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Blood substitutes and Universal Blood
April 17, 2009
Sanela Begic, Philip Cameron, Christine Deangelo,
Vera Kemmer and Jake Taylor
Introduction
The term “blood substitutes” refers to substances meant to fulfill the role of natural blood or blood components. These can be divided up into erythrocyte (RBC or red blood cell) and leukocyte (WBC or white blood cell) substitutes, plasma substitutes, blood volume expanders, and platelet substitutes. Current research has included artificial platelets using a fibrinogen coat over various particles, producing “universal” blood cells by either masking or removing erythrocyte antigens, hemoglobin or perfluorocarbon-based red blood cells, and erythrocytes grown from stem cells.
In the past, blood transfusions have been plagued by issues such as blood group incompatibility with accompanying transfusion reactions, storage difficulties and shortages, donor recruitment issues and costs, human error, transfusing blood-borne diseases, and cultural or religious objections. Artificial blood components ideally would be free from all bacterial and viral contaminations, and antigens contributing to transfusion reactions. Artificial blood would be easily eliminated, and readily acquire oxygen from the lungs and transport the oxygen to the tissues. It would have a long shelf-life, be inexpensive to manufacture, and necessitate relatively few storage requirements. This is what scientists are currently trying to accomplish with their research in this area.
- Index
- Background
- History
- Universal Blood Products
- ECO RBCs
- Stealth RBCs
- Hemoglobin-Based Red Cells
- Conjugated Hemoglobin
- Cross-Linked Hemoglobin
- Recombinant hemoglobin
- Surface-Modified Hemoglobin
- Perfluorocarbon-Based Red Cells
- Blood products grown from stem cells
- Plasma Volume Expanders
- Platelet Substitutes
- 1. Platelet-Derived Products
- 2. Fibrinogen Based Products
- Direction of Future Research
- Definitions
Blood is required to perform many functions in the body. The primary function of blood is to carry and supply oxygen to tissues1>. Blood removes waste products from the body such as carbon dioxide, lactic acid, and urea while it also delivers hormones and nutrients to tissues. Blood performs immunological functions, helps to buffer and maintain body pH, and coagulates during injury. Centrifugation of a fresh blood sample will separate the blood into its components. Once separated, the blood components can be harvested separately and used in transfusion medicine for various purposes 2 .
Blood may be transfused as whole blood or as one of its components. The AABB3, can separate a unit of donated blood into four components which include red blood cells, plasma, platelets and cryoprecipitated antihemophilic factor (AHF). Red blood cells are transfused into patients with chronic anemia resulting from disorders such as kidney failure, malignancy, or gastrointestinal bleeding, those who have congestive heart failure, are elderly or debilitated and those with acute blood loss resulting from trauma or surgery. Red blood cells may be treated and frozen for extended storage (up to ten years)4
www.nsbri.org/HumanPhysSpace/index.html
Plasma is usually not transfused, but fractionated into specific products such as albumin (chief protein constituent), specific clotting factor concentrates and IVIG (intravenous immune globulin). Fresh Frozen Plasma (FFP) is plasma frozen within eight hours after donation and can be stored for up to seven years. It is used to treat bleeding disorders or for plasma replacement. Cryoprecipitated AHF is the portion of plasma that is rich in clotting factors and is used to prevent or control bleeding in individuals with hemophilia and von Willebrand’s disease. Platelets or thrombocytes help the clotting process and are used to treat a condition called thrombocytopenia (shortage of platelets) and in patients with abnormal platelet function. Granulocytes, a specific type of white blood cell are transfused in patients with infections that are unresponsive to antibiotic therapy. Plasma derivatives are concentrates of specific plasma proteins that are prepared from pools (many units) of plasma and are heat-treated and/or solvent detergent-treated to kill certain viruses including HIV and hepatitis B and C.
>Transfusion medicine as we know it today did not come easy. Several time honored events have paved the way for practices currently in use.39 Before any ideas could be developed and even before knowledge of today’s techniques could be realized, the idea of “blood” in general fascinated people. From as far back as history can take us, man-kind has had a passion towards the mysterious dimension of blood. It wasn’t until 1492 that those thoughts resulted in actions. At the request of his physician, Pope Innocent VIII is said to have received a transfusion from the blood of three ten year old boys. The actions proved to be disastrous as all three boys died and so did the Pope. However unique the measure was, the idea of blood transfusion was adopted.39
It wasn’t until English physician William Harvey and his work on circulation and heart functions in 1628 that a true breakthrough occurred. His knowledge led to answers of the functions of blood and proved the importance of the mysterious substance.The idea of transferring one person’s blood to that of another for beneficial purposes was still unimaginable however. The year 1665 proved to be the first successful transfusion on record involving humans; however, it was not what you might think - the transfusion was not from human to human but rather from sheep to human.39 This was the work of Jean-Baptiste Denis in France, which ultimately was deemed unsuccessful as the results could not be replicated on a regular basis.40 The enchanting desire to experiment with the deviant substance was in full swing as the common practice of bloodletting proved to be common practice. The idea of thinning one's blood to get rid of evil desires and to help one get well verified its ineffective use as death was often the end result. In 1818, British obstetrician James Bundell performed the first successful transfusion of human to human blood when he treated his patient of a postpartum hemorrhage using her husband’s blood. Although the techniques of today’s practice were not used, the concept was realized.40 The work of Karl Landsteiner, an Austrian physician, was the biggest breakthrough for transfusion medicine as we know it today. Landsteiner documented the idea of ABO blood typing and developed the idea of the first three blood types A, B and O.1 His work later won him the Nobel Prize for Medicine. Landsteiner was not done there; he later discovered, along with some of his colleagues, the Rhesus blood group system which was the culprit to many transfusion reactions that were occurring at the time. Landsteiner's work was instrumental in blood transfusion medicine and paved the way for all the things we see today.
The ABO group system is based on the existence of two different antigens, antigen A and antigen B – differentiated by the immunodominant terminal sugars of oligosaccharides found on glycolipids or glycoproteins - on the >membrane.41 Blood lacking both of these antigens is defined as a group O blood type and is identified as the universal blood type, meaning that it can be donated to anybody. On the other hand, blood that caries either one or both antigens of the ABO system can cause a fatal hemolytic transfusion reaction in the incompatible recipient who has antibodies to the corresponding antigen present on donor cells.
http://www.biologycorner.com/resources/blood_type.jpg
Because only 5 percent of non-Asians are group AB and thus the universal recipient, a majority of patients require either type specific or universal blood type blood products. Therefore, there is a great demand for and also a constant shortage of the group O blood type. Two major approaches in an effort to provide a larger supply of universal, group O blood type red cells involve chemical modifications of other blood types such as group -A and -B:
Enzymatic removal of either immunodominant monosaccharide that determines group A, which is a terminal a-1,3-linked N-acetylgalactosamine (GalNAc), or the corresponding monosaccharide that determines group B, an a-1,3-linked galactose (Gal). Group O cells lack either one of these monosaccharides and terminate with a-1,2-linked fucose (Fuc) residues, designated as H-antigen.41
PEGylation or covalent linking of cell surface molecules with polyethylene glycol ( PEG) in order to mask the blood group antigens from their antibodies.42
http://www.hematology.org/publications/hematologist/JA07/images/Linenberger.gif
The goal of this approach to remove blood type specific sugars and to generate universal-ABO red blood cells or ECO-RBCs. Accidental transfusion of ABO-incompatible red blood cells (RBCs) is a leading cause of fatal transfusion reactions. ECO concept is the only one for which human clinical trials have been performed43 and it bears great promise. 
The idea to enzymatically remove blood type identifying sugars from the end ofthe ABH carbohydrate chain, attached to red blood cell membrane, and thus create universal red blood cells is not novel. It has been introduced over two decades ago by Goldstein et al.44 and co-workers at the New York Blood Centre. These A, B, or AB red blood cells that have been converted to O red blood cells are also referred to in the literature as ECO red blood cells. They are equivalent to O type and thus are an “ABO-universal blood”. They are not truly "universal" blood, because other blood systems have not been considered, and such RBCs can still react with some human sera.
The proof of the principle was achieved when first ECO RBCs were transfused into healthy group –A and –O human volunteers. A successful Phase II clinical trial in patients was reported in 2000 by Kruskall et al.45 Transfused RBCs were produced from group B RBCs using recombinant enzyme, coffee bean a-galactosidase. Phase I and II clinical trials showed normal survival, no clinical reactions, no rise of anti-B titers, and negative direct antiglobulin tests (DAT) post-transfusion.41
>Difficulties encountered were: acidic pH of 5.5 needed for the optimum enzyme activity but at the same time not best for RBCs, large quantities of enzyme needed for conversion (1-2 g enzyme per unit of group B RBCs) making the process expensive, and finally no success converting group A RBCs due to either more complex structure of an antigen or enzyme inefficiency. In addition to group B ECO RBCs, conversion of A2 subgroup was accomplished using chicken liver A-zyme (1g of enzyme per unit of RBCs at pH 5.5), but conversion of A1 RBCs was only partial.41
http://3.bp.blogspot.com/_DZH2cmCoois/RhJ22dl5mQI/AAAAAAAABtI/KTptkgp8hM8/s400/ABO_antigens_glycosidases.bmpIn the search for solutions , ZymeQuest and their collaborators have screened large panels of 2,500 bacterial and fungal isolates to search for a more efficient enzyme, with substrate specificity for the more complex branched blood group A and B structures and a neutral pH optimum.47 Their study has successfully identified two prokaryotic glycosidase gene families with a-N-acetylgalactosaminidase (GH109) and a-galactosidase (GH110) activities, with neutral pH optima, and high activity and specificity for blood group A and B substrates, respectively High yield of both enzymes is achieved using E.coli. ECO RBCs have survival expectations equivalent to native group O RBCs in non-ABO matched individuals, as previously reported for group-B ECO RBCs.41
>An automated and cost-effective process at neutral pH is feasible for use in transfusion medicine, and is currently in progress.41 Because of similar properties of novel recombinant enzymes, conversion to RBCs that type as blood group O can be made in the same buffer system. Anticipated consumption of the recombinant enzyme during this process will be about 30- A-ECO (~60mg), and 1000-fold less B-ECO (~2mg) than previously reported in protocol for group-B ECO RBCs conversion using Coffee bean a-galactosidase.41 This process holds great promise of achieving a goal of universal-ABO RBCs production, which would ultimately boost the scarce supply and also improve safety of blood transfusion.
Future Directions : The company ZymeQuest, based in Beverly, MA, has licensed novel enzymes and developed a machine that can simultaneously treat eight units of blood in 90 minutes.48,49 The company is hoping to have the blood processing instrument on the European market in 2011 and few years later in the United States .48,49
The goal of this approach is to mask ABO/Rh and other red cell antigens46 and to generate universal red blood cells or stealth-RBCs. This would be of special use to patients receiving repeated blood transfusions, with subsequent development of alloimmunization to various antigens, such as sickle cell patients.42
PEG is a non-ionic polyether which is present in a variety of molecular weights (MW = 100 - 8000000 g/mol) and various configurations such as linear, branched, and star. Its basic structure is: HO-(CH2CH2O)n-CH2CH2OH. PEGs are not charged and are water soluble. Each ethylene oxide unit interacts via hydrogen bonding with three water molecules, and the terminal hydroxyl group is involved in coupling reactions. Due to this water attracting property of PEG , pegylated proteins are covered with a shell of PEG , and each molecule of PEG in turn is surrounded with a hydration sphere (Fig. 2) The original idea was that this kind of protection in the form of a double-shell could obstruct binding of antibody to its corresponding antigen.
Different reagents have been used for linking PEG to RBCs such as: cyanuric chloride, propionic acid, and benzotriazole carbonate. Technique of using different MWs of PEG , together with different configurations, and various coupling reagents, has been employed by different research groups. In 2007, Garraty át. al.46 published some of the problems encountered in these studies.
Such problems included:
o incomplete masking of antigens as measured by routine blood bank procedures including the antiglobulin test (AGT) and flow cytometry
O non -specific uptake of proteins and subsequent reactions in the monocyte monolayer assays (MMA>), which suggested a shortened survival of RBCs
o presence of IgM and IgG antibodies to PEG in some sera or to a neoantigen created by PEG treatment
Most of these problems have been eliminated by using 2nd generation of PEG derivatives such as branched PEG RBCs, XPEG RBCs, and PEG RBCs made by using thiolation mediated chemistry.
Still, one very important concern left was whether or not PEG is immunogenic when bound to RBCs, and if so if the antibodies are clinically significant. Results of an animal study on a rabbit model published by Sroda át al.50 showed that PEG is immunogenic and that these antibodies shortened RBC survival. In 2007, Armstrong át al.51 published their findings on pegylated asparaginase ( PEG -ASNase), providing additional evidence that supports immunogenicity and clinical significance of PEG antibodies. The presence of anti- PEG is associated with shorthened survival or rapid clearance of PEG -ASNase in patients treated for acute lymphoblastic leukemia ( ALL ), rendering their treatment ineffective.
The most likely explanation of the presence of PEG antibodies in healthy donors is exposure to PEG in the environment. Immunogenicity of PEG bears a doubt that ‘stealth RBCs’ will ever have a future use in humans. Perhaps new generations of PEG can bring some hope to this approach.
The heart of oxygen carrying ability lies in a molecule called hemoglobin. Since any loss of red blood cells and thus a loss of hemoglobin results in decreased oxygen-carrying capacity, the initial research in red cell substitutes was with this molecule. Hemoglobin is separated from the cell in a saline solution and the leftover debris is filtered away, leaving only the hemoglobin.>21 Since free hemoglobin (or stroma-free hemoglobin), a tetramer, is toxic to tissues and quickly degenerates into dimers and monomers, research has focused on stabilizing the tetramer form by chemically binding it and making it less harmful. Other molecular substances associated with the binding and release of oxygen as well as vasoactivity (>P50) were also kept in mind while doing hemoglobin-based substitute research. These substances include 2,3-DPG (2,3-disphosphoglycerate or 2,3-bisphosphoglycerate, BPG) with correlation of the oxyhemoglobin dissociation curve (see also P50), and nitric oxide. What scientists have come up with are hemoglobin-based oxygen carriers (>HBOCs) which include polymerized, conjugated, cross-linked tetrameric, recombinant, and surface-modified hemoglobin. Although the oxygen-carrying ability of some of these products was surprisingly good, some of the failures with HBOCs have included renal failure, blood clots which leads to heart attacks and strokes, gastrointestinal distress including an increase in pancreatic enzymes, and systemic vasoconstriction. 9 Research is now aimed at making hemoglobin-based products with none of these drawbacks and just as much efficacy.
Polymerized hemoglobin is made with reagents such as glutaraldehyde which links the surface amino acid groups together. In April 2001, there were three polymerized blood products currently undergoing phase III clinical trials>: Hemolink, which was made from human hemoglobin polymerized with o-raffinose, produced by Hemosol (now Therapure Biopharma, Inc.); PolyHeme, which is manufactured by Northfield Laboratories; and Hemopure, which is a bovine hemoglobin-based product made by Biopure. Each of these products has a shelf-life (without need for refrigeration or freezing) of anywhere from as little as 12 months to up to 3 years. One of the pros to this hemoglobin product is it is the only product (as of April 2001) that had not caused vasoconstriction after infusion.10 One drawback to using bovine hemoglobin is it may carry the additional risk of zoonotic infection and prion diseases. Companies that produce this kind of hemoglobin product say this product is even safer than hemoglobin taken from humans due to rigorous monitoring.21
Polyheme. Image courtesy of Northfield Laboratories. 31
In 2001 and 2002, Hemosol had received approval from the FDA to begin clinical trials with their product, Hemolink, in patients undergoing coronary artery bypass grafting (CABG) and re-do CABG surgeries. Also in 2002, Hemolink was approved for chemotherapy-induced anemia trials. The side effects experienced by Phase I and II clinical trial patients included vasoconstriction in hypertensive patients, and jaundice and hypertension. In 2003, the trials of this product were put on hold due to increased myocardial infarction rates with Hemolink recipients. In Phase III trials, similar side effects were noted, this time including pancreatic enzyme elevations. Further Phase III trials have been suspended and production of Hemolink has ceased.
>PolyHeme is a cross-linked glutaraldehyde made from expired human blood products. Opponents to this product say it lacks 2,3-DPG, but the company adds PLP (pyridoxal phosphate) which is an organic phosphate that works like DPG. Northfield Labs claims their product may even be better at unloading oxygen than real red blood cells.>21 PolyHeme is targeted at patients with life-threatening bleeding especially when red blood cells are not available for any number of reasons (noncompatibility, remote location, etc.). In 2002, 75% of patients with hemoglobin levels of less than 1 gm% following traumatic injury, who then received PolyHeme, survived and recovered as compared with 16% in the control group.37 
As of 2005, PolyHeme (">PolyHeme trials) and Hemopure were still undergoing clinical trials. Polyheme had completed Phase II and has moved on to Phase III trials as intraoperative red cell replacement products. Hemopure was in Phase II trials involved with orthopedic and general surgery patients.20 One major drawback with both of these products is the short halflife at less than 24 hours. These red cell substitutes would only be a temporary fix until allogeneic transfusions could be given or the patient would require another transfusion of the substitutes, increasing the odds of having adverse side effects as the dose increases. However, between April 2001 and February 2005, 88% of patients receiving Hemopure were able to avoid allogeneic transfusions. It seemed to be well tolerated and adverse reactions were rare, with only 5% showing an increase in systolic blood pressure greater than 30 mmHg. This increase was managed with pharmaceuticals and was not noted to be of clinical significance. Biopure's Hemopure was undergoing trials in South Africa in 2006 and was being tested for use in emergency situations. With the success of these trials, Hemopure is now licensed as an "oxygen bridge">38 for commercial use in South Africa. It is to be used for acute perioperative anemia with a maximum dose of 7 units. However, in December 2006, the Blood Products Advisory Committee of the FDA voted against recommending that the US Navy proceed with late-phase clinical trials of Hemopure.37 At this time, Biopure is addressing the FDA's questions regarding the efficacy and safety of their product.
Another bovine-based, polymerized hemoglobin oxygen carrier is HemoTech, which is being manufactured by HemoBioTech. This was first proposed by Texas Tech researchers. The production method is based on reacting pure bovine hemoglobin with three chemicals: o-adenosine 5'-triphosphate (o-ATP), o-adenosine, and reduced glutathione (GSH). This process chemically modifies the hemoglobin to create "beneficial activities."32The o-adenosine can counteract the hemoglobin properties that cause narrowing of blood vessels as previously seen with other red cell substitutes. The GSH with adenosine attached to it has been shown to reduce the inflammation that is often caused by free hemoglobin. HemoBioTech believes that their product has vasodilatory effects as well due to the adenosine.
>Animal testing of this product had been ongoing in the late 1980s and into the early 1990s, showing HemoTech's nontoxicity and effectiveness as a substitute oxygen carrier. The first human clinical trial was conducted in 1990 in Zaire. This study involved nine children with sickle cell anemia. They received HemoTech at a volume of approximately 25% of their total blood volume over a period of 2 hours. This study showed overall improvement in the children, as well as a decrease in sickle-cell-induced pain, most likely due to the vasodilatory effects of HemoTech. HemoBioTech concluded that the Phase I clinical trial showed non-toxicity and biological activity in humans including erythropoiesis.33
HemoZyme is known as a "second generation" HBOC. This product is made by SynZyme Technologies, LLC. It is a polymerized polynitroxyl hemoglobin product, developed for blood loss in surgeries and hemorrhagic shock. SynZyme is hopeful their product will be approved for surgical use and then plans to go ahead and get approval for using HemoZyme in trauma situations along with hemorrhagic shock in both combat and civilian situations. They claim their product can be used to prevent or treat ischemia, reperfusion, and inflammatory injuries as well as multiorgan failure which often accompanies hemorrhagic shock.78
Conjugated hemoglobin includes a product called Hemospan, manufactured by Sangart>. This is human hemoglobin molecules extracted from outdated human red blood cells (RBCs) linked with polyethylene glycol (PEG) to decrease free hemoglobin's toxicity. The research in this area has focused on hemoglobin lipid membranes with the introduction of PEG, resulting in a greater halflife circulation time.14 It has a low hemoglobin concentration but a high oxygen affinitiy and high viscosity. The premise is that the oxygen will be released especially and specifically in areas of hypoxic tissue, thus avoiding hyperoxia of normal tissue and the toxic effects thereof.38 It is believed that hyperoxia of normal tissues is one of the causes of the vasoconstriction seen with other HBOCs. Hemospan comes in a powdered form and is mixed into liquid form just prior to use. In 2004, it was undergoing Phase Ib/II trials involving orthopedic surgeries and no adverse events had been attributed to its use.29 Phase III trials were completed in the early part of 2008 on over 1000 patients including orthopedic surgery patients in Europe. Although these trials were regarded as successful, they failed to show improved outcomes because of the patient population studied. Sangart is currently searching out possible populations on which to test Hemospan's ability to improve patient outcomes over allogeneic RBCs.
Another conjugated hemoglobin is made with dextran. This product is Dextran-Hemoglobin (DxHb), manufactured by Dextro-Sang Corporation. Dextran by itself has proven to be an effective plasma volume expander and has thus shown its biocompatibility. DxHb is made by conjugating human hemoglobin to dextran, which is a poly1->6-alpha-D-glucose with 1->3-alpha-glucopyranosyl branch residues which are randomly placed. This conjugation has prevented excretion of the hemoglobin by the kidneys as well as kidney damage. It also does not enter the lymph system, thus preventing tissue edema. In animal studies, survival after exchange-transfusions with DxHb is 75-100%.35
Cross-linked hemoglobin is based on attaching small bridges of sugar molecules to hemoglobin dimers, then making a stable tetramer. One such product is a diasprin cross-linked tetramer made from expired human red cells called HemAssist. Baxter Corporation collaborated with the United States Army to manufacture this product. The crosslinking substance is 3,5-bis(dibromosalicyl)fumarate or DBBF, which links together the two alpha chains of the hemoglobin molecule. This prevents dissociation of the tetrameric form of hemoglobin to dimers, thus eliminating renal toxicity.>28 The Phase III trials of this product were unsuccessful, however, as the study showed increased mortality in trauma situations, most likely due to dose-related increases in blood pressure and a decline in heart rate.20 The increased blood pressure was most likely due to vasoconstriction which had been shown with previous HBOCs. Further trials of HemAssist have been suspended.
Recombinant hemoglobin is a cross-linked hemoglobin made by genetically altered organisms such as the bacteria Escherichia coli or Saccharomyces cerevisiae, a yeast. Baxter Corporation teamed up with Somatogen and made Optro, which is a recombinant hemoglobin produced by E. coli. A few parts of the amino acid sequence of human hemoglobin are replaced to prevent dissociation into dimers and to help maintain adequate oxygen affinity.22 This altered human hemoglobin coding segment of DNA is then inserted into the bacteria or yeast DNA. It is made in a way similar to the making of insulin and can be produced in mass amounts. Optro underwent Phase II trials on cardiac surgery patients and Phase III trials on traumatic shock patients. Trials were discontinued due to adverse effects with Optro causing vasoconstriction, GI distress, fever, chills, and backaches.
Surface-Modified hemoglobin is made with large molecules such as polyethylene glycol (PEG), which is bound to the surface lysine groups. There are two companies, Enzon and Apex Bioscience (bought by The Medicines Company), that are producing these hemoglobin products. One of the disadvantages of this product was the increased vasoconstriction that had been reported with other artificial red cell substitutes. However, Enzon’s product was small enough that it could be used for treatment of patients with stroke and/or cancer due to its small size. These small molecules could pass through constrictions that natural red blood cells could not, thereby reoxygenating tissues previously cut off from oxygen supplies. Cancer patients were benefited by this product’s ability to bring oxygen to tumor cells which made them more susceptible to current cancer therapies, i.e., radiation and chemotherapy. Apex Bioscience has been working on hemoglobin-based red cells to be given to patients with septic shock and resultant hypotension.10 Their product is a pyridoxylated hemoglobin polyoxyethylene conjugate (PHP) which is undergoing Phase III trials in patients with shock in association with a systemic inflammatory response syndrome.37
Research into microcapsules which encapsulate free hemoglobin has been done in the past. Companies had been trying to work with nylon, collodion, gelatin, gum arabic, and silicon but they were quickly cleared by the reticuloendothelial system.22 One method is to purify the hemoglobin and then encapsulate it in a lipid mixture with phospholipids, cholesterol, and alpha-tocopherol. This product has a longer shelflife than other HBOCs and perfluorocarbons as well as less vasopressor effects and reperfusion injury due to the encapsulation of the other enzymes associated with red blood cells.38
The development of an immediate synthetic oxygen-carrying support that can have the potential of supplementing red blood cells could result in a revolution in transfusion medicine. Using an entirely different approach, oxygen delivery can be achieved using organic chemicals and high gas solubility.64 A perfluorocarbon (PFC) emulsion is a candidate for an artificial blood substitute.65 PFCs are chemically and biologically inert artificial fluorinated organic fluids that are immiscible in water and have a high solubility for oxygen. A pure PFC solution has the ability to sustain life for an extended period of time.63 However, PFCs are oil-like fluids, and oil and water do not mix; therefore, they cannot carry water-soluble salts and metabolic substrates.64 In order for PFCs to be good oxygen carrying compounds they must be dispersed in an aqueous solution such as albumin or other high electrolyte concentration solution. Where hemoglobin uses localized chemical bonds between the dioxygen molecule and heme, PFCs have a physical separation with oxygen characterized by loose, non-directional van der Waals interactions. The difference between the two is the ability for PFCs to uptake oxygen. This is clearly present in oxygen uptake curves where hemoglobin is sigmoidal in shape and the PFC’s is linear.66 A PFC dissolved oxygen is immediately available for tissues and is not bound up by carbon monoxide, nitric oxide or any other chemical reagents. Additionally, PFC dissolved oxygen is preserved as temperatures decrease, as opposed to hemoglobin. The many benefits for the use of such a product utilizing PFC emulsion over hemoglobin for oxygen delivery doesn’t stop there. A PFC emulsion is blood type free and therefore would be free of any alloantibodies or autoantibodies, providing a decreased risk for transfusion reactions. PFCs can be stored longer than native red cells in addition and a PFC emulsion has high performance to deliver oxygen throughout the peripheral circulation because of its lower viscosity as compared to blood viscosity.66 The potential to have immediate oxygen-carrying capacity in the case of a trauma would be very advantageous for both the patient and the administrator.
Development of such a PFC emulsion requires many steps. First one needs to select the correct PFC and develop an emulsion that is stable and injectable. The PFC would require such characteristics as low vapor pressure, rapid excretion, and the ability to form stability in emulsions. The overall process should be easily synthesized in large scales and the overall cost should be low. These are not common characteristics of PFC emulsion oxygen transport molecules.66 Despite these stringent conditions there have been attempts to develop such products.
The first PFC developed was Fluosol-DA (Green Cross Corp, Osaka, Japan). The FDA approved Fluosol in 1989 for use in high-risk coronary balloon angioplasty. It holds the distinction of being the only synthetic oxygen carrier to be approved for human clinical use in the United States. Fluosol suffered many problems that couldn’t be resolved which ultimately led to its removal from the market. First, Fluosol delivers insufficient amounts of oxygen, requiring patients to require supplemental oxygen. Second, Fluosol is not excreted in a favorable manner which allows for remnants to be present in the body for months and months. Third, the side effects of Fluosol are not tolerable for many patients.
The side effects include flu-like symptoms as well as an overall decrease in platelet counts. Lastly, the product stability is very poor. For Fluosol, it is stable for about 8 hours, which creates logistical difficulties during practical use.67
Another product that emerged was a product called Oxygent, developed by Alliance Corp out of San Diego, California. Oxygent is an improved second-generation concentrated PFC emulsion based on perflubron (perfluorooctyl bromide; C8F17Br) with a shelf life of up to 2 years.67 Supplemental oxygen inhalation is still required by the patient as the oxygen capacity is still less than normal blood. Studies have shown that Oxygent in conjunction with acute normovolemic hemodilution (ANH) reduces the need for RBC transfusion in 492 patients undergoing major noncardiac surgery.67
However, in the United States, trials with patients undergoing cardiopulmonary bypass have been discontinued due to a possible increase in the amount of strokes by patients using Oxygent as a treatment regimen. Studies have been discontinued due to the developmental costs as well as the lack of funding. Currently there are no major trials ongoing in the United States that involve PFC’s as a synthetic oxygen-carrying alternative or the use of PFC emulsions . There is currently no true substitute for the many functions of human red blood cells, and synthetic products will not replace the need for blood donation in the foreseeable future.
Blood products grown from stem cells
>Stem cell research has opened up alternatives to the traditional blood transfusion process. Rather than collect blood from a donor and match it to the recipient’s blood type, erythrocytes may be grown either from embryonic stem cells or hematopoietic progenitor cells. The latter approach would seem more practical as hematopoietic cells are already committed to becoming blood cells and are easily harvested from bone marrow or cord blood.16 Additionally, this approach could sidestep ethical issues associated with the use of embryos. Despite this, in 2008, Lu et al reported success using embryonic stem cells to grow mature erythrocytes on a large scale, producing 1010-1011 cells, with a 60% enucleation rate, thus eliminating the possibility that these erythrocytes might cause tumors in transfusion recipients.15,17 However, the cells produced by Lu and colleagues show a phenotype more consistent with fetal or embryonic erythrocytes than adult blood cells. The problem of creating cells with an adult phenotype remains to be solved. In addition, while the scale of production exceeds earlier work, a unit of packed red blood cells contains around two trillion cells, several orders of magnitude greater than what Lu, et al achieved. Further research will be required to increase yields, eliminate potentially tumorigenic nucleated cells, and determine the half-life and immunogenicity of erythrocytes grown from stem cells.17
Stem cells may also offer an option for the replacement of leukocytes. Current technology cannot provide an artificial substance able to match the complex function of white blood cells. The various types of white blood cells are able to recognize and eliminate many different bacteria, viruses, and other foreign materials. Using stem cells, leukocytes may be grown and transfused. Alternatively, stem cell therapy could be used to grow new white cells in vivo. Researchers at City of Hope Medical Center in Duarte, California, have done exactly that. Genes allowing T-cells to identify and resist the main known variants of the HIV virus in vitro were introduced into a quantity of the patients’ own stem cells. These stem cells were then introduced in a bone marrow transplant to treat AIDS-related lymphoma. Early data indicate the resistant cells are persisting in transplant patients. 18
Plasma volume expanders are inert fluids that are used to increase the volume of circulating blood. Volume expanders do not carry oxygen, but they allow the red blood cells to circulate freely throughout the cardiovascular system. When a patient experiences blood loss due to surgery or trauma, two factors are considered: oxygen delivery to tissues by red blood cells and maintenance of circulating blood volume. Inadequate circulating blood volume, or hypovolemia, can cause collapse of the blood vessels and should be avoided. Intravenous administration of plasma volume expanders corrects hypovolemia temporarily until other suitable blood products can be administered. If half of the red blood cells are lost due to injury or trauma, the remaining 50% of red blood cells can deliver sufficient oxygen to tissues in a resting patient, provided there is adequate blood plasma volume to carry the red blood cells through the microvasculature.
There are two basic categories of plasma volume expanders: crystalloids, which are a solution of water and salts (electrolytes and/or sugars); and colloids, which contain protein, starch or gelatin.5There are several different types of crystalloids and colloids that have different properties, qualities and uses. Crystalloids may be normal saline (0.9% isotonic solution), hypertonic saline, or Ringer’s Solution which contains calcium, potassium chloride, sodium chloride, and sodium lactate in water.5 Colloids may be albumin, hydroethyl starch (HES, pentastarch, hetastarch), gelatin or Dextran.6 Albumin is the predominant plasma protein and remains the standard against which other colloids are compared. However, albumin, pooled from human donors, is in short supply and expensive. The use of PEG-albumin (polyethylene glycol) in preoperative hemodilution was shown to be more effective in maintaining capillary perfusion than with an HES product.23 Hetastarch provides equivalent plasma volume expansion to albumin, but has been shown to alter clotting parameters (prolonged APTT and PT). Colloids have been shown to increase osmotic pressure, but the effects are short-lived. Lower molecular weight colloids exert a larger initial osmotic effect, but are rapidly cleared from the circulation. Larger molecules exert a smaller osmotic pressure that is sustained longer.7 Crystalloids do not contain any macromolecules. They spread rapidly over the intravascular and interstitial space due to their lack of intrinsic colloid oncotic pressure. To achieve a comparable volume effect like colloid solutions, a fourfold infusion volume of crystalloids is necessary.8 Crystalloids may be used in addition to colloid solutions to compensate for interstitial fluid deficit.
There are problems associated with the use of colloids for plasma volume expansion. There are several starch based colloids in use today such as Pentastarch (Pentaspan),11> (PentaLyte),25 and Hetastarch (Hespan),12 all of which are hydroethyl starch (HES) products. Use of HES products is contraindicated for patients with allergies to starch, congestive heart failure, or for those patients with renal dysfunction or on dialysis. Disturbances in coagulation and hematocrit have also been reported. In blood coagulation, HES as well as Dextran and gelatin all can induce specific decreases of von Willebrand factor and factor VIII.24 In a comparative review of different plasma substitutes on blood coagulation, all artificial colloids could potentially induce increased bleeding tendency after infusion of very large volumes. Therefore, crystalloid solutions such as plasma or albumin could be considered for use. Injection of HES enhances the microcirculation and leads to better oxygen transport into tissue and muscle. Because of this, HES was identified as a doping agent in February 2002.27 HES products with high molecular weight (200 kD) are associated with high rates of renal failure. Because of these problems, HES products are currently in a third generation of development based primarily on Mean molecular weight (Mw) in kD (kiloDaltons), Molar substitution (MS = the molar ratio of the total number of hydroxyethyl groups to the total number of glucose units) and C2-C6 ratio13.
Acetyl Starch (ACS) is a new synthetic colloid solution. In a comparison study with HES 6%, preliminary results indicate that ACS and HES are equally effective plasma volume expanders, and ACS has fewer coagulation side effects 26 than HES. Clinical trials are continuing.
A unit of platelet concentrate, as currently in use, is pooled from the blood of several donors, and must be stored in bags permeable to oxygen and carbon dioxide at room temperature for not more than five days. Because one unit requires blood from multiple donors, and because of the short shelf-life, platelets for transfusion tend to be in short supply. Again, because one unit may come from multiple donors, platelets may be more likely to transmit disease.16 Attempts to overcome these shortcomings have been varied.
It has been shown that platelets may be preserved for up to ten years when held at -80°C in 6% dimethylsulfoxide (DMSO). While cryopreserved platelets are the only alternative to fresh platelets currently in clinical use, they have some disadvantages. First, they are considerably more expensive. Secondly, the preservation process appears to alter the morphology of the cells, although protecting agents have been developed to limit this effect and decrease the need for DMSO.55
Treatment of with psoralen and UV light has been used to inactivate bacteria and viruses in liquid-stored platelets, and has been approved for clinical use in Europe. Platelets treated in this manner appear to retain normal function, but have a shorter survival in vivo. 55
An infusible platelet membrane (IPM) product called Cyplex has been researched by Cypress Bioscience. Cyplex is derived from lysed platelets and consists of microparticles with a low HLA content. Major advantages of Cyplex are that it can be made from outdated donor units, and it has a shelf life of over two years at 4°C. The hemostatic effect of Cyplex appears to be dependent on the presence of some residual native platelets in the recipient’s blood, with the IPM product promoting aggregation at the injury site. The same is believed to be true of fibrinogen-based platelet alternatives. It remains to be seen whether Cyplex will be fully developed.52,55
Rehydrated Lyophilized (RL) platelets were initially examined as an alternative to fresh platelets in the 1950’s by Klein and colleagues. Initial attempts did not preserve hemostatic function, and were of no clinical benefit. In 1975, Allain et al. showed that cross-linking surface membrane proteins and lipids with 3.6% paraformaldehyde for 48 hours at 4°C produced a product still capable of agglutiniating in the presence of vonWillebrand Factor (vWF) and ristocetin. Studies by Blajchman, Bardossy and Read in 1994 showed correction of bleeding times in thrombocytopenic rabbits nearly equal to that seen with fresh platelets. Using a canine cardiopulmonary bypass (CPB) model, Woodman and Harker (1990) found that a large dose of RL platelets given just prior to removing the CPB pump produced a correction in bleeding time lasting for at least three hours post-op. Additionally, test animals had been splenctomized prior to surgery, thus minimizing the possible role of endogenous platelets.56> RL platelets were able to halt ear and jugular vein bleeding in pigs undergoing complete blood replacement with the hemoglobin-based oxygen carrier HBOC-201.56,60
Questions have been raised regarding the safety of RL platelets, particularly how they can provide hemostasis without causing intravascular thrombosis. According to ICD-9 statistics, secondary (thrombin-mediated) hemostatic responses are responsible for the majority of vascular occlusions, while primary (glycoprotein Ib and vWF-mediated) responses are rarely the cause. While RL platelets show primary responses nearly equal to fresh platelets, thrombin-mediated aggregation is below 50% of that produced by fresh platelets. Additionally, RL platelets are rapidly cleared from the circulation by macrophages in the spleen (t1/2 ~ 10 min), thus greatly reducing the prothrombotic potential.57,58 RL platelet preparations are sterile, as it has been found that bacteria and viruses added to the platelet suspension are killed by the fixing process. Additionally, unpublished findings of Hemocellular Therapeutics Corporation, a joint venture of East Carolina University and The Univerisity of North Carolina, show that HLA antigens are largely destroyed by the fixation process.56 It should be noted that because the extremely short half-life allows for local hemostasis at the site of a wound, but not for prophylaxis, the FDA evaluates RL platelets not as a platelet substitute, but as a hemostatic agent.>58
Platelet substitutes not derived from platelets involve conjugating fibrinogen, or portions of it, to other particles. In 1999, Levii and colleagues reported some success using human albumin microparticles conjugated to fibrinogen in rabbit models of thrombocytopenia. These albumin microcapsules, named Synthocytes, were able to correct bleeding times in rabbits chemotherapy (Busulfan) and antibody (goat anti-rabbit platelet) induced thrombocytopenia. Chemotherapy rabbits were also subjected to surgical wounds. Correction of bleeding from both ear vein lacerations and surgical wounds lasted at least three hours. In addition, no significant difference was noted in jugular vein thrombus growth in rabbits injected with saline, non-coated albumin, and Synthocytes.>53 A similar preparation, Thrombospheres (developed by Hemosphere) has a smaller mean diameter and may have a longer duration of action.55
Okamura and coworkers were able to correct bleeding times of bulsulfan-induced thrombocytopenic rabbits using H12 (a peptide sequence occurring at the carboxyl end of the fibrinogen gamma chain) conjugated to poly(ethylene glycol) modified polymerized albumin (PEG-polyAlb). The hemostatic effect of this H12-PEG-polyAlb lasted at least six hours, and was calculated to be at least 31-fold greater than that of a similar volume of platelets.54
Wong and Chang in 2007 cross-linked fibrinogen to polymerized hemoglobin, creating a product with both platelet-like properties and oxygen carrying abilities. Polyhemoglobin has been tested in clinical settings, and in a trauma situation where large amounts are infused, hemodilution can occur, necessitating the administration of platelets and/or clotting factors. Tested in a hemodiluted setting, polyhemoglobin-fibrinogen blood clotted faster and clots adhered better than blood containing polyhemoglobin alone. Fibrinogen could also be conjugated to other HBOCs to yield other oxygen carrying platelet substitutes.61
Direction of future research and clinical trials
A future volume expander coming from the BioTime company is HetaCool 30. HetaCool is in the animal testing phase and is used for complete blood volume replacement during hypothermic surgery. Hypothermic surgery is a new field where the body temperature is chilled down to near freezing temperatures and the blood is totally replaced. There is no date set from the BioTime company as to when this product might be brought to market.
According to the Journal of the American Medical Association (JAMA),>36 there is a 30% increase in the risk of death and nearly a three-fold increase in the risk of myocardial infarction when all HBOC trials were pooled. They also noted that 38% of the trials were not reporting the number of myocardial infarctions that were occurring during the clinical trials. They argue that blood substitutes are not necessary, that blood products are generally widely available and the shortage of blood has been exaggerated. They also feel that with new techniques to identify viral or bacterial contamination and the increased ability to identify compatible cross-matched blood, the supposition that artificial blood is superior to donor blood with respect to disease transmission or transfusion reactions is unsubstantiated. Until the mechanisms and potential toxicities of hemoglobin-based oxygen carriers are further determined, JAMA would like Phase III trials on these types of red cell substitutes discontinued.36
Despite JAMA's objections, there are still areas where red blood cell substitutes would be of great benefit. These areas include at battlefield hospitals, in areas where blood-borne disease is rampant such as HIV in Africa, with individuals who refuse human blood products such as Jehovah's Witnesses, and in remote locations including when blood loss would normally lead to death. Both PolyHeme and Hemopure have shown promise and clinical trials of these two products are expected to continue. Further research will be done on the use of Hemopure with regard to its use for acute reversible tissue ischemia. Clinicians have already described improved wound outcome with this product.38
There is another polymerized stroma-free HBOC called OxyVita. It is made by a company called OxyVita (formerly associated with IPBL Pharmaceutics, Inc.) which just started up in 2003. It is currently in the research stages of development. OxyVita's capabilities will be undergoing extensive testing and its applications in the clinical setting are yet to be defined.77
The Dendritech Corporation, a company based in Michigan, is developing nano-polymers called >dendrimers>to be used as artificial red cells. Dendrimers are completely artificial. They have been used in drug delivery systems and are currently being developed for gene therapy and contrast agents for MRI. The idea is to take dendrimer polymers and put them in an aqueous carrier solution, which would then be able to transport dissolved oxygen. Using these for red cell substitutes is still in the early stages of research. In 2005, the US Army awarded $275,000 to Dendritech for further development of dendrimers as blood cell substitutes.38
Though only in the theoretical stage, nanotechnology has been proposed as an alternative to blood products. Robert A. Freitas, Jr., research fellow at the Institute for Molecular Manufacturing in Palo Alto, CA, has written detailed descriptions of diamond-based mechanical erythrocytes he calls Respirocytes. In addition, he has described mechanical platelets and phagocytes, called Clottocytes and Microbivores, respectively.62
2,3 DPG = 2,3-bisphosphoglycerate or 2,3-diphosphoglycerate, a three-carbon isomer found in human red blood cells; it enhances the red blood cells' ability to release oxygen to tissues that need it most.
>APTT = activated partial thromboplastin time.
conjugation= occurs in organic compounds, when atoms are covalently bonded with alternating single and double bonds.
dendrimers = polymers in which the atoms are arranged in branches and subbranches with a central carbon atom backbone.38
>doping agent = performance enhancing drug used primarily to improve athletic performance.
emulsion= a mixture of two liquids that normally are unmixable; one liquid is dispersed throughout the other. Example: Oil and water. Perfluorocarbons are emulsified in lipids or oils to make perfluorocarbon emulsions.
glutaraldehyde> = a dialdehyde reagent which reacts with any of the 42 surface amino acid groups on hemoglobin.27
>HBOC = hemoglobin-based oxygen carriers, any oxygen therapeutic that uses hemoglobin as its primary oxygen-carrying unit.
hemodilution = the concentration of erythrocytes and formed elements in the blood is lowered due to an increase in plasma volume.
hydroethyl starch = plasma substitute made from nonionic starch, sodium chloride and water.
nitric oxide (NO) = a gas that is important for cell signaling; contributes to vessel homeostasis by inhibiting vascular smooth muscle contraction and platelet aggregation.
oncotic pressure = form of osmotic pressure exerted by proteins in blood plasma that pulls water into the circulatory system.76
P50 = the amount of oxygen (partial pressure level) at which the hemoglobin molecules are 50% saturated. The P50 of normal human red blood cell-encapsulated hemoglobin is about 26-28 mmHg.
polymerization= reacting monomers together to get three-dimensional shapes or chains called polymer chains.recombinant = DNA sequences that are combined together in such a way that would not normally occur in nature. Example: Using E. coli to manufacture hemoglobin.
PT = prothrombin time.
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