Artificial Blood

Artificial Blood Abstract: Concern about potential infective agents in donated blood has stimulated the recent development of blood substitutes. A blood substitute (also called artificial blood) is a substance used to mimic and fulfill some functions of biological blood. Artificial blood is a product made to act as a substitute for red blood cells. Chemically cross-linked hemoglobins are ready for routine use. New generations of modified hemoglobin are being prepared to modulate the effects of nitric oxide and oxygen radicals, and artificial red blood cells are also being developed. The two most common types of artificial blood are HBOCs (hemoglobin-based oxygen carriers) and PFCs (perflourocarbons). Artificial blood is being designed for the sole purpose of transporting oxygen and carbon dioxide throughout the body. These are helping doctors and surgeons avoid the risks of disease transmission and immune suppression and also address the chronic blood donor shortage. Scientific community has begun to explore the possibility of using stem cells as a means of producing an alternate source of transfusable blood. Other potential techniques are also being considered. These are Dendrimers (as a substitute for Oxygen Carrier), Biodegradable micelles, Placental umbilical cord blood, Hemerythrin, Respirocytes etc. Artificial Blood can carry oxygen in situations where a person's red blood cells can't do it on their own. The term oxygen therapeutic is for Artificial Blood/Blood Substitute only. Unlike real blood, artificial blood can be sterilized to kill bacteria and viruses and also the Shelf life can be increased to make it ideal for emergency and battlefield situations. There are adverse effects and extensive clinical trials are being conducted to test the safety and efficacy. The mode of action of Artificial Blood Substitutes will define application.

Artificial Blood Substitutes Hemopure 1. What is Hemopure? Hemopure is a Hemoglobin-based oxygen carrying solution (HBOC). It is also known as Hemoglobin Glutamer-250 (bovine) or HBOC 201. 2. Manufacturing: Hemopure is made of chemically stabilized, cross-linked bovine (cow) hemoglobin situated in a salt solution. Hemoglobin by itself is toxic to the kidneys because it contains stroma lipids, which are contaminated with endotoxins. If the stroma lipids are removed, however, then the Hemoglobin has too high an affinity for oxygen, which means less oxygen off-loading to the tissues. In order to assure that the Hemoglobin is not toxic, but still therapeutically useful, it must be stabilized. Stabilization can be achieved through a number of methods, but Hemopure stabilizes the hemoglobin by cross-linking it. This is done by cross-linking the two alpha, and the two beta subunits. This then stabilizes the alpha-beta dimers, which in turn makes the hemoglobin molecule more stable, and also reduces its affinity for oxygen, making it easier to deliver oxygen to the tissues. 3. Safety Measures: Since the Hemoglobin used in Hemopure is derived from cows, It is made sure that Hemopure is: free of pathogens, infectious agents (BSE), and chemically pure, including:    

Using only very tightly monitored herds, where the country of origin, food supply and health are closely supervised. The use of external experts who closely regulate the strict manufacturing process of Hemopure to determine the capability to remove potential pathogens. Strict observance of the global industry and regulatory standards. Extensive clinical trials.

4. Advantages of Hemopure vs. RBC’s

Hemopure is smaller in size (up to 1,000 times smaller than a typical red blood cell) and has less viscosity than human red blood cells (which contain hemoglobin). This means that it can carry more oxygen at a lower blood pressure than red blood cells. Also, because of its smaller size, it can carry oxygen through partially obstructed or restricted blood vessels, where RBC’s cannot reach, as seen in the diagrams below.

Inadequate tissue oxygenation resulting from occluded arteries can result in heart attack, angina, or transient ischemic attack, which is a precursor to stroke. Traditionally, these conditions have been treated by blood transfusions, but RBC’s are often too big to pass through the occlusion, which is why Hemopure can potentially be very helpful.

5. Where is Hemopure Now? Hemopure is currently approved in South Africa for the use of surgical patients who are anemic, thereby reducing or eliminating the need of blood transfusions for these patients. It is currently in Phase III Clinical Trials in South Africa and Europe. In the U.S. Hemopure, is currently under review by the F.D.A. and is conducting animal studies. In March, 2003, the U.S. Naval Medical Research Center (NMRC) signed a collaborative research and development agreement with Biopure to help fund and conduct a trial on the effects of Hemopure in out-of-hospital resuscitation of patients with severe hemorrhagic shock. This trial, named “Restore Effective Survival in Shock” (RESUS) and over $14 million in Congressional, Navy, Army, and Air Force funding has been given so far to support the trauma development program for Hemopure. Hemopure has also been approved for compassionate use.

Oxyglobin Solution 1. What is Oxyglobin Solution? Oxyglobin solution is the first and only oxygen therapeutic to be both US FDA and European Commission approved for veterinary use. The solution consists of chemically stabilized bovine hemoglobin in a balanced salt solution and contains no red blood cells. The cross-linked hemoglobin, several tetramers bound together, works by circulating in the plasma and supplying oxygen to tissues. 2. Advantages of Oxyglobin There are many advantages of Oxyglobin over a regular allograft blood transfusion. Oxyglobin could be stored for up to 3 years at room temperature and does not require any heating or cooling before transfusion. The blood of the recipient does not need to be typed, for Oxyglobin is compatible with all blood types. All Oxyglobin products have been tested for and removed of potential contaminants such as viruses, bacteria and TSE agents. 3. Safety Measures It is made sure that Oxyglobin Solution is: free of pathogens, infectious agents (BSE), and chemically pure, including:    

Using only very tightly monitored herds, where the country of origin, food supply and health are closely supervised. The use of external experts who closely regulate the strict manufacturing process of Oxyglobin Solution to determine the capability to remove potential pathogens. Strict observance of the global industry and regulatory standards. Extensive clinical trials.

4. Where Oxyglobin is now? Currently, Oxyglobin can only be used in canines and not in humans. The current supply of Oxyglobin is low, for the company is spending most of its resources on Hemopure, a blood substitute for human use.

PolyHeme 1. What is PolyHeme? Polyheme is a hemoglobin based oxygen carrier and, as the only blood substitute to reach a Phase III trial, represents the leading technology in this field, Polyheme originally began as a military project following the Vietnam War and has since shown great potential for both military and civilian use. 2. Manufacturing: Polyheme utilizes human hemoglobin as the oxygen carrying molecule in solution, and the extraction and filtration of this hemoglobin from red blood cells is the first step in production. Then, using a multi-step polymerization process, the purified hemoglobin is associated into tetramers and, as the final step, is incorporated into an electrolyte solution. The polymerization of the hemoglobin represents the critical step in this process because, as demonstrated by failed attempts at blood substitutes, when hemoglobin remains disassociated, it tends to take up nitric oxide, causing vasoconstriction. Also, free hemoglobin can be taken up by the kidney causing liver dysfunction and failure.

3. Advantages: Artificial blood substitutes in general have inherent advantages over the donor system in place today. However, a blood substitute has yet to be developed that can effectively and safely mimic all the functions of natural blood. Polyheme has come the furthest of all blood substitutes currently in trials, and if approved for general use, has great life-saving potential. 1) Donor blood is often in short supply and expires after only 42 days. Polyheme has the possibility to be manufactured to stock hospitals across the country, so that the supply is never limited, and carries a shelf life of 12 months. 2) When natural blood is available for transfusion, both the donor blood and the recipient blood must be carefully matched, a process that in the best facilities takes 20 minutes but can take up to an hour. Polyheme is universally compatible, meaning any blood type can accept it, and can be transfused immediately. That time difference can easily save the lives of severely hemorrhaging patients who otherwise would have died. Also, since Polyheme is thinner than normal blood, it can be transfused more rapidly and can safely be transfused in large volumes. 3) Polyheme is manufactured in such a way so as to bring the risk of disease transmission to virtually zero. While the risk of contracting disease from donated blood has been decreasing with improved tests, there is still a chance. Certain tests done on donated blood only indicate the presence of antibodies to a particular antigen. These antibodies, depending on the phase of the disease, may not yet be completely formed during the period when the blood is donated and then transfused. Also, new pathogens such as prions, which are responsible for bovine spongiform encephalitis, or mad cow disease, are not yet able to be detected in blood. Polyheme completely circumvents these risks.

4. Possible Problems Polyheme was developed just as a temporary solution to blood loss. As a military project, the focus was to develop a blood substitute to keep trauma patients alive in remote areas where donated blood is not available, until they can reach more sophisticated facilities. While it can effectively replace blood function, Polyheme cannot necessarily do it for extended periods of time, having a circulation half-life of only 24 hours. Conditions requiring blood for longer than the circulation time of Polyheme would require repeated transfusions of Polyheme or later replacement with donor blood. While this product, given this condition, still carries enormous life-saving potential, the short circulation time is still a problem, or at least something to be improved upon. Another factor that can limit the effectiveness of Polyheme is the fact that it is manufactured using human hemoglobin. While this hemoglobin can be reclaimed from expired red blood cell products, it does not completely eliminate the need for donors because there must be a source of the outdated erythrocytes. The use of human hemoglobin could limit the supply and manufacturing potential of Polyheme, and while it has a shelf life much longer than donated blood, it will also be used more quickly due to the short circulation time. All this adds up to a limit on the supply of Polyheme.

Hemospan 1. Hemospan is designed to:      

Alleviate the current severe shortages of blood for transfusions. Completely avoid the transmission of infectious disease. Be universally compatible and available for administration within minutes. Be stored indefinitely, allowing stockpiling for use in emergencies, trauma and disasters. Utilize efficient oxygen transport resulting in lower effective doses, lower cost per patient, and greater safety. Have a simplified production process and high yield resulting in lower costs and reduced raw material requirements.

2. Manufacturing: Hemospan is produced in powder form, allowing it to be stored for years. The powder can then be mixed into liquid form and transfused immediately, regardless of a patient's blood type. Specifically, Hemospan is said to demonstrate a high oxygen transport capability with a low hemoglobin content. The starting material for Hemospan is unmodified hemoglobin from outdated human red blood cells. However, the source could be any form of hemoglobin - human, animal, or recombinant. The genius in its development appears to come in its combination of the human red blood cells with polyethylene glycol (PEG) to eliminate the toxicity associated with free hemoglobin. PEG polymers are readily available, synthetic materials that are attached to the surface of hemoglobin by way of simple chemical reactions. When PEG is attached to the surface of hemoglobin, a thin layer of water is formed around the protein. This surrounding water layer, it turns out, is essential for three reasons - it protects the hemoglobin from the immune system, increases the effective size of the molecule (thereby increasing circulation time), and produces a viscosity that is similar to a native red blood cell.

Dextran-Hemoglobin

1. What is Dextran-Hemoglobin? Dextran-Hemoglobin is another hemoglobin based oxygen carrier.

2. Manufacturing: Dextran hemoglobin is prepared by conjugating human hemoglobin to dextran, a branched polysaccharide made of many glucose molecules joined into chains. The process utilizes coupling hemoglobin to bromo-Dextran, resulting in a very high yield. 3. Advantages: Dextran conjugation has many advantages: o o o o

Dextran is readily available in a variety of molecular weights, and its production is fairly standardized It has been used as a clinical plasma volume expander, so its biocompatibility and safety have already been established Dextran is completely metabolized and excreted from the body after a brief storage period in cells It can be chemically modified by a variety of methods to form defined and stable compounds

4. Uses: Dextran has been used in drug delivery systems, and to prolong the plasma half-lives of several compounds. Therefore it has been established already that dextran can increase the therapeutic efficacy of various proteins.

In animal models dextran-hemoglobin has been the first soluble blood substitute to sustain complete recovery of animals that have had almost complete replacement of erythrocytes. Dextran-hemoglobin’s good performance as an HBOC is a result of:    

Long residence time in plasma, with a half-life of up to 57 hours, compared to 22 hours for the first-generation of HBOCs Non-clearance through renal route, which protects the kidney function and structure Slow extravasation which limits tissue edema Long residence time leads to lower frequency of HBOC infusions required to maintain adequate hemoglobin. Paired with low concentration of 6% hemoglobin leads to greater treatment economy and lower imposed metabolic iron load

5. Where Dextran-Hemoglobin is now? Although Dextran-Hemoglobin has so far mostly been tested in animal models, results have been very promising, and a clinical trial in humans is expected to begin by the end of this year in Thailand. A trial on dogs in Thailand is currently underway, and once that trial is finished the human trial will begin. The company hopes that regulatory approval will be cheaper and easier to acquire in Thailand, and that this approval, if granted, will aid the transition into the US and European markets. While this technology seems to be an improvement over first generation HBOCs, it still has some limitations. Because it relies on human hemoglobin, an adequate blood supply is still needed. However, the product still has certain key advantages  

Blood has a maximum shelf-life of only 42 days and Dextran-Hemoglobin can be made from an older blood supply that can no longer be used for transfusion In the long run, it is anticipated that hemoglobin supplies will be met through the scientific production.

Oxygent 1. What is Oxygent? Oxygent is a solution used as an intravascular oxygen carrier to temporarily augment oxygen delivery to tissues. 2. Manufacturing: Right now, the goal of the development of Oxygent is simply to reduce the need for donor blood during surgery, but this product clearly has the potential for additional future uses. Perfluorocarbons surrounded by a surfactant called lecithin and suspended in a water based solution give Oxygent its oxygen carrying capacity. The Oxygent particles are removed from the bloodstream within 48 hours by the body's normal clearance procedure for particles in the blood. Namely, the lecithin is digested intracellularly and the PFC's are exhaled through the lungs. 3. Advantages Oxygent is entirely manmade, containing no human or animal blood or blood components. This gives Oxygent a number of advantages over its HBOC competitors: 

HBOC’s rely on human or animal hemoglobin which limits their manufacturing capacity. Oxygent has no such check and has a production capacity of 10 times that of its closest competitor.

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Oxygent cannot transmit viruses or other infectious agents, and it is able to be heatsterilized to prevent bacterial infection without any harm coming to the solution.

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The oxygen-carrying particles in Oxygent are roughly 1/40 the size of red blood cells giving them the ability to get around blockages in blood vessels that would hinder or stop the bulky erythrocytes.

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The small size also makes perfluorocarbons more efficient oxygen carriers than hemoglobin, with one unit of Oxygent having the equivalent oxygen carrying capacity of 1-2 units of red blood cells.

Oxygent shares some advantages over donor blood with its HBOC competitors as well: 

Oxygent is universally compatible.

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Oxygent has a shelf life of 2 years compared to 42 days for donor blood.

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Oxygent does not need to be refrigerated.

Dendrimers

1. What is Dendrimer? Dendrimers are as a substitute of oxygen carriers especially for battlefield resuscitation applications. 2. Advantages: Some of the advantages of dendrimer technology include        

Biocompatibility Non-toxicity Non-irritability Thermodynamically stable in the body Ability to reach places in the body inaccessible to more massive red blood cells High solubility in water. High oxygen solubility Efficient rate of oxygen transfer into an aqueous phase

The greatest advantage of dendrimer technology is possibly the potential for very cost-effective manufacturing. Because dendrimers are not derived from human or animal sources and manufacturing techniques are relatively simple and thoroughly established, if a dendrimer was identified that was an effective hemoglobin substitute the cost of manufacturing would be dramatically less than that of current HBOCs and even transfused blood.

Artificial Blood Design Characteristics of Artificial Blood product 1. It must be safe to use and compatible within the human body 2. It must be able to transport oxygen throughout the body and release it where it is needed. 3. It must be shelf stable.

Raw Materials To produce hemoglobin synthetically, manufacturers use compounds known as amino acids. These are chemicals that plants and animals use to create the proteins that are essential for life. There are 20 naturally occurring amino acids that may be used to produce hemoglobin. All of the amino acid molecules share certain chemical characteristics. They are made up of an amino group, a carboxyl group, and a side chain. The nature of the side chain differentiates the various amino acids. Hemoglobin synthesis also requires a specific type of bacteria and all of the materials needed to incubate it. This includes warm water, molasses, glucose, acetic acid, alcohols, urea, and liquid ammonia. For other types of hemoglobin-based artificial blood products, the hemoglobin is isolated from human blood. It is typically obtained from donated blood that has expired before it is used. Other sources of hemoglobin come from spent animal blood. This hemoglobin is slightly different from human hemoglobin and must be modified before being used.

The Manufacturing Process The production of artificial blood can be done in a variety of ways. For hemoglobin-based products, this involves isolation or synthesization of hemoglobin, molecular modification then reconstitution in an artificial blood formula.

Hemoglobin Synthesis 

To obtain hemoglobin, a strain of E. coli bacteria that has the ability to produce human hemoglobin is used. Over the course of about three days, the protein is harvested and the bacteria are destroyed. To start the fermentation process, a sample of the pure bacteria culture is transferred to a test tube that contains all the nutrients necessary for growth. This initial inoculation causes the bacteria to multiply. When the population is great enough, they are transferred to a seed tank.

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A seed tank is a large stainless steel kettle that provides an ideal environment for growing bacteria. It is filled with warm water, food, and an ammonia source which are all required for the production of hemoglobin. Other growth factors such as vitamins, amino acids, and minor nutrients are also added. The bacterial solution inside the seed tank is constantly bathed with compressed air and mixed to keep it moving. When enough time has passed, the contents of the seed tank is pumped to the fermentation tank.

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The fermentation tank is a larger version of the seed tank. It is also filled with a growth media needed for the bacteria to grow and produce hemoglobin. Since pH control is vital for optimal growth, ammonia water is added to the tank as necessary. When enough hemoglobin has been produced, the tank is emptied so isolation can begin.

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Isolation begins with a centrifugal separator that isolates much of the hemoglobin. It can be further segregated and purified using fractional distillation. This standard column separation method is based on the principle of boiling a liquid to separate one or more components and utilizes vertical structures called fractionating columns. From this column, the hemoglobin is transferred to a final processing tank.

Final Processing Here, it is mixed with water and other electrolytes to produce the artificial blood. The artificial blood can then be pasteurized and put into an appropriate packaging. The quality of compounds is checked regularly during the entire process. Particularly important are frequent checks made on the bacterial culture. Also, various physical and chemical properties of the finished product are checked such as pH, melting point, moisture content etc.

Once fermented, the hemoglobin is purified and then mixed with water and other electrolytes to create useable artificial blood.

Production of Red Blood Cells from Stem Cells Production of blood from the hematopoietic stem cells removed from umbilical cord between the mother and fetus of humans after birth

Artificial Blood Analysis

Applications of Artificial Blood      

Perioperative uses in surgery Resuscitation after traumatic blood loss Increase oxygen delivery to ischemic tissues Septic shock Patients with multiple antibodies to red-blood-cell antigens who cannot receive blood transfusion Anemia: to supply iron and to stimulate bone marrow to produce blood cells

References Winslow RM. Current status of oxygen carriers ('blood substitutes'): 2006. Vox Sang. Aug 2006 Chen JY, Scerbo M, Kramer G. A review of blood substitutes: examining the history, clinical trial results, and ethics of hemoglobin-based oxygen carriers. Clinics (Sao Paulo) Spahn DR. Blood substitutes. Artificial oxygen carriers: perfluorocarbon emulsions. Crit Care. 1999 Frietsch T, Lenz C, Waschke KF. Artificial oxygen carriers. Eur J Anaesthesiol. Sep 1998 Goodnough LT, Scott MG, Monk TG. Oxygen carriers as blood substitutes. Past, present, and future. Clin Orthop. Dec 1998 Hess JR. Blood substitutes. Semin Hematol. Oct 1996 Ketcham EM, Cairns CB. Hemoglobin-based oxygen carriers: development and clinical potential. Ann Emerg Med. Mar 1999

Hemopure: http://www.biopure.com

Oxyglobin: http://www.cyclingnews.com/news.php?id=news/2004/mar04/mar27news

Polyheme: http://www.wired.com/news/medtech/0,62955-1.html?tw=wn_story_page_next1 http://www.sdreader.com/php/cover.php?mode=article&showpg=1&id=20050728

Hemospan: http://www.sangart.com http://www.jacobsschool.ucsd.edu/cover_story/2003/Nov-Dec/NovDecPage3.html

Dextran-Hemoglobin: http://www.dextrosang.com/

Oxygent: http://www.newscientist.com/article.ns?id=dn4760 http://www.allp.com/Oxygent/FAQ1201.htm http://www.body1.com/news/index.cfm/6/18/1

Dendritech: http://www.dendritech.com/