tappenden (2007), the fundamental objective of these two classifications of artificial blood substitutes, both in the elective and trauma setting, is to lessen the huge demand for allogeneic blood transfusions and sustain tissue oxygenation. The U.S. military have been at the centre of efforts made towards the development of artificial blood substitutes because of the huge demand of blood in the battlefield scenario coupled with the need to have blood units with a longer shelf-life to minimize blood wastage during war. Statistics reveal that almost two-thirds of the 1.3 million units of blood resources dispatched during the long-standing Vietnam conflict were wasted, while in excess of 100,000 units during the first Gulf War were disposed (Tappenden, 2007). The need to develop artificial blood substitutes have been further reinforced by the wide-range of risks and difficulties associated with allogeneic blood transfusions (Fleming et al, 2007).
Brief History of Blood Transfusions & Blood Substitutes
Kim & Greenburg (2009) acknowledge that the history and aetiology of blood substitutes is intimately related to that of blood transfusion and can be traced back to ancient human civilizations, implying that the concept of using artificial blood instead of human blood for transfusing patients is not new. In 1656, Sir Christopher Wren proposed that ale, wine, scammony, and even opium could be employed as blood substitutes, and even went further to infuse the materials into dogs to examine their effects (Squires, 2002; Kjellstrom, 2003). In 1854 while treating patients with Asiatic cholera, Edward Hodder and Thomas experimented with cow’s milk, and concluded that injection of the product into the circulation in place of blood was an absolutely viable, secure and legitimate procedure (Winslow, 2006). Another study conducted in 1883 by Sydney Ringer demonstrated “…that the excised ventricle of the frog would beat for some hours if supplied with an aqueous solution of sodium, potassium and calcium salts” (Winslow, 2006a p. 9). It is important to note that these and other early attempts to use blood substitutes substantially informed modern practice in artificial blood substitutes.
Contemporary scientific attempts to replace human blood started in the early 1900s with the discovery of human blood group antigens by Karl Landsteiner, which he classified into A, B, C (later christened O), and AB groups (Squires, 2002; Sarker, 2008). This discovery was critical to understanding why early blood transfusions had failed. Other scientific studies conducted in the early 1900s, according to Kim & Greenburg (2009), led to an informed comprehension of the oxygen transport/delivery role of the red blood cells and a “…new recognition that allogeneic blood transfusion can only be successful if a donor and a recipient have a matching blood type” (p. 813). Ottenberg in 1913 became the first scientist to successfully apply blood group serology to transfusion practice, but blood transfusions continued to remain severely restricted even after the scientist’s description of blood compatibility testing in large part due to twin challenges of absence of suitable anticoagulants and lack of storage methods (Squires, 2002; Winslow, 1996). These particular authors note that the two world wars precipitated progress on these two challenges, eventually allowing blood transfusion to become a standard procedure in medical practice.
The many challenges presented by allogeneic blood transfusions, and which have been discussed comprehensively in this paper, prompted researchers to start evaluating other viable alternatives that could replace or supplement transfusions based on human blood. Squires (2002) notes that researchers and institutions interested in developing these alternatives mainly concentrated on imitating the oxygen-carrying capability of haemoglobin. According to Simoni (2009), the ability of haemoglobin (Hb) to sustain life even in the absence of red blood cells has been distinguished since the 1930s. This observation, which is credited to Amberson, bred much hope for a breakthrough in the form of an oxygen-carrying blood substitute. In this context, as observed by Simoni (2009), “…a blood substitute was never thought to be a substance that could replace blood in all its functions, but an emergency resuscitative fluid that is capable of restoring blood volume and transporting oxygen” (p. 92).
The heavily invested efforts directed to the development of an artificial blood substitute mainly based on haemoglobin as the only natural oxygen carrier (Simoni, 2009), with Squires (2002) reinforcing this fact by noting that early attempts to develop artificial blood substitutes in the decade of the 1970s laid much focus on cell-free solutions of human haemoglobin. Kluger (2010) notes that “…approaches to developing HBOCs dealt with the fact that haemoglobin can bind and release oxygen but does not survive outside the red cell” (p. 538). However, problems which included short half-life of Hb in solution, unusually high oxygen affinity, and undesirable clinical outcomes such as depression, abdominal pain, hemoglobinuria and high instances of renal toxicity soon exhibited (Squires, 2002), prompting some researchers to contemplate the use of perfluorochemicals to act as oxygen carriers (Simoni, 2009). The perfluorochemicals have their own pitfalls as will be demonstrated in the review of past and current research in blood substitutes, thus it is only fair to say that efforts towards the development of these substitutes have not been largely successful due to a number of emergent challenges which have so far proved difficult to solve.
Current Risks & Difficulties in Blood Transfusion
Safety and adequacy continues to inform paradigms of nearly all blood donor screening programs worldwide (Stramer, 2007). Although transfusion of donor blood is a routine and safe practice, there exist several risks and difficulties with this procedure, and which continues to feed the impetus to develop artificial blood substitutes. In terms of difficulties, research has demonstrated that human red blood cells have exacting storage prerequisites devised to extend clinical effectiveness and curtail risk of bacterial contamination (Squires, 2002). Simoni (2009) argues that “the packed red blood cells must be kept refrigerated and have a shelf life of only 42 days” (p. 93). This observation considerably limits the availability of blood particularly in disaster sites or when countries engage in war. Research has also demonstrated that during storage the erythrocytes lose their respiratory capability as a result of the depletion of 2,3-diphosphoglycerate (DPG), which is critical in the sustenance of haemoglobin’s capacity to release oxygen (Simoni, 2009). Evidence has also been adduced to the fact that the total depletion of 2,3-DPG happens by three weeks of storage, causing adverse postoperative complications and higher mortality when infused into the recipients.
Donor blood require ‘typing’ and ‘cross-matching’ because they harbour blood group antigens, making it exceedingly difficult to use such blood in times of natural catastrophes, accident scenes and in the battlefront (Simoni, 2009). The increased demand and diminished supply of donor blood have been noted worldwide, making artificial blood substitutes attractive for the short-term replacement of red blood cells lost during surgery (Squires, 2002). According to available statistics, the World Health Organization (WHO) estimate the global annual demand for human blood to stand at 100 million units, and the U.S., which annually uses just about 12 million units of blood, projects a shortage of 3-4 million units per year by the year 2020 (Simoni, 2009). According to Tappenden (2007), England and Wales jointly require an estimated 8,000 units of blood per day, and the stock of O negative blood in the UK banks can only last for 5.1 days. It is imperative to note that the projected deficit of donated blood does not take into consideration the more acute demand for blood in natural catastrophes and during wars, thus the emergent need to develop viable blood substitutes.
In terms of risks, research has demonstrated that various potentially lethal diseases can be transmitted through allogeneic blood transfusions. Although countries have made heavy investments towards the development of blood testing programs (Sarker, 2008), the possibility of contracting blood-borne diseases still lurks (Simoni, 2009). It has been well demonstrated that the human immunodeficiency virus (HIV), hepatitis C virus, hepatitis B virus, West Nile virus, variant Creutzfeldt-Jacob disease (vCJD), and some bacterial infections could be transmitted through allogeneic blood transfusions (Stramer, 2007; Lefrere & Hewitt, 2009). Squires (2002) notes that before the development of comprehensive testing tools for HIV, the risk of transfusion-associated acquired immunodeficiency syndrome stood at just about 38 per 100,000 recipients of human blood, while Stramer (2007) observes that even with the benefits of using complicated and sensitive antibody testing procedures to screen blood for HIV, lookback analysis in the U.S. have led to the identification of 4 breakthrough HIV cases originating from 3 blood donations. In addition, research have demonstrated that “repeated transfusions may produce immunosuppressive effects and in some recipients allergic and haemolytic transfusion reactions and even graftversus-host disease” (Simoni, 2009 p. 93). These transfusion risks coupled with the difficulties of relying on donor blood provides the impetus for the development of disease-free artificial blood substitutes.
Benefits of Blood Substitutes
Tappenden (2007) acknowledges that artificial oxygen carriers have been developed to obviate the inadequacies of packed red cells, implying that these blood substitutes carry immense benefits to mankind if researchers succeed in controlling the side effects that have so far been associated with them. One obvious advantage of blood substitutes, according to Squires (2002), is that cross-matching tests to determine compatibility are not required as they are compatible with all blood types. Tappenden (2009) notes that this benefit prevents “the risk of ABO incompatibility associated with human error which still occurs despite rigorous checking of packed red cells in 1:34,000 units transfused” (p. 3). According to Squires (2002), an error in cross-matching allogeneic blood may result in haemolytic transfusion reaction in the recipient, the basis of rare but considerable transfusion-associated morbidity and mortality. Another advantage comes from the fact that cell-free haemoglobin can be sterilized by ultra-filtration and low heat to destroy or inactivate infectious bacteria and other agents, a strategy that cannot be applied to red blood cells (Squires, 2002).
Another noteworthy benefit of artificial blood substitutes is that since they are compatible with any blood group type, “they can be used in patients with alloautoantibodies such as those with sickle cell disease” (Tappenden, 2009 p. 3). It has been noted that some certain groups of patients, such as those professing the Jehovah Witness faith, are unable to accept allogeneic blood transfusions or human and animal proteins such as haemoglobin (Squirres, 2002). Such patients can benefit from perfluorocarbons as the only option if a blood transfusion is required as research has demonstrated that perfluorocarbon emulsions have an outstanding capability to transport oxygen and carbon dioxide without essentially binding to these gases. It is imperative to note that through partial liquid ventilation of the lungs, these emulsions have been effectively employed to premature newborns with a heightened respiratory distress syndrome (Squires, 2002). While normal human blood has a limited shelf life when cooled at 4oC and 5 hours at room temperature (Sojka & Sojka, 2007), blood substitutes have an extended shelf life of 1-3 years at room temperature, implying that they can be accumulated for use during natural catastrophes and military crises, or in trauma settings (Tappenden, 2009).
Even though many health institutions have put in place effective blood testing and screening procedures, the risk of disease transmission through allogeneic blood transfusion is ever present (Sarker, 2008; Henkel-Hanke & Oleck, 2007). In contrast, “blood substitutes provide a disease-free source of blood, which is of great benefit to countries with a high HIV/AIDS population, where disease-free blood is a limited resource and HIV and hepatitis transmission in blood remains widespread” (Tappenden, 2009 p. 3). This is a particularly important benefit to the least developed world, where government health institutions are least equipped to undertake effective blood screening procedures, and where HIV cases are rampant. South Africa, for example, has licensed the clinical use of the haemoglobin-based oxygen carrier Hemopure (HBOC-201) in part due to the high rate of HIV infections among the population (Tappenden, 2009; Fleming et al., 2007; Henkel-Hanke & Oleck, 2007).
Artificial blood substitutes are highly effective in oxygen delivery when compared to transfused allogeneic blood (Sarker, 2008). Tappenden (2009) reinforces this observation by noting that while transfused allogeneic blood may take up to 24 hours to attain optimal oxygen transfer capability due to 2,3 DPG depletion, artificial blood substitutes are known to attain optimal oxygen capacity immediately. In terms of immunological effects, it has been hypothesized that artificial oxygen carriers do nit prime the circulating neutrophils unlike transfusion with allogeneic blood, consequently curtailing the occurrence of multi-organ failure which may exhibit with use of allogeneic blood after long-standing storage. The availability of artificial blood products coupled with their ease of usage (as no cross-matching is required) not only ensures that blood is immediately available for infusion, but also guarantee that recipients can receive blood in pre-hospital settings, battlefield scenarios, and in inaccessible locations where allogeneic blood may be unavailable (Tappenden, 2009; Fleming et al., 2007; Sarker, 2008; Sojka & Sojka, 2007).
There is a real possibility that the cost of artificial blood substitutes may fall below that of packed red blood cells as they are not tied to many cost variables normally associated with allogeneic blood, such as donor enrolment, phlebotomy, management, admission, blood screening and testing, and refrigeration costs. Lastly, research carried on a rat model demonstrated that haemoglobin-based oxygen carriers “can reach post-stenotic and poorly perfused tissues with plasma flow where erythrocytes cannot due to their smaller size” (Tappenden, 2009 p. 4). Although haemoglobin-based oxygen carriers are 1/1000th the size of red blood cells, their cellular oxygen delivery is three times that of the erythrocytes.
An Ideal Blood Substitute
Greenburg (2009) argues that “blood is indisputably essential for life, and specifying the attributes of a solution to replace the life force of blood is a daunting task” (p. 415). As such, an appreciation and thorough understanding of the composition and structure of blood in the context of its multiple functions is generally perceived as an indispensable precondition for listing design specifications of an ideal blood substitute. While it is necessary to have a deeper understanding of the functions of blood (Greenburg, 2009), researchers must also study past experiments on artificial blood substitutes to come up with ways by which to circumvent the many shortcomings already attributed to blood substitutes developed to date (Akira, 2009). Such an effort, according to Greenburg (2009), will not only make it possible to define the characteristics needed for an ideal blood substitute, but it will guide concerted efforts aimed at refining the substitutes to meet international health standards and guarantee safety for recipients.
Among the major functions of blood, the provision of tissue perfusion with oxygen transported by the erythrocytes and the sustenance of vascular volume are critical for survival as demonstrated by the undesirable clinical outcomes usually associated with loss of vascular volume in trauma cases or in operative surgery that may result in loss of red blood cell mass (Greenburg, 2009). Consequently, an ideal blood substitute must not only ensure the maintenance of vascular volume, but must also effectively transport and deliver oxygen to tissues (Squires, 2001). Indeed, as acknowledged by Greenburg (2009), the role of haemoglobin and intravascular volume replacement has been the main object of research in an ideal blood substitute given the importance of the need to perfuse tissue with oxygen. However, many of the blood substitutes in various stages of clinical testing are still plagued by the unusually high affinity to oxygen (Baudin-Creuza et al, 2008; Sojka & Sojka, 2007).
According to Greenburg (2009), “blood provides cellular and molecular elements of the coagulation and immune systems and, by providing the vascular volume, serves as a communication pathway for hormonal and cytokine signalling and the delivery of nutrients and removal of metabolic waste products” (p. 415). These are vital functions that any ideal blood substitute must address. An ideal blood substitute must also contain elements and components that functions to not only sustain life and homeostasis, but also to participate in the protective responses to injury and the restorative processes usually associated with normal human blood (Baudin-Creuza et al, 2008; Greenburg, 2009). As observed by Simoni (2009), the colloid osmotic pressure of an ideal blood substitute must not exceed that of plasma if its life and homeostasis maintenance functions are to be preserved. As such, an ideal blood substitute must have a Bohr Effect (regulation of oxygen affinity by pH), must retain usual oncotic pressure and low viscosity, must maintain usual vasculature and endothelium, and must have the capacity for oxidative stability without causing damage to vital organs (Hirsch & Harrington, 2000).
Researchers argue that an ideal blood substitute must have the capacity to overcome the challenges that have so far been associated with haemoglobin-based oxygen carriers, namely short half-life of Hb in solution, abnormally high oxygen affinity, high incidences of renal toxicity, hemoglobinuria, abdominal pain and other detrimental clinical outcomes (Squires, 2002). An ideal blood substitute must also possess the characteristic of universal compatibility to all blood groups, not mentioning that it must be pathogen-free, non-hazardous, non-immunogenic and non-pyrogenic (Simoni, 2009). The exhibition of such features, according to Akira (2009), will demonstrate a clear advantage of artificial blood carriers over red blood cells. In addition, an ideal blood substitute must have an extended shelf-life, preferably at room temperature, must be easily available, and must have the capacity to survive in circulation for a number of weeks without losing its core functionalities. Indeed, many ongoing studies on the development of artificial blood substitutes rely on these preconditions for guidance and direction. With potential sales projected at about $12 billion annually according to available figures, the market for feasible artificial blood substitutes present one of the largest economic possibilities for any medical product currently under development (Simoni, 2009).
Past & Current Research in Blood Substitutes
Although much hope was raised in the 1980s and 1990s about the possibility of developing oxygen-carrying solutions based on either haemoglobin (Hb) or perfluorocarbon (PFC) emulsions that could be used as alternatives to erythrocytes for transfusion (Winslow, 2006b), available research demonstrates that there has been no true substitute for the human red blood cells, and that the various synthetic products currently under development may not have the capacity to replace the need for blood donation and allogeneic blood transfusion in the foreseeable future (Ness & Cushing, 2007). Haemoglobin-based oxygen carriers, in particular, bears many features that would serve as a useful adjunct to erythrocytes in clinical settings, but no research has so far succeeded in alleviating the multiple side effects and undesirable clinical outcomes of the products when administered to blood recipients (Hirsch & Harrington, 2000). However, ongoing studies are positive that these technologies have the potential to radically remodel the practice of transfusion medicine (Ness & Cushing). Essentially three designs serve as prototypes for oxygen therapeutics though current research has expanded its tentacles beyond the three designs, which include haemoglobin-based oxygen carriers, perflourocarbon emulsions, and haemoglobin-based oxygen carriers encapsulated within liposomes (Hirsch & Harrington, 2000). This section aims to critically evaluate the pathways of past and current research into the development of the three designs under two broad categories – haemoglobin-based oxygen carriers and perflourocarbon emulsions.
Although the concept of developing an artificial blood substitute has been around for over a century, “…only in the last 30 years have significant advances been made toward clinically useful products that are shelf-storable universal oxygen carriers with minimal toxicity” (Kim & Greenburg, 2006 p. 538). Hirsch & Harrington (2000) acknowledge that as early 1937, research had demonstrated that “infusion of red blood cell lysates (i.e., the contents of the erythrocyte freed from the membrane) or purified haemoglobins into animals could oxygenate tissues, but resulted in severe renal toxicity as a result of haemoglobin tetramer dissociation” (p. 115). Henkel-Hanke & Oleck (2007) note that oxygen therapeutics started in the 1930s when Amberson and colleagues successfully established that haemoglobin solutions acquired by lysing red blood cells could transport oxygen in animals. In the 1940s, Amberson advanced his research by infusing a haemoglobin solution into a recipient for whom all donor blood had been exhausted, but the patient developed undesirable clinical outcomes related to heightened blood pressure and died shortly after the infusion (Sakai et al, 2009).
In 1949, a study conducted by Amberson and colleagues on 14 anaemic patients demonstrated that multiple infusing with a haemoglobin solution resulted in restoration of blood volume, enhanced oxygen-carrying capability, and stimulation of haematopoiesis (Henkel-Hanke & Oleck, 2007). However, this study also demonstrated that stromal remnants in the unpurified haemoglobin solutions resulted in high toxicity, which was associated with undesirable clinical outcomes such as vasoconstriction, renal failure, and abdominal pain. This observation has also been reinforced by Hirsch & Harrington (2000), who report that early studies undertaken to evaluate the viability of unpurified haemoglobin as a blood substitute revealed that the product could not be used in isolation since it resulted in renal injury, while stromal contaminants from red blood cell lysates also proved pathogenic. It is imperative to note that similar results had been reported in animals given haemoglobin solutions several decades earlier (Kjellstrom, 2003).
To circumvent the challenge of severe renal pathophysiology associated with haemoglobin extracted from RBC, which researchers had noted arises from renal filtration of the dimmers (Hirsch & Harrington, 2000), subsequent research focussed on developing cross-linking methods to produce a prototype cell-free haemoglobin-based oxygen carrier (Hae & Greenburg, 2004). This observation is reinforced by Chang (2006), who reports that early research into an effective haemoglobin-based oxygen carrier demonstrated that dimmers in nonencapsulated haemoglobin bind oxygen non-cooperatively, in addition to exhibiting an unusually high oxygen affinity. It is of importance to note that haemoglobin exists in tetrameric form within the red blood cell, but each tetramer splits into two dimmers when haemoglobin is extracted from the erythrocyte (Kjellstrom, 2003). This researcher notes that beside unfavourable kidney effects, the dimmers also apply a high oncotic pressure in addition to realising oxygen at low P50.
Research in the 1980s finally succeeded in developing crosslinking agents that are used to engineer a multiplicity of haemoglobin-based oxygen carriers, free from stromal contaminants (Thomas, 2008). Previous research had demonstrated that cross-linking of haemoglobin molecules could successfully prevent their breakdown into dimmers (Kjellstrom, 2003). In the early 1990s, three main types of crosslinked haemoglobins were developed, namely “polyhaemoglobins (intermolecular crosslinking), conjugated haemoglobins (haemoglobin conjugated to inert markers such as dextian derivatives), and intramolecular cross-linked haemoglobins used to stabilize the tetramer” (Hirsch & Harrington, 2000 p. 116).
The most promising of the crosslinked haemoglobins, according to Hirsch & Harrington (2000), were the intratetrameric crosslinked haemoglobins, which used reagents that enhance the formation of bridges within the central cavity. The researchers report that “the first generation intratetrameric crosslinked haemoglobins under serious consideration as HBOCs [were] human HbA diaspirin crosslinked at the α99 Lys residues” (p. 117). Purification of huge quantity for analytical use purposes was done in the 1990s by the Army Letterman Institute of Research (ALIR) and then in cooperation with the Baxter Healthcare Corporation, after which Baxter further modified the production techniques and succeeded to present an independent product that was identical in design (Hirsch & Harrington, 2000; Goodnough, 2003).
The first generation intratetrameric crosslinked haemoglobins were faced with a number of challenges, including the accessibility to the extravascular space, incidences of increased blood pressure when used, and vasoconstriction that was presumed to arise from the binding of nitric oxide (NO) by haemoglobin-based oxygen carriers entering the extravascular space (Hirsch & Harrington, 2000; Goodnough, 2003). This implies that the first generation intracrosslinked tetramer failed to satisfy the safety preconditions of an ideal blood substitute. Subsequent research therefore focussed on findings means by which haemoglobin entrance into subvascular space could be curtailed (Sakai et al, 2009), and researchers succeeded in these attempt developing larger molecular weight polymerized derivatives by “using intermolecular crosslinkers to produce octamers (2 tetramers) and higher multimeric structures” (Hirsch & Harrington, 2000 p. 117). For example, Northfield Corporation used a glutaraldehyde cross-linking mechanism to develop their deoxy HbA, which reached phase II clinical trials before encountering the challenge of attenuated peripheral vasoconstriction by glutaraldehyde modification of recombinant human haemoglobin (Winslow, 2000a; Winslow, 2000b; Chang, 2003). Researchers linked this problem to increased molecular weight as well as a mechanism dependent on surface modifications. In 1998, the U.S. terminated further clinical trials of Baxter Healthcare Corporation’s HemAssist (diaspirin crosslinked haemoglobin) product after a higher mortality rate in the treatment group was reported (Kjellstrom, 2003; Hirsch & Harrington, 2000). Europe also suspended further clinical trials of the product, which had already progressed to Phase III, due to safety concerns.
Other research efforts led to the development of a stroma-free Hb (SFH), a purified Hb solution free of red cell membrane which could reversibly bind and deliver oxygen, but was also found to have two major shortfalls – unusually high affinity to oxygen and too short intravascular circulation half-time (Kim & Greenburg, 2006; Sakai et al., 2009). The high oxygen affinity, in particular, was perceived as unfavourable to optimal oxygen transportation and offloading to tissues. According to these Kim & Greenburg (2006), “…a cellular free SFH has a high oxygen affinity than native intra-erythrocytic Hb because 2,3-diphosphoglycerate (DPG) normally present in the red cells is lost during purification” (p. 540). However, SFH can still be employed as a universal resuscitation fluid for patients with any blood type due to the fact that it does not possess any antigenic cell membrane.
Northfield Laboratories has developed PolyHeme, which was evaluated in phase III clinical trials in North America and Europe (Henkel-Hanke & Oleck, 2007). It is imperative to note that PolyHeme uses human-derived haemoglobin that has been polymerized with glutaraldehyde, then pyridoxylated (Kim & Greenburg, 2006). Thomas (2008) acknowledges that glycoaldehyde, glutaraldehyde and ring-opened o-raffinose reagents have been used to polymerize haemoglobin by institutions such as Northfield Corporation and Hemosol, Inc According to Henkel-Hanke & Oleck (2007), “the P50 of PolyHeme is 26 to 32 mm Hg, and the haemoglobin concentration is 10g/dL” (p. 210). These authors note that the product is able to achieve a half-life of 24 hours after intravenous administration, and a shelf-life of up to 1 year when stored at 2oC to 8oC. Phase III clinical studies of PolyHeme demonstrated that the product has the capacity to enhance oxygen delivery and decrease mortality, and a number of research articles (Henkel-Hanke & Oleck, 2007; Stowell, 2005; Goodnough, 2003) reveal that pre-hospital phase III evaluation was undertaken in 20 U.S. Level 1 trauma centres to investigate the safety and efficacy of PolyHeme in improving the survival rate of severely injured, bleeding patients when employed before entering a health facility. Northfield Laboratories sought for a Biologic Licence for the product in August 2001. It is important to note that PolyHeme has been approved by FDA for compassionate use and it presently awaiting regulatory approval for clinical use (Stowell, 2005; Lowe, 2006).
Kim & Greenburg (2006) reports that there have been attempts to engineer red blood cells to assist patients who with rare red blood cell antigens, and therefore present challenges in identifying matching blood donors. It is important to remember that blood types are categorized based on the presence of AB and Rh antigens on the erythrocyte membrane, implying that a simple ABO=Rh blood typing is enough to match appropriate donor blood for most transfusions (Kim & Greenburg, 2006). However, this is not usually the case in patients with rare red blood cell antigens. In equal measure, compatibility problems normally occur in patients who are subjected to chronic transfusions due to the alloimmunization against minor red cell antigens (Thomas, 2008; Ferguson et al, 2008; Fleming et al, 2008).
In the light of the above challenges, a number of researchers have directed their efforts towards the development of universal donor red cells. One group of researchers reported by Kim & Greenburg (2006) neutralized or masked the red cell surface antigens using polyethylene glycol (PEG) while relying on previous research findings which demonstrated that covalent binding of PEGs the erythrocytes does appear to mask the red blood cell surface antigens, thereby availing the potential of transfusion of heterologous or even xenogenic red blood cells. In another recent study that used the same technology to circumvent the challenge of blood incompatibility, type A or B human red blood cells modified with methoxyPEG demonstrated diminishing anti-A or anti-B antibody binding. Researchers have also used a permutation of thiolation and acylation mediated PEGylation to mask both A and RhD antigens (Thomas, 2008). The overall finding of these engineered red blood cell approaches is that whereas these ‘stealth’ or ‘embalmed’ erythrocytes may obviate blood incompatibility challenges, they still need to overcome other underlying issues such as limited supply, storage, possibility of disease transmission, and intravascular survival (Kim & Greenburg, 2006).
There have been efforts directed at using bovine haemoglobin as a starting point for the construction of a polymeric blood substitute (Hirsch & Harrington, 2000). One advantage of bovine Haemoglobin over human haemoglobin is that it does not need 2,3-diphosphoglycerate to transport and release oxygen (Napolitano, 2009). Also, bovine haemoglobin has a naturally low oxygen affinity and can be directly polymerized without prior modification to achieve the required oxygen affinity as well as circulation time (Kim & Greensburg, 2006). In the development of Hemopure, a bovine haemoglobin-based artificial oxygen carrier, a chloride shift is employed, which augments the Pgg of the product to 43 mm Hg, thereby enhancing oxygen transportation and delivery to tissues (Henkel-Hanke & Oleck, 2007). Hemopure, also identified as HBOC-201 or polymerized bovine haemoglobin, has been endorsed in South Africa for use in perioperative management of anaemia in adult elective surgical patients, and to reduce the reliance of allogeneic blood transfusions due to a high HIV prevalence rate (Ferguson et al, 2008). In 2002, Biopure Corporation, the makers of Hemopure, filed its Biologic Licence Application (BLA) with FDA to be allowed to market the HBOC-based product in the U.S. for use in orthopaedic surgical patients, but the drug agency replied in 2003, requesting for more information regarding certain clinical and preclinical data (Fleming et al, 2007).
As reported by Henkel-Hanke & Oleck (2007), “several studies have demonstrated the ability of Hemopure to increase oxygen diffusion capacity and decrease the need for allogeneic blood transfusion” (p. 210). In mid-2000s, a phase III clinical trial involving 688 orthopaedic surgical patients was completed, with results showing that Hemopure spared the need for allergenic blood transfusion in 59.4 percent of the 350 recipients of the product (Henkel-Hanke & Oleck, 2007). These authors note that self-limited undesirable clinical outcomes occurring more recurrently in the experimental group included gastrointestinal, hepatobiliary, skin, and cardiovascular disorders, but such outcomes are not surprising considering the fact that dysphagia, jaundice, skin disorders, tachycardia, and transient hypertension are known to be associated with the use of Hemopure. Comparable rates of serious undesirable clinical outcomes and mortality were noted in the groups studied. It is imperative to note that Hemopure has been approved for compassionate use in humans in the U.S., but is yet to receive regulatory approval for clinical use (Henkel-Hanke & Oleck, 2007).
Kim & Greenburg (2006) report that “more recently, a HBOC based on polymerized bovine Hb with MW of 20 megadaltons has been produced using ‘zero link’ methods” (p. 540). Among the advantages of this HBOC product, clinical trials demonstrated that it neither extravasates into the interstitial tissues nor trigger hypertension when administered intravenously into animals (Napolitano, 2009). However, studies using bovine Hb and other animal-based Hb as the basis for developing a polymeric blood substitute have been faced with a multiplicity of challenges, which include potential immunogenicity and transmission of animal borne syndromes such as bovine spongiform encephalopathy (Kim & Greenburg, 2006), and Mad Cow Disease (Hirsch & Harrington, 2000; Thomas, 2008).
Recent advances in recombinant DNA technologies have enabled the development of native or exclusively customized haemoglobins from microorganisms (E. coli, yeast, etc.), transgenic plants, or animals (Kim & Greenburg, 2004). These authors report that prestabilized recombinant human haemoglobin was developed “in E. coli and S. cerevisiae using an expression vector containing two mutant human globin genes, one with a low oxygen affinity and another tandemly fused a-globins” (p. 818). Baudin-Creuza et al (2008) note the expression of Hb in microorganisms is beneficial in preventing the challenges related to acellular tetrameric Hb. Research reveals that first generation recombinant a-a crosslinked human haemoglobin-based oxygen carriers produced in E. coli did progress to clinical trials in the U.S. and Europe, but were suspended due to vasoconstriction and other undesirable effects (Kim & Greenburg, 2004). In early 2000s, a second generation product with abridged vasoactivity was constructed and underwent preclinical studies. A meta-analytic review of various research articles (Kim & Greenburg, 2004) reveals that human haemoglobin has also been developed in transgenic animals, such as mice and pigs, by injecting human a and b globin gene constructs into newly fertilized eggs extracted from the animals. The resultant embryo is then let to develop in a surrogate mother until the newborns are delivered, implying that the red cells of the transgenic animals born thus contain authentic human haemoglobin. According to Kim & Greenburg (2004), “harvesting and purification of desired Hb product from these animals is, however, more complicated since the red cells contain hybrid Hbs as well as the animal’s own Hb and human Hbs” (p. 819). The efficacy and economic viability of these methods are yet to be fully revealed.
Recently, scientists have attempted to develop haemoglobin-based artificial blood substitute based on nanotechnology (Baudin-Creuza et al, 2008; Chan, 2006). These researchers acknowledge that “nanoparticles with well defined surface characteristics may be easily prepared using new polysaccharide-poly (alkyl cynoacrylate) copolymers” (p. 1452). The researchers further note that with many biological properties of the natural or chemically modified polysaccharides, such as bio-adhesion, tissue addressing, transport functionality and antithrombic capacity, the nanoparticles already in various stages of development represent a major leap forward in the design of a new blood substitute. Chan (2003) reports that nano-dimension artificial red blood cells, which have the capacity to retain their circulating haemoglobin level at twice the duration of PolyHb, are already been developed using a composite biodegradable polymeric membrane comprising co-polymer with polylactic acid.
Although perfluorocarbons are largely perceived as simple organic compounds in which all hydrogen atoms have been replaced by fluorine, some compounds that contain halogens are also included in the list of perfluorocarbons (Hirsch & Harrington, 2000). According to the researchers, “these compounds are insoluble and must be emulsified with albumin or phospholipids” (p. 116). Kim & Greenburg (2004) also note that perfluorocarbons must be diffused in a plasma-like aqueous emulsion such as albumin or in physiologic electrolyte solution to be effective as an artificial oxygen carrier. These authors also note that a number of perfluorocarbons in various stages of development use various emulsifying agents such as Pluronic-68, egg yolk phospholipids, and triglycerides, and also use various colloidal agents such hydroxyl ethyl starch (HES) to balance colloidal osmotic effect. As is the case with haemoglobin-based oxygen carriers, the development of perfluorocarbons is largely directed by the need to curtail or eliminate transfusion of autologous blood (Togny et al, 2008).
Seminal research on certain types of perfluorocarbons revealed that these emulsions are not only completely inert, but they have the capacity to dissolve large quantities of gases (Lowe, 2006). This view is reinforced by Kjellstrom (2003), who argues the perfluorocarbon emulsions are excellent carriers of oxygen and carbon dioxide. According to Henkel-Hanke & Oleck (2007), development of perfluorocarbons “began in the 1960s, when Clark and Gollan found that mice submerged in oxygenated silicone oil or liquid fluorocarbon could exchange oxygen and carbon dioxide in the liquid” (p. 207). Previously, Sloviter and colleagues had examined the use of microscopic emulsion of fluorocarbon oils suspended in aqueous solutions, and found that emulsions were smaller in diameter and non-rigid, thus could be better utilized to transport oxygen to animal tissues when compared to microscopic particles of silicons or organic fluid (Chang, 2002). However, subsequent studies during the 1060s revealed that the success noted in animal models could not be replicated in humans because the only perfluorocarbon accessible had a long-lasting presence in the reticuloendothelial system (Napolitano, 2009).
Although the first clinical trials using perfluorocarbon-based products in humans were reported in 1978 (Kjellstrom, 2003; Henkel-Hanke & Oleck, 2007), research into the product shifted towards developing techniques by which the prolonged presence of the fluorocarbon polymer in the reticuloendothelial system could be prevented (Chang, 2002). A meta-analytic review of several research articles (Henkel-Hanke & Oleck, 2007; Hirsch & Harrington, 2000; Chang, 2006) demonstrates that incidences of mortality from chemical pneumonitis IV were associated with the prolonged presence of fluorocarbon polymer in the reticuloendothelial system. However, intensive research efforts by Green Cross Corporation (Japan) led to the development of Fluosal-DA 20 in 1976, which has been approved by FDA for use in distal perfusion during percutaneous transluminal coronary angioplasty (Kim & Greenburg, 2004). Fluosal-DA 20 is a dilute fluid of a combination of two perfluorocarbons – perfluorodecalin and perfluorotriprophylimine, and employs egg yolk phospholipid and Pluronic-68 as emulsifying agents (Hirsch & Harrington, 2000; Kim & Greenburg, 2004). Colin & Cushing (2009) and Henkel-Hanke & Oleck (2007) report that Fluosal-DA 20 remains the only artificial oxygen carrier to have attained FDA approval for clinical use in the United States, but its production was stopped in 1994 when autoperfusion angioplasty catheters were introduced. Subsequent studies revealed that Fluosal could not dissolve much oxygen due to the fact that emulsions contain much less perfluorochemicals per volume when compared with pure liquids (Kim & Greenburg, 2004), was excreted slowly and some metabolites remained in the body for months (Cohn & Cushing, 2009), and was also associated with biological adverse effects, including temporary decrease in platelet count and fever (Kluger, 2010).
With lessons learned from the limitations of Fluosal and other early perfluorocarbon developments, research efforts were directed towards the development of a second generation of perfluorocarbon emulsions. These studies, as acknowledged by Cohn & Cushing (2009), focused on the development of high perfluorocarbon content for maximum efficacy in oxygen delivery to tissues, and developing a perfluorocarbon that could be removed by plasmapheresis, thereby making intraoperative use more feasible. These concerted efforts led to the development and testing of a number of perfluorocarbon emulsions, namely Oxygent, Oxycyte, Oxyflour, Perflubron, and Pertoran (Cohn & Cushing, 2009). According to Hirsch & Harrington (2000), “…Alliance Pharmaceutical Corporation reported 17 clinical studies completed in Phase I and Phase II with their perfluorocarbon Oxygent” (p. 117). The results of these clinical trials showed that Oxygent, which uses egg yolk phospholipid as the sole emulsifying agent (Kim & Greenburg, 2004), was well tolerated in both Europe and the U.S., and no noteworthy adverse hemodynamic or haemostasis effects were exhibited. Nevertheless, fever exhibited within 4-6 hours after administration, as an outcome of macrophage-mediated clearance, and a reduction in platelet count was reported two to four days after use (Hirsch & Harrington, 2000). These challenges raise germane issues particularly related to the circulatory half-life and dose limitations, but Oxygent still appeared as one of the most promising artificial oxygen carriers in recent times (Kluger, 2010; Lowe, 2006). Cohn & Cushing (2009) report that “a large European multicentre phase III study has shown that Oxygent, in conjunction with acute normovolemic haemodilution, reduced the need for red blood cell transfusion in 492 patients undergoing major noncardiac surgery” (p. 402). However, further clinical trials involving Oxygent has been halted due to high development costs and serious adverse neurologic outcomes, including stroke and thrombocytopenia (Henkel-Hanke & Oleck, 2007).
Several other perfluorocarbon-based artificial oxygen carriers have either received FDA approval for clinical use, are currently in phase III clinical trials, or studies into their development have been terminated (Goodnough, 2003). Synthetic Blood International (US) developed Oxyflour, which basically consist of perfluorodichlorooctane suspended in emulsion (Cohn & Cushing, 2009). These authors note that Oxyflour has a substantially higher oxygen delivery capability when compared to Fluosal, and also has an extended stability at room temperature. Preclinical investigations of the emulsion indicated that it did not trigger pulmonary hyperinflation, and was also readily eradicated from the body’s vital organs, including the lung, liver and spleen. AS reported by Cohn & Cushing (2009), “early-phase clinical trials were successfully completed with only mild thrombocytopenia and flu-like symptoms reported in healthy human volunteers” (p. 403). However, a major financing hitch ended the development of this product.
Henkel-Hanke & Oleck (2007) note that a perfluorocarbon-based prepapration known as Perflubron (Liquivent), developed by Alliance Pharmaceuticals (US), has already achieved FDA approval for use as a contrast agent for MRI, but not as an artificial blood substitute (Cohn & Cushing, 2009). S-9156, developed by Sonus Corporation (US), is known to dissolve adequate amounts of oxygen, thus can be used for ultra small volume resuscitation application (Kim & Greenburg, 2004). Another perfluorocarbon-based emulsion known as Perftoran was endorsed by the Russian Ministry of Health for clinical use in 1999 (Chang, 2002), and is already in use in Russia and Mexico (Cohn & Cushing, 2009). Perftoran, however, is unlikely to be used in the U.S. in the near future due to lack of clinical evidence in America as well as the timeline involved for FDA approval. Currently, all perfluorocarbon clinical trials have been stopped in the US, but research is still ongoing about the capacity of the emulsion to be used as oxygen therapeutic to maintain safe oxygen levels during operative surgery (Henkel-Hanke & Oleck, 2007; Ferguson et al, 2008; Fronticell et al, 2007).
Conclusion & Future Directions
Although this paper acknowledges that there exists other artificial blood substitutes under development and which have not been mentioned here due to limited space, the pathways in past and current research into haemoglobin-based oxygen carriers and Perfluorocarbons have been well espoused. It is a well known fact the shortage of donor blood and the risk of disease transmission during transfusion still advocates for comprehensive studies focussed on the development of readily available blood substitutes (Squires, 2002; Chang, 2003). However, it is imperative to realize that there is still a long and engaging way to go before artificial blood can effectively replace human blood in routine clinical transfusions. It is also important to remember that artificial oxygen carriers are not blood substitutes as current research is yet to demonstrate how these carriers can replace red blood cells (Stowell, 2005; Togny et al, 2008; Cox, 2010).
Current research have been successful in increasing the dwell times of many artificial blood substitutes, but their development is still constrained by high production costs and difficulties with acquiring and processing adequate amounts of these products (Stowell, 2005). Future research efforts must therefore aim at coming up with ways by which development and productions costs of artificial oxygen carriers can be kept down, and how these products can be produced in sufficient amounts. The greatest breakthrough, however, can only be attained if researchers are able to develop an artificial blood substitute free from any adverse clinical outcome, and which can successfully function in the same level or more efficiently than the normal human red blood cells. Such a product, according to Henkel-Hanke & Oleck (2007), would allow for surgical procedures with even greater blood loss to be performed, and at the same time curtailing the need for allogeneic blood transfusion. Future research also need to be directed at comprehensively identifying the appropriate indications, uses, benefits and risks of the artificial oxygen carriers already in various phases of development so as to enable better management and use in the event that they receive approval.
There exists an urgent need to develop new generations of artificial blood substitutes so as to broaden the potential areas and scope of application (Togny et al, 2008; Fronticell et al, 2007; Malchesky, 2008). This paper has demonstrated that some blood substitutes that were initially developed to fill in the gap of human red blood cells ended up being used in the management of other health conditions. Future research, therefore, need to be guided by the need to discover new areas where the artificial blood substitutes currently under development could be effectively used in the treatment and management of various health complications. Future research efforts must also explore naturally-occurring materials as possible candidates of artificial oxygen carriers as such materials, if discovered, may go a long way to reduce the development costs involved, and to some extent the undesirable effects noted in the use of crosslinked products and other synthetic compounds (Akira, 2009; Malchesky, 2008). However, we must acknowledge the fact that even if we start to undertake these studies seriously and immediately, it will definitely take some time for viable artificial blood substitutes to be ready.
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