Artificial Cells, Blood Substitutes, and Biotechnology, 34: 537550, 2006 Copyright Q Informa Healthcare ISSN: 1073-1199 print/1532-4184

online DOI: 10.1080/10731190600973725

Toward 21st Century Blood Component Replacement Therapeutics: Artificial Oxygen Carriers, Platelet Substitutes, Recombinant Clotting Factors, and Others
Hae Won Kim and A. Gerson Greenburg
Brown University Medical School and The Miriam Hospital, Providence, Rhode Island, USA

Abstract: In this brief overview, recent progress and current status of blood substitute research and development is summarized. Current blood substitute development efforts are focused on red blood cell substitutes but substitutes for platelets and other blood components are also in progress. Red cell substitutes currently in various stages of development are semi-synthetic or synthetic oxygen carriers that include stealth or masked red cells, hemoglobin-based oxygen carriers and perfluorocarbon-based oxygen carriers. Artificial platelets (or platelet substitutes) are in early stages of development and include human platelet fragments or particles of synthetic=semi-synthetic materials or recombinant human serum albumin coupled with platelet surface receptor fragments. Of note, some recombinant clotting factors (Factors VII, VIII, IX) have already been successfully developed and licensed for treatment of hemophilia. In addition, some future approaches and prospects of blood component replacement therapeutics are discussed.
Keywords: Artificial oxygen carriers; Blood substitutes; Platelet substitutes; Recombinant clotting factors; Red cell substitutes

Over the last century, allogeneic donor blood transfusion has evolved as lifesaving treatment for patients with conditions of serious blood deficiencies (e.g., acute anemia, severe thrombocytopenia, hemophilia). To date, millions of patients have received blood transfusion and countless lives have been saved as a result. However, allogeneic blood transfusion carries a risk of disease transmission (e.g., hepatitis, AIDS, syphilis,
Address correspondence to Hae W. Kim, Biopure Corporation, 11 Hurley Street, Cambridge, MA 02141. E-mail:HWKim@Biopure.com 537

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malaria, and other bloodborne diseases). Today, in North America, Western Europe, and other advanced countries, allogeneic donor blood transfusion is generally safe due largely to the availability of highly sensitive assays such as nucleic acid amplification assay to screen for AIDS and other infectious diseases. In the U.S. today, the risk of HIV infection through a single transfusion is approximately 1=1,000,000 [1]. Still, risks of infection remain due to a marker-negative window period for donors or donors infected with new emerging pathogens (e.g., vCJD, West Nile virus, A5N1 and other avian flu virus) for which screening assays have not yet been implemented. A recent report [2] estimates that, in some sub-Saharan Africa countries, over 30% of population ( 25 million) is infected with HIV. In these countries, HIV infection rate through transfusion is second only to that from sexual activities. Increase in HIV infection is not limited to sub-Saharan Africa. New HIV infections continue to rise worldwide; over the last two years, the steepest increase in new HIV cases occurred in parts of Asia and Eastern Europe. Many more people in the world are infected with hepatitis (especially the more serious B and C type) and other bloodborne diseases. Blood transfusion transmissible infections are prevalent in third world countries due to limited resources to screen donor blood and dependency on paid or family donors. Of the 81 million units taken worldwide in 20012002, approximately 6 million transfusions were conducted without proper testing [2]. Most of these unscreened transfusions occurred in countries with low and medium human development index, a comparative index developed by the United Nations Development Program based on life expectancy, educational attainment and adjusted income [2]. Aside from the risk of disease transmission, there is a worldwide chronic shortage of allogeneic donor blood. Considering this shortage and the fact that donated red cells can only be stored refrigerated for about 5 weeks and much shorter times for platelets and clotting factors, the potential for an acute shortage is high should an event requiring transfusion of mass casualties arise (e.g., natural disasters, wars). In addition, allogeneic donor blood can only be used for recipients with compatible type specific antigen=antibody status. For these reasons, substitutes for red cells and other critical blood components that can overcome these limitations continue to be highly desirable and are actively pursued. The idea of developing a blood substitute has been around for over 100 years; 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. There are many excellent reviews published recently regarding the history and principles of blood substitute development [35]. Therefore, this article will only briefly discuss various approaches, focusing primarily on the most recent developments and some future prospects.

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CURRENT STATUS OF BLOOD SUBSTITUTE DEVELOPMENT 1. Artificial Oxygen Carriers (Red Blood Cell Substitutes) Because red blood cells deliver oxygen essential to tissues and organs, in acute anemia caused by traumatic hemorrhage, lost oxygen carrying capacity must be promptly restored or the situation could be fatal. Allogeneic donor blood (red cell) transfusion is effective but has limitations mentioned above. For this reason, the search for a red cell substitute has been ongoing for decades but only recently have a few candidates reached active clinical testing [4]. 1.1. Engineered Red Blood Cells Blood types are classified based on the presence of AB and Rh antigens on the red cell membrane surface. In most transfusions, simple ABO=Rh blood typing is sufficient to match appropriate donor blood. Occasionally, however, patients with rare red cell antigens present difficulty in identifying matching blood donors. Compatibility problems occur more often in patients who must receive chronic transfusions (e.g., sickle cell anemia or thalassemia patients). In such patients, alloimmunization against minor red cell antigens makes it difficult to find matching donors. There have been efforts to create universal donor red cells. In one such approach, the red cell surface antigens are neutralized or masked using polyethylene glycol (PEG) or its derivatives [68]. Covalent binding of PEGs to RBCs does appear to mask the RBC surface antigens, thereby opening possibilities of transfusion of heterologous or even xenogenic RBCs. In one recent study [8], Type A or B human RBCs modified with methoxyPEG showed decreased anti-A or anti-B antibody binding. Furthermore, methoxyPEG modified sheep RBCs were resistant to phagocytosis by human peripheral blood monocytes. In another approach, a combination of thiolation and acylation mediated PEGylation was reported to mask both A and RhD antigens [6]. In another approach, red cell A or B antigens were enzymatically removed by exoglycosidase treatment, but this was only partially successful [9]. These stealth red cell approaches may obviate incompatibility problems but have yet to deal with issues of limited supply, storage issues, potential disease transmission, and intravascular persistence. 1.2. Hemoglobin=Heme-based Oxygen Carriers (HBOC) Stroma-free Hb (SFH), a purified Hb solution free of red cell membrane stroma as cell-free Hb in a physiologic solution, can reversibly bind and

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deliver oxygen. Because there is no antigenic red cell membrane in SFH solution, it can be used as a universal resuscitation fluid for recipients with any blood type. This SFH, however, was found to have two perceived shortfalls: SFH was perceived to have too high an oxygen affinity (P50 of 1015 mmHg versus 2628 mmHg for normal red cell Hb) and too short an intravascular circulation half-time (T1=2 < 1.5 hr) to be useful. Acellular free SFH has a higher oxygen affinity than native intra-erythrocytic Hb because 2,3-diphosphoglycerate (DPG) normally present in the red cells is lost during purification. The high oxygen affinity was perceived as detrimental to optimal oxygen offloading to tissues. In addition, acellular tetrameric Hb (a2b2) in solution readily dissociates into ab dimers that are easily filtered through the kidneys and excreted in the urine. The HBOCs currently in development or in clinical testing are Hbs chemically or genetically engineered to produce desirable oxygen offloading characteristics and an extended circulation halftime. Key approaches of HBOCs currently in development as red cell substitutes are briefly described below.

1.2.1. Polymerized Hbs. Intravascular retention times of HBOCs can be increased by intermolecular crosslinking (polymerization) of stabilized Hbs using bi- or poly-functional crosslinkers. For example, PLP-Hb (Hb modified with pyridoxal-50 -phosphate, a DPG analog with a monofunctional aldehyde) can be polymerized using glutaraldehyde, a bifunctional nonspecific crosslinker, to produce poly(PLP-Hb) with t1=2 of over 30 hours following a partial exchange transfusion in adult baboons [10]. Hemopure (HBOC-201) (Northfield Laboratories, Chicago, IL), a poly(PLP-Hb) product, is being tested in a pivotal Phase III prehospital trauma trial in the U.S. Of note, this clinical trial is being conducted without consent under the waiver provision. An alternative approach to circumvent the high oxygen affinity and limited availability of human SFH is to utilize naturally low O2 affinity bovine Hb as a starting material [11]. Bovine Hb has a naturally low oxygen affinity (P50 3032 mmHg) compared to human Hb. Bovine Hb is not DPG dependent and can be directly polymerized without prior modification to achieve both desired oxygen affinity and circulation time [12]. PolyHeme1 (Biopure Corp., Cambridge, MA), a bovine Hb polymerized with glutaraldehyde, is being tested in Phase II=III clinical trials in the U.S. and Europe [13]. More recently, a HBOC based on polymerized bovine Hb with MW of 20 megadaltons has been produced using zero link methods. This HBOC product was reported not to extravasate into the interstitial tissues and has been shown not to cause hypertension following

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intravenous administration into animals [14]. Porcine Hb polymerized with glutaraldehyde is being developed in China [15]. Potential immunogenicity and transmission of animal borne disease such as bovine spongiform encephalopathy (BSE) could be of concern with these animal Hb-based products. 1.2.2. Conjugated Hbs. Another way to increase t1=2 of a HBOC is to conjugate Hb with a macromolecule. Human or bovine Hb conjugated with PEG appears to protect the molecule from renal excretion [1618]. Recently, human Hb conjugated with maleiimide PEG (Hemospan1, Sangart Corp. San Diego, CA) has been developed [19]. This product is reported to have improved hemodynamic and circulatory properties and is being tested in Phase II clinical trials in the U.S. One to one conjugate of bovine Hb and human serum albumin (bHb-HSA) has been prepared with average MW of 157 kDa [20]. When intravenously administered to 30% and 60% acutely hemorrhaged rats, this product maintained blood pressure without pressor effect. Also, a novel conjugate of human Hb and hydroxyethyl starch (HRC101) has been produced and is in early stages of evaluation [21]. 1.2.3. Hemoglobin Vesicles (HbV). Human or animal SFH encapsulated in phospholipid vesicles (liposomes) was developed as a potential red cell substitute but encountered difficulties due to high Hb oxidation rate and a short intravascular half-life. Recently, totally synthetic heme imbedded between two lipid bilayers (lipid-heme vesicles) has been developed [22]. A stable, fat microsphere suspension has been achieved by emulsifying triglycerides with lipid-heme as a surfactant. These lipid-heme products are reported to have heme concentration and reversible O2 binding properties close to that of normal blood. 1.2.4. HBOCs with Built-in Anti-oxidants. To reduce oxygen radical mediated damages, human or bovine Hb conjugated with superoxide dismutase and catalase has also been developed [2325]. More recently, Hb and red cell enzymes (e.g., met Hb reductase, SOD, CAT and others) encapsulated in nanometer size biodegradable polymer (polylactide or polyglycolides) vesicles have been developed [26]. Unlike the lipid vesicles, these nanocapsules could be prepared permeable to glucose and anti-oxidant enzymes to prevent Hb oxidation. 1.2.5. Albumin-heme Conjugate (rHSA-heme). Recently, synthetic porphinatoiron (II) complexes conjugated to a recombinant human serum albumin (rHSA) have been developed [27]. In this albumin-heme hybrid

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approach, as many as 8 porphinatoiron(II) complexes could be absorbed to a rHSA molecule. These albumin-heme hybrids showed reversible oxygen binding under physiologic conditions. Of these, rHSA-FecycP(Im) appears to be promising as a red cell substitute since it has a P50 similar to native erythrocytic Hb and a longer intravascular circulation time ( >36 hours in anesthetized rats) [28]. It would be of some advantage to use a natural plasma protein like HSA as a component of an oxygen carrier solution as it is without undue adverse physiologic effects. 1.2.6. Recombinant=Transgenic Hbs. With recent advances in recombinant DNA technologies, native or specifically modified Hbs have been produced from microorganisms (E. coli, yeast, etc.), transgenic plants or animals [2933]. An earlier HBOC product based on recombinant human Hb produced in E. coli was discontinued due to notable hypertensive effect, as also observed with certain other HBOCs [29]. The hypertensive effect was more often observed with non-polymeric HBOCs but the exact cause has not been positively identified. Among the several proposed mechanisms, Hb scavenging of endothelium derived nitric oxide (NO), a potent vasodilator, appears to play a key role. An improved version of this recombinant HBOC that has 2030 times lesser NO scavenging rate has been developed and was shown not to increase blood pressure in rat studies [30]. More recently, polymerized recombinant human Hb with certain characteristics of bovine Hb has been developed as a candidate for red cell substitute [33]. Like bovine Hb, the oxygen affinity of this product is regulated by the [Cl ] than DPG. Hb polymers of MW as high as 1 million Daltons was produced by introducing cysteine residues on the Hb surface that form intermolecular disulfide bonds. Similarly, polymerized Mb has also been produced. In in-vivo animal experiments, the product did not elicit vasoconstriction. The polymeric Hb products have higher oxygen affinities than native Hb but are considered to deliver adequate amounts of oxygen to tissues as the infarct size was reduced following infusion in mice with experimental cerebral ischemia [33]. However, safety, effectiveness and economics of these approaches are yet to be revealed. 1.2.7. Annelid Hbs. Naturally polymeric acellular invertebrate Hbs have recently been proposed as a new class of oxygen carriers for use as a red cell substitute [3436]. Arenicola marina (lugworm) and Lumbricus terrestris (earthworm) have extracellular Hb of  3600 kDa. These giant Hbs are hetero-multimeric proteins with 156 and 144 heme containing globin chains, respectively, along with numerous linker peptides [34,36]. These Hbs display a quaternary structure of two superimposed hexagonal

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layers. Interestingly, these giant Hbs are reported to be resistant to autooxidation and subunit dissociation under physiologic conditions. In animal experiments, except for a slight immune response, no apparent signs of serious toxicity was observed with both Hbs [34,35]. It appears that these naturally occurring polymeric Hbs deserve further exploration in terms of safety and effectiveness as clinically useful oxygen carriers. 1.2.8. New Approaches to Hb Encapsulation and Entrapment. Recently, Hb encapsulated in polymer vesicles (polymersome encapsulated Hb or PEH) has been developed as a potential oxygen carrying red cell substitute [37]. Polymersomes were prepared using bioinert amphiphilic diblock copolymers. The average diameter of PEHs was greater than 100 nm but had a higher Hb loading capacity than those of liposome encapsulated Hb (LEH) preparations. In addition, unlike liposomes, polymersomes did not induce Hb oxidation. The oxygen affinities of PEHs were reported to be comparable to that of human erythrocytes. These results indicate that PEHs may have the potential to be developed as clinically useful oxygen carriers. In a separate approach, liposome encapsulated actin-Hb dispersion was shown to prolong circulatory halftime to greater than 72 hours [38]. Hemoglobin entrapped in a nanoscale silica gel or polymeric matrices has also been proposed as potential oxygen carriers [39,40]. For example, Hb entrapped in wet nonporous silica gel with or without presence of allosteric effectors has been explored [39]. Although not suitable for intravenous perfusion, silica gels are inert and optically transparent allowing a full characterization of the functional and structural properties of entrapped Hb by spectrophotometric methods. Interestingly, the oxygen affinity of Hb-silica gel preparations could be modulated over a wide range by varying entrapping protocol. Using this approach, it is plausible that a new type of HBOC could be prepared if a fully biocompatible matrix is found. With this HBOC, oxygen affinity could be tuned to meet the specific patients clinical condition. In another report, bovine Hb was entrapped in temperature or pH sensitive hydrogel nanoparticles [40]. Crosslinked within the polymer matrix of nanogel particle, tetrameric Hb does not dissociate into dimers and could avoid direct contact with body tissues while preserving oxygen binding=delivery. These approaches are still in early stages in development but may produce useful products in the long run. 1.3. Perfluorocarbon Based Oxygen Carriers (PFBOCs) Current PFBOCs are generally stable emulsions of one or more perfluorocarbons in aqueous media using various emulsifying agents (surfactants)

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such as Pluronic-681, egg yolk phospholipids, and triglycerides. In some cases, a colloidal agent (e.g., hydroxyl ethyl starch or HES) is added to balance colloidal osmotic effect. After extensive exploratory experiments with various perfluorochemicals, several perfluorocabon emulsions have been developed as potential oxygen carrying red cell substitutes and are in various developmental stages. The first PFBOC developed was Fluosol-DA1 (Green Cross Corp., Japan), a 20% (w=v) co-emulsion of perfluorodecalin and perfluorotripropylamine with egg yolk phospholipid and Pluronic-681 as emulsifying agents. It was approved for clinical use but withdrawn soon after introduction. Because emulsions contain much less perfluorochemicals per volume compared with pure liquids, the amount of oxygen they could dissolve is also less. For example, breathing ambient air and with the normal arterial and venous oxygen tensions (100 and 40 mmHg, respectively), Fluosol-DA1 could deliver only 0.4 ml oxygen per 100 ml. To meet the metabolic oxygen demand, the fraction of O2 in the inspired gas (FiO2) patients were required to breathe was 100% oxygen (i.e., FiO2 1.0), a situation to be avoided clinically due to the adverse effects of elevated oxygen concentration on the lungs (e.g., oxygen toxicity). Recently, Oxygent1 (Alliance Corp. San Diego, CA), a stable 60% (w=v) emulsion of perfluorooctyl bromide (perflubron) has been developed using egg yolk phospholipid as the sole emulsifying agent. Under normal arterial and venous oxygen tensions, Oxygent1 can unload as much as 1.3 ml oxygen per 100 ml, a remarkable improvement in oxygen delivery capacity. Yet, the oxygen delivery capacity of Oxygent1 is less than 30% of normal blood (5 ml O2=100 ml blood at 15 g Hb=dl) and may still require oxygen enriched air breathing to ensure adequate oxygen delivery. This product has been tested in Phase II=III clinical trials in the U.S. and other countries but the trials were suspended due to higher than expected rate of stroke in treated patients [41]. More recently, Perftoran(r), a 20% w=v co-emulsion of perfluorodecalin and perfluoromethyl cyclohexyl piperidin with 4% proxanol-268 as an emulsifying agent, has been tested in elective cardiac surgery patients [42]. The study patients underwent an acute normovolemic hemodilution with either Perftoran or control hemodiluent. The Perftoran group was reported to have no serious complications, had higher arterial oxygen levels and needed less allogeneic blood transfusion than patients with control hemodiluent [42]. In addition, a new class of PFBOCs formulated as stable microbubbles has recently been introduced. In one approach, subcapillary size microbubbles formed after intravenous infusion of 2% dodecafluoropentane emulsion is reported to deliver a sufficient amount of O2 to tissues in pigs bled to 50% blood volume [43]. Of note, with this preparation, an

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extremely small dose (0.4 ml=kg) may provide lifesaving oxygen transport in potentially fatal hemorrhagic shock. In another approach, exceptionally long-lived microbubbles have been obtained using perfluroalkylated phosphatidylcholine as a shell component and perfluorohexane a filling gas component [44]. These long-lived microbubbles may have a potential as an intravascular oxygen carrier as well as an ultrasound image contrast material. 2. Platelet Substitutes When blood vessels become damaged, activated platelets aggregate on to the damaged area providing an active surface on which fibrin and other activated clotting factors attach. The resulting complex forms a clot plug preventing further blood loss and allow vessel repair. For severe thrombocytopenia, platelet transfusion is required (generally platelet count is less than 20% of normal value) to prevent hemorrhage. Transfusion of platelet concentrate is a current treatment for severe thrombocytopenic conditions. However, platelet concentrates from donor blood can only be stored for 24 hours, thus its availability is rather limited. In addition, platelet concentrate is obtained from pooled blood from multiple donors and may pose a higher risk of disease transmission. To alleviate availability, frozen platelets and freezedried platelets are being tested [45]. Although promising, these products still carry risk of disease transmission. Therefore, to solve the availability and safety problem, platelet substitutes are being developed. Platelet substitutes or artificial platelets that are currently in development are either particles derived from human platelets or semisynthetic=synthetic particles that express platelet surface receptors or their ligands (e.g., GPIb, GPIIb, GPIX, vWF, fibrinogen). Recombinant human albumin capsules or polymers coated with fibrinogen or recombinant GPIa=IIa were shown to correct experimental thrombocytopenia [46,47]. 3. Recombinant Clotting Factors Hemostatsis is a physiologic regulatory mechanism that senses vascular damage and controls bleeding to prevent further blood loss and ward off invading pathogens. Hemostasis is achieved by the concerted interactions of vascular tissues, platelets and plasma coagulation factors. Blood clot (stabilized crosslinked fibrin) forms via sequential activation of intrinsic (contact) or extrinsic (tissue factor) clotting mechanisms. People with congenital (hemophilia A or B) or acquired deficiencies in coagulation factors develop coagulopathies. For patients, frequent transfusion

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of fresh frozen plasma (FFP) and=or cyoprecipitate fraction may be necessary to replace deficient coagulation factors. Since early 1970s, commercial factor VIII and FIX concentrates from pooled human plasma became available for treatment of hemophiliacs. However, pooled plasma could transmit infectious pathogens, although these risks have decreased substantially over the last decade due to improvements in donor screening, improved test assays, and viral inactivation. In addition, chronic transfusion of blood products causes immunologic complications including serum sickness and acquired antibodies against specific coagulation factors. These problems have lead to development of recombinant clotting factors as safer alternatives to donor blood derived factors. With the advancement of recombinant DNA and expression technology in recent years, recombinant coagulation factors aimed at treating hemophiliac patients have been developed. Production of recombinant coagulation factors or any protein is complicated by the requirement of efficient host-cell expression system and need for post-translational modifications. For example, FVII, FIX and prothrombin all require post-translational c-carboxylation for biologic activity requiring use of mammalian cell cultures such as Chinese hamster ovary cells or baby hamster kidney cells [48]. FVIII is a high MW protein ( 300 kDa), which also requires post-translational glycosylation. Therefore, production of these factors are complicated and less economical than proteins that can be produced in bacteria or yeast. Of note, some recombinant human clotting factors (e.g., Factor VII, VIII, IX) have already been successfully produced and licensed for treatment of hemophiliac patients.

4. Artificial Antibodies Antibodies (immunoglobulins) are essential components of blood that protect the host against invading pathogen and infection. Antibodies raised in animals or animal sources against specific pathogen are already in wide use to prevent or treat serious infectious diseases (e.g., vaccines against meningitis, small pox, influenza). However, although animal antibodies are effective, they could cause severe allergic reactions, such as anaphylactic shock. To avoid such reactions, human antibodies are desirable and should be developed. Recent advances in genetic engineering technology enabled us to produce completely human antibodies for therapeutic purposes. For example, using a combinatorial infection technique (phage technique), a very large human Ig-Vgene library approaching 1011 in diversity was created with human germline V-gene segment and synthetic joining segments [49]. From this library, antibodies against a plethora of antigens could be produced with high affinities. Human

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antibodies could also be obtained from transgenic animals that carry a restricted set of human immunoglobulin genes. With immunization and immortalization, human hybridoma with high affinities could be obtained. Therapeutic antibodies with two antigen binding specificities (bi-specific antibodies and diabodies) can also be produced. A diabody directed against surface Ig idiotype of lymphoma and the T-cell CD3 was shown to be effective in recruiting T-cells to kill lymphoma cells in vitro [50]. With the rapid advances in genomics, proteomics, and rational design methods, it may soon be possible to create on-demand artificial antibodies with desired specificities and affinities. Such technology would help develop prevention and treatment modalities against fast evolving highly virulent infectious pathogens (e.g., A5N1 and other avian flu viruses).

5. Stem Cells for Blood Component Replacement Therapies With recent advancement in understanding stem cell biology and in-vitro culture technology, it is conceivable that someday universal or patient specific blood cells (e.g, erythrocytes, platelets, leukocytes, and others) will be available for anemia and other blood cell deficiencies. To achieve that goal, factors that control optimal growth, proliferation and differentiation into desired blood cell lineage must be identified. Once such factors are known, largescale culture methods must be developed to produce sufficient amount of desired blood cells needed for clinical therapy. Embryonic stem cells may be used to produce blood cells as they are pluripotent to become every cell type. However, for the purpose of blood component replacement therapy, use of hematopoietic stem cells (HSC) and progenitor cells derived from bone marrow, chord blood or other sources may be more practical as they are already committed to become blood cells and relatively easy to identify and harvest. The HSC approach could also avoid ethical debate associated with use of embryo. The HSCs are multipotent to become different blood cells under appropriate conditions. Recently, some regulatory factors (e.g., cytokines and growth factors) involved in HSC differentiation into a specific blood cell lineage have been identified [51]. However, at present there appears to be insufficient knowledge and technology to produce sufficient blood cells in culture for clinical therapy. In addition, current media formulations used in stem cell cultures are complex, variable and expensive and may not be suitable for largescale productions. Whether the technology will allow us to produce sufficient amount of functional blood cells at an affordable cost is uncertain. Despite these uncertainties and difficulties, stem cell cultures offer a unique potential for producing entire repertoire of cellular components of blood. Clearly, more research needs to be done in this area.

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PROSPECTS Progress toward clinical, usable red cell substitutes has been slow due to technical difficulties in large-scale production and some adverse effects observed in clinical trials. Despite these hurdles, a couple of HBOCs are currently being evaluated in the advanced stages of clinical testing. If no serious adverse effects emerge, one or more of these products may be approved for clinical use in the U.S. within the next few years. If these red cell substitutes do become available, the way transfusion medicine is practiced today may undergo drastic changes, since red cell substitutes do not require blood typing and crossmatching tests and can be stored for extended periods without refrigeration. It will greatly improve care of acutely anemic patients who require immediate transfusion but are situated where blood is not available (e.g., at site of accident, natural disaster, or battlefield). Red cell substitutes may be used as a hemodiluent in elective operations to avoid or minimize allogeneic blood transfusion. They can also be used as oxygen therapeutics in ischemic rescue, organ preservation and other applications. The cost of HBOC transfusion will be lower in the long run; if not immediately, then allogeneic blood transfusion as processing= storage=administration costs will be substantially lower. The lower cost, ease of use, and universal compatibility of red cell substitutes will greatly improve care of acutely anemic patients. Over 80% of the worlds population live in regions where prevalence of transfusion transmissible diseases (e.g., AIDS, hepatitis, syphilis) is high but insufficient resources are available to procure and administer safe blood or blood products [2]. In addition, many promising new approaches=ideas are on the horizon to develop substitutes for red cells as well as other components of blood (e.g., platelets, clotting factors). It may not be too unrealistic to expect that, before the end of the 21st century, we may have developed safe, effective, and affordable substitutes for all key components of blood. Considering the many advantages, blood substitutes will benefit many patients across all social, economic, and geographical divisions and contribute greatly to the global healthcare arena. REFERENCES
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