IV
EXPANDING THE ALTERNATIVES



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--> IV EXPANDING THE ALTERNATIVES

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--> Frozen Red Cell Technology C. Robert Valeri Although I was invited here today to talk about frozen blood, it is not actually the blood that is frozen, but just specific cells in the blood, so what I will brief you on today is the status of red blood cell (RBC) freezing. The most common approach to the freeze-preservation of red blood cells in the United States today is accomplished primarily using high concentrations (40% weight/volume [w/v]) of intracellular glycerol as a cryoprotectant and storage with mechanical refrigeration at -80°C. Originally, there was a great effort to use low concentrations (20% w/v) of glycerol and freezing at -150°C, which was achieved with liquid nitrogen. I understand that this costly system is now being phased out and that the Europeans, primarily the British Army, are presently interested in using extracellular hydroxyethyl starch as the cryoprotectant because postthaw washing would not be required. Glycerol is the most widely used cryoprotectant, but the previously frozen red blood cells must be washed before transfusion to remove the glycerol. Postthaw washing is the major logistic problem with freezing red blood cells with glycerol, whether you use high or low glycerol; postthaw washing to reduce glycerol to less than 1 gram percent is necessary. Although red blood cells frozen with hydroxyethyl starch do not require washing, this method is not a popular one. The types of red blood cell products we have been freezing at the Naval Blood Research Laboratory over the past 20 years through the support of the U.S. Navy are as follows: Nonrejuvenated: The major frozen red cell inventory, autologous and rare blood. Indated-Rejuvenated: Red cells with improved oxygen transport function. Outdated-Rejuvenated: Red cells salvaged from outdated blood supply. Quarantine: All cryopreserved red blood cells can be used as a source of quarantined allogeneic red blood cells. We freeze nonrejuvenated red blood cells shortly after blood collection while the oxygen transport function of the red blood cells is still maintained. For obvious reasons, this method is used primarily for the freeze-preservation

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--> of autologous red cells and rare red cells. We have also been rejuvenating O-positive and O-negative donor red blood cells prior to freezing to restore or improve their oxygen transport function, which deteriorates during liquid storage. The reason that only O-positive and O-negative red cells are rejuvenated and frozen for allogeneic use is that the problems associated with thawing, washing, and postwash storage, which I will discuss, make the practice not feasible for other than O-positive and O-negative red blood cells. Our laboratory became interested in the rejuvenation process during the Vietnam War when we recognized that there were large quantities of outdated O-positive and O-negative red blood cells in Vietnam. We developed a procedure for rejuvenating not only outdated red cells but indated red cells as well. Rejuvenation of indated red cells improves the oxygen transport function of the red cell, a very important benefit in specific clinical situations. Previously frozen red cells are used in at least the following seven ways: Autologous: anticipated surgery. Autologous: potential future use, insurance to minimize use of allogeneic blood. Quarantined allogeneic O-positive and O-negative red cells can be frozen for greater than 6 months, during which time the donor can be retested for infectious disease markers. Rare type and selected red cells can be saved. Red cells with improved oxygen transport function are especially useful in coronary artery and cerebrovascular disease, cardiopulmonary bypass surgery, and hypothermia. Patients with immunoglobin A (IgA) deficiency and paroxysmal nocturnal hemoglobinuria (PNH). Rare and autologous red cells can be refrozen after thawing. The most important of these uses is the quarantine of allogeneic frozen red cells, i.e., the use of freeze-preservation as a means of avoiding the potential for transmission of disease through an allogeneic transfusion. It is now possible to quarantine frozen donor red blood cells for at least 6 months to retest the donor for pathogens that were undetectable at donation. As Table 5 shows, however, transfusion of long-frozen red blood cells within 24 hours of thawing is feasible even after storage for up to 21 years.

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--> TABLE 5 Experience with Long-Term Frozen Storage of Red Blood Cells Transfused Within 24 hours of Thawing Method Material Preserved Frozen Storage Limit -150°C liquid nitrogen, hydroxyethyl starch (HES) Nonrejuvenated RBCs Limited data -150°C liquid nitrogen, 20% (w/v) glycerol Nonrejuvenated RBCs At least 8 yrs -80°C mechanical refrigeration, 40% (w/v) glycerol (a) nonrejuv RBCs; (b) indated rejuv RBCs; (c) outdated rejuv RBCs At least 21 yrs; At least 14 yrs; At least 14 yrs -80°C mechanical refrigeration, 6% DMSO Platelets At least 2 yrs -80°C mechanical Plasma At least 7 yrs The major problem that the military frozen blood banking system faces today is the limited postthaw storage period. At the present time, the FDA approves a postthaw storage period of only 24 hours because of fear of contamination. In our laboratory, we use a sodium chloride-glucose solution during deglycerolization and postthaw storage, and we have data collected over the past 20 years that indicate that washed previously frozen red blood cells can be stored safely at 4°C in a sodium chloride-glucose solution for at least seven days. Moreover, when we used an additive solution such as ADSOL (AS-1), Optisol (AS-5), or Nutricel (AS-3), all currently approved for liquid storage, we observed acceptable 24-hour posttransfusion survival values, acceptable residual hemolysis, and no contamination, even after 2 weeks of postthaw storage at 4°C. Our primary goal now is to help develop a closed system for the deglycerolization procedure to insure sterility for two weeks at 4°C and gain FDA approval for a longer postthaw storage period. I want to stress again that what I am talking about are allogeneic O-positive and O-negative red blood cells only. I would like to comment briefly here about the frozen blood banking system deployed by the Department of Defense that uses a -80°C mechanical freezer with an air-cooled dual-cascade compressor requiring 230 volts. We have gained FDA approval for storage of nonrejuvenated red blood cells

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--> frozen with 40% w/v glycerol in these freezers for 10 years. Actually, we have studied nonrejuvenated frozen red blood cells after 21 years of frozen storage and have published data showing satisfactory results. We have also frozen both indated and outdated rejuvenated red cells which have been approved by FDA for storage at -80°C for 10 years. These are red blood cells that are biochemically modified to restore or improve their oxygen transport function before freezing with 40% w/v glycerol. These frozen red blood cells have been stored in -80°C mechanical freezers for at least 14 years with satisfactory results. The U.S. Navy has -80°C mechanical freezers aboard ships as well as at mobile hospitals. These freezers maintain supplies of frozen platelets as well as the frozen O-positive and O-negative allogeneic red blood cells with normal or improved oxygen delivery capacity. Platelets are frozen with dimethylsulfoxide (DMSO), an FDA-approved cryoprotectant. Frozen platelets can be stored at -80°C for at least two years. We are also able to freeze pluripotential mononuclear cells for use in bone marrow transplantation: these cells can be frozen with 10 percent DMSO and stored at -80°C for 1.5 years. We have also received FDA approval for the storage of fresh frozen plasma at -80°C for seven years. To summarize the data: the -80°C mechanical freezer can be used by the Department of Defense to store frozen red cells for 21 years, frozen platelets for 2 years, frozen pluripotential mononuclear cells for 1.5 years and fresh frozen plasma for 7 years. Dr. Fratantoni has asked me to comment on justification for the use of frozen red blood cells, since as we heard earlier from Dr. McCullough, it is quite costly. I have already listed the various uses for frozen red blood cells. I believe the importance of these uses justifies the additional cost for freezing O-positive and O-negative allogeneic red blood cells, selected red blood cells, rare red blood cells, and autologous red blood cells. We have found that patients undergoing specific surgical procedures require red blood cells with the ability to deliver more oxygen to tissue at high oxygen tensions. Before the storage limit of liquid preserved red blood cells was extended to 42 days and before mandated testing of blood products for the infectious disease markers for hepatitis B and C and HIV, there was an interest in freezing autologous red cells. Although the fear of transmission of disease is not as great as it was prior to 1985, freezing O-positive and O-negative donor red blood cells for a 6-month quarantine period would allow time for the donor to be retested to document the presence or absence of infection at the time of retesting. I would now like to describe to you one of the latest work efforts in our laboratory which I consider to be important. As you have heard, there is a large supply of outdated Type A and Type B red blood cells. We have been investigating a procedure introduced several years ago by Jack Goldstein at the

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--> New York Blood Center to enzymatically convert B and A red blood cells to O red blood cells. Studies are in progress to assess enzymatically converted B and A red blood cells to O red blood cells which are then biochemically modified and frozen with 40% w/v glycerol and stored at -80°C in mechanical freezers. I'll close by describing probably one of the most important areas of investigation at our laboratory at the present time: the development of closed automated deglycerolization methods. The FDA mandated that a sterile docking device must be used to dock the thawed units to the washing system. The FDA has also recommended that the deglycerolization solutions be passed through 0.22 micron filters, which will require an automated system. To accomplish these goals, the U.S. Army and the U.S. Navy are both working with three commercial companies which have contracts to develop a closed deglycerolization system. The success of this project would allow postwash storage of deglycerolized red blood cells for at least two weeks at 4°C. Questions/Comments Pinya Cohen: What is the cost of initially freezing the red blood cells, then of deglycerolizing them later? C. Robert Valeri: I would say that the total cost of providing previously frozen red blood cells is three times the cost of liquid preservation. Pinya Cohen: What I am wondering is the need for extending the frozen storage at -80°C from 10 years to 20 years. C. Robert Valeri: The extension of frozen storage from 10 years to 20 years would avoid the need to discard the frozen glycerolized red blood cells after 10 years. It means that you have to replace the supply in the freezers, which is costly for the government. If you could store the red cells for 20 years, the costs would be less. In our experience the cost for freezing, thawing, and washing is three times the current cost of collecting and storing red cells in the liquid state. Pinya Cohen: Do you have a breakdown of cost of thawing and washing independent of the freezing? C. Robert Valeri: Yes. What we did several years ago, which I really didn't emphasize, was to design a method to freeze the red cells in the same bag in which they were collected With this method, the freezing of the red cells represents a relatively small percentage of the total cost. The major costs

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--> associated with freezing are those for the instruments used in processing and the disposables and solutions. So, actually, about 20 percent of the total cost is related to the freezing of the red cells. Pinya Cohen: So, from that perspective then, the only advantage to expanding it from 10 years to 20 years or whatever has nothing to do with the deglycerolization. It has to do with getting new blood to put into the system. C. Robert Valeri: Yes. Celso Bianco: Do you know any system or any place that uses frozen blood as part of managing their inventory, except for big emergencies or within the military? C. Robert Valeri: I can't answer that. I don't know. Alvin Drake: Wasn't there a period when the Cook County Blood Bank had a student of Charles Huggins, who tried to run a 100 percent frozen blood operation? C. Robert Valeri: Yes. That was Gerald Moss. When he returned from Vietnam, he went to Cook County and for a period of one to two years, ran the Cook County Blood Bank using frozen deglycerolized red cells. Alvin Drake: Why would they do that? C. Robert Valeri: If you read the early papers on the work by the Navy, the purpose was not to use frozen red blood cells exclusively, but to use them as a supplement to the liquid blood banking system. Alvin Drake: He had some kind of disinfection procedure? C. Robert Valeri: The original concept by James Tullis was that when glycerol was added and removed from red blood cells during the freezing procedure, the virus associated with hepatitis was eliminated. The primary goal of frozen deglycerolized red blood cells in those days was to provide hepatitis-free red blood cells. Joseph Fratantoni: The use of deglycerolized red blood cells to prevent the transmission of hepatitis was not confirmed by several published reports. C. Robert Valeri: Harvey Alter, Harold Meryman, and others reported that

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--> infected chimpanzees whose red cells were frozen, thawed and washed were shown to transmit hepatitis. In addition, R.K. Haugen from Miami, Florida, has published a paper in the New England Journal of Medicine which reported that the use of frozen deglycerolized red cells was associated with the transmission of hepatitis. So, as Dr. Fratantoni points out, published papers show that frozen, deglycerolized red blood cells do transmit hepatitis.

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--> Logistical Concerns in Prepositioning Frozen Blood Michael J. Ward The Armed Services Blood Program Office, where I have been director for a little over three years, and the Department of Defense are the primary users of frozen blood, so what I want to do today is give you just a little bit of an overview of the Armed Services Blood Program before turning to the topic of frozen blood itself, because our perspective is unique. The reason we use frozen blood differs from that of anyone else in this audience. I think that needs to be said up front. I am going to focus on four areas very quickly for you: our mission, our distribution system, the frozen blood program, and finally, how we do it. Our office coordinates the blood programs of all three military departments, each of which has a separate FDA license. We make certain that quality blood products, blood substitutes, and blood services are provided. Over the years we have put a lot of dollars into research and development to make sure that we had what we needed to assist health care providers on the battlefield. Many of you have reaped the benefit of those dollars invested. We have done that for one reason, and that is to make sure our beneficiaries have what they need in peace, peacekeeping, what is now called Operations Other Than War, such as in Haiti and Somalia, and, of course, full-blown war. We have Army, Air Force, and Navy blood donor centers, but we also have contingency contracts with the American Red Cross and the American Association of Blood Banks (AABB). Those contracts were activated once during Operation Desert Shield in December, before the attack on the Iraqi forces in Kuwait. We have a number of memoranda of understanding with different organizations throughout the United States to supply some of the blood products for our peacetime needs. All of those blood products, whether collected in-house or provided through memoranda of understanding, are tested in complete accordance with FDA and AABB requirements, and in terms of contingency support, they are shipped to what we call an Armed Services Whole Blood Processing Laboratory

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--> Alvin Drake: Isn't there machinery to do that? Michael Ward: Not yet. The problem is there is no market for it. If this group were to say that the answer to the blood problem in America is that it all needs to be frozen, you would suddenly have a lot of companies very interested in producing a machine that will do what you want. There are some companies out there now, but not the major companies, because it is economics. If a private company cannot see the economic feasibility of doing it, the Department of Defense isn't going to foot the entire bill. There has to be some venture capital that goes into it. However, we are confident that once this new system comes out and we have the 21 days to work with, then when we see signs that there is going to be a military action, we will begin deglycerolizing and then stockpiling the refrigerators with liquid blood. C. Robert Valeri: With regard to Al Drake's question about the desirability of thawing and processing in quantities larger than a unit at a time, are you going to get both units from the same individual or are you going to pool units across donors prior to freezing? Right now everybody is actually collecting a single unit of red cells from a donor. Alvin Drake: Well, if you are going to give me six units in a hurry, I can't believe you are going to get them from the same person, so it won't make much difference if it gets pooled before storage or pooled in me. Michael Ward: We can deglycerolize more than one unit in a disposable package, and we do do that. The problem, of course, is that you then get into problems with cross contamination. America wants its sons and daughters treated just like you treat them in these civilian hospitals, and that is our goal: to provide the same quality care that they could get if they went to a civilian facility. Eve Lackritz: If I understand correctly, when you are in a war situation, you import all your blood from the United States. The Saudis didn't donate? Michael Ward: That is correct. There are some reasons for that. To be politically sensitive, not all of the blood that is available in the world is as good as the blood that we can provide. We want to make sure that all the blood we use meets the same standards, the same level of testing. When we ship a unit of blood, we want the standard of medical care to be identical to that provided in the United States. That way we are assured that all the testing that we require has been done and that they are getting a quality product.

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--> Eve Lackritz: You said you keep an aliquot of blood on all your frozen blood. Do you also keep aliquots from all your regular blood? Michael Ward: We do, as all blood banks do, but because of the shelf life, 35 to 42 days, those are only kept for a short period of time. Eve Lackritz: You just keep that extra aliquot for retesting? Michael Ward: That is right. Only in case we need to do additional tests.

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--> Extended Liquid Storage of Red Blood Cells John Hess I was asked to talk about the feasibility and utility of extending the shelf life of red blood cells (RBC). In a sense we could have seven-week red blood cells now. Two years ago, the Federal Republic of Germany licensed PAGGS-mannitol as an additive solution for the seven-week storage of red blood cells collected in standard citrate-phosphate-dextrose (CPD). This solution does not meet the letter of the U.S. requirements for licensure because the in vivo recovery of those cells was 74.6 percent and the U.S. rules require 75 percent. However, the Germans licensed this solution in the context of a national constitutional crisis over sending German soldiers to join the United Nations relief expedition in Somalia and their perception of a national need for better blood storage solutions to be able to provide international blood support. Claes Högman and his colleagues,7 who developed the standard European six-week storage solution of saline, adenine, glucose and mannitol (SAGM), have produced an improved version called Research Additive Solution 2 (RAS2) that unequivocally stores packed red cells for seven weeks. Red cell survivals were almost 80 percent after seven weeks of storage. In form, function, and format this solution is similar to the additive solutions that are used in the United States today. It delivers packed cells in 100 ml of additive solution. This solution formulation, which is already owned by one of the major blood bag suppliers in the United States, could be licensed and manufactured, tested, and made available in this country within a period of two years. Even longer storage is possible. Tibor Greenwalt and his associates have published a description of a solution called Experimental Additive Solution 25 7   Högman, CF, L Eriksson, J Gong, AB Högman and JM Payrat (1995). Shall red-cell units stand upright, lie flat or be mixed during storage—in vitro studies of red cells collected in 0.5 CPD and stored in RAS2 (erythrosol(R)). Transfusion Science, 16(2): 193–199.

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--> (EAS-25) that allowed 73 percent survival of packed red cells at nine weeks.8 The problem with this particular solution is that it requires 200 ml of the additive in the packed red cell unit. This means that the hematocrit of the resulting "packed" cells will only be about 40 percent. Further, the resulting RBC units still contained about 1 percent glycerol. They may not be safe in certain massive transfusion situations. Such a situation would require rethinking how we use red cells. The U.S. Army is currently supporting Greenwalt's testing of our new variants of this solution. About a decade ago, Harry Meryman and his colleagues9 showed that human red blood cells can be stored in hypotonic solutions for periods as long as 35 weeks and yield recoverable cells. This technology has been licensed and Bayer has produced a set of derivative solutions. AS-24 is one. The solution performed well in studies done in vitro. We have a cooperative research and development agreement with Bayer to test it in human beings in our lab. I have reviewed the products pending in the immediate future. Certainly, the potential that Meryman has shown for longer-term storage suggests that there would be a major benefit to understanding the red cell storage lesion. In all likelihood we can extend blood storage even longer. Extending storage is a useful thing to do. If we had longer liquid red cell shelf life, we could reduce outdating. If we are outdating somewhere between a half million and a million units annually and we can extend the shelf life by one to two weeks, then realistically we can recover somewhere on the order of a hundred to several hundred thousand units of blood a year. That means that over the course of our lifetimes, we can expect to save 10 million units of blood, a billion dollars. Thus, improved storage would pay for itself. Extending the shelf life to seven or eight weeks would mean that blood collected before students went home for Thanksgiving would still be available in January. This has the potential to reduce some seasonal shortages. My own background as a Battalion Surgeon in Korea, a Support Command Surgeon in Thailand, Director of Health in American Samoa, and a Public Health Service officer on an Indian reservation in the Dakotas, has taught me that the problems of blood storage are worst in isolated places. Those problems are with maintaining inventory. The small size of the population you are serving makes swings in inventory longer and the outdating problems worse. Increased storage life means the ability to rotate stocks between a 8   Dumaswala, UJ, NL Bentley and TJ Greenwalt (1994). Studies in red blood cell preservation. 8. Liquid storage of red cells in a glycerol-containing additive solution. Vox Sanguinis, 67(2): 139–143. 9   Meryman, HT, ML Homblower and RL Syring (1986). Prolonged storage of red cells at 4 degrees C. Transfusion, 26(6): 500–505.

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--> central supplier and the outlying area. It means it is possible to keep blood on hand in outlying areas but still bring it back and utilize most of it. There is another benefit of improved red cell storage. Blood deteriorates at a fixed rate in a storage solution. If you can improve that storage solution, then the quality of the cells at any given time will be better. This benefits everybody who gets transfused. It is not known if better cells will ever be a material contribution to the economics of transfusion, but they certainly improve care. Finally, one of the limits of autologous use in this country is the limited ability to draw enough units to meet some kinds of surgical demand. Extending the liquid storage of red cells will improve the availability of autologous blood to people who want it and, therefore, free up allogeneic blood for other uses. I would like to end this with a plea to support improved storage. Groups such as this Forum can help in several ways. One is to declare that extending storage is useful. Then, researchers and companies who seek resources to improve storage will find them more easily. Second, groups such as this Forum can certainly encourage good science. Understanding the red cell storage lesion can potentially lead to major benefits. Third, we should encourage appropriate development. There are many schemes for increasing the storage period of red blood cells. Only some of them are compatible with most uses. What we don't need is more different kinds of blood products. If we are going to improve the storage length of blood, it needs to be compatible with all of the present uses. Finally, please encourage regulatory approval. Encouraging our colleagues who regulate to hold conferences that address these issues and to publish points to consider is useful. Questions/Comments Celso Bianco: How do you plan to deal with the problems of bacterial contamination? John Hess: I don't know of any evidence that bacterial contamination at 42 days or 56 days is worse than at 38 days. Steve Wagner and colleagues10 have recently published a nice review of this whole area, but the problem is uncommon with red blood cells, and when it occurs it is most commonly seen at about 27 days. If a unit is going to be contaminated by psychrophiles 10   Wagner, SJ, LI Friedman and RY Dodd (1994). Transfusion-associated bacterial sepsis. Clinical Microbiology Reviews, 7: 290–302.

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--> (bacteria capable of growing at 0–4°C) at 42 days or beyond, it is already grossly contaminated at 27 days. We are looking at the idea of testing for bacterial contamination and have supported work on devices such as "through the bag" sensors. As you know, we all currently rely on visual inspection at the time of use as our screen for bacteria. People have ideas of measuring the oxygen content by scanning spectrophotometry, and have devised sensors for carbon dioxide, for pH, and for ammonia that can be glued on the outside of the bag. As the ammonia diffuses through it develops a diazo dye that turns black. A calibrated series of these dots that turned black at weekly intervals might be useful for picking up bacterial contamination. We are looking at these, although we don't have any answers yet.

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--> Overview of Blood Substitutes Joseph Fratantoni Blood substitute is the name that most people tend to use for materials that are designed to replace red cells. Another name is artificial blood. The military has referred to the material that they would like to develop as a resuscitation fluid, reflecting their primary need to deal with hemorrhagic shock. Probably red cell substitute is the most appropriate name, but from habit we will probably be calling them blood substitutes more often. The generic name is actually oxygen carriers, and while we talk about using red cell substitutes for hemorrhagic shock or perhaps perioperative hemodilution, there have been other uses. I will talk a bit about one material that was approved for use in perfusing the myocardium during angioplasty, but there are some other possible uses that have been proposed should an oxygen carrier that is safe and effective be developed. A major problem that we face in evaluating these materials is determining efficacy. It is clear with a number of preparations that they can carry oxygen. We know that there is limited intravascular half-life. Determining criteria for clinical benefit is really a problem. It is difficult to determine when someone needs red cells or when someone benefits from red cells, and it is even more difficult to determine when that person needs a red cell substitute or would benefit from a red cell substitute. With many of the preparations, the major problems to date have been related to safety. However, you really can't separate the safety problems from the efficacy problem because you will be talking about giving patients a substitute for a product which already is, as we all know, rather safe and effective. It might be worth thinking for a minute about what the red cell does before trying to replace it. It permits a high hemoglobin concentration by shielding hemoglobin from the high viscosity and oncotic pressure of the vasculature. It allows the hemoglobin to circulate longer, instead of being cleared rapidly by the kidneys. We have also learned in the last several years that the red cell protects the body from hemoglobin as well as vice versa. Hemoglobin is both a

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--> pharmacologically active material and a very tender molecule. Red cells maintain the hemoglobin in a functional state. It is worth recalling that all land-dwelling vertebrates have their oxygen carriers wrapped in a membrane. Trying to strip that membrane away and infuse hemoglobin is a tricky job. The use of hemoglobin itself is one basic approach, using either human or non-human hemoglobin or the human hemoglobin gene expressed in a recombinant system. It is then modified by cross-linking the subunits of hemoglobin, polymerizing the material so that it circulates for a longer time and it is more stable, conjugating it to macromolecules or encapsulating it within a liposome. The product would then be a modified or encapsulated human or nonhuman hemoglobin. The use of fluorocarbons is a second approach. They are manufactured and then modified by emulsifying them. The final product is a perfluorocarbon emulsion. Baxter is working with a cross-linked preparation that is derived from human red blood cells. The Army is working with a very similar product, Biopure, in a joint venture with Upjohn, using bovine hemoglobin from shed bovine blood that is chemically modified. Northfield Laboratories has human material that is chemically modified and polymerized. Somatogen, in a joint venture with Eli Lilly, has human recombinant material that is expressed in Escherichia coli and is additionally modified and cross-linked at the oxygen binding site. Enzon has a polyethylene glycol-conjugated material. One mechanism for hemoglobin problems that have been seen, which have included hypertension and a number of vasospastic and perhaps musculoskeletal problems, is that hemoglobin not in the red cell is free to diffuse outside the intravascular space and into the vessel wall, where the endothelial cell is releasing an endothelium-derived relaxing factor, now known to be nitric oxide. Nitric oxide will interact with G proteins to form more guanosine monophosphate (GMP), which in turn leads to vascular relaxation. When hemoglobin gets within this vascular wall (and it can do that when it is not encapsulated in a red cell), it will bind nitric oxide even more avidly than it binds oxygen. Without nitric oxide to stimulate GMP formation you don't get vasorelaxation; instead, you get vasoconstriction, which could explain the hypertension and some of the muscular effects that are being seen. Another possible problem is whether or not hemoglobin is going to deliver oxygen as advertised. Winslow and his co-workers, in studies with hamsters progressively hemodiluted with a hemoglobin solution, have measured the amount of oxygen that is actually delivered to tissue. Hamsters were infused with either hemoglobin or with Dextran. As you hemodilute with Dextran, you get a little more oxygen carried as the dilution decreases viscosity, and then there is a sharp drop in oxygen as dilution increases.

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--> The surprise in this experiment was that with hemoglobin dilution you also get an increase in oxygen delivery for awhile and then a drop. The exact reason for this is still not clear. These results are submitted for publication and I think still being discussed rather intensively. One mechanism that is proposed is that oxygen from hemoglobin is being consumed by the vessel wall. Whatever the mechanism, if this sort of phenomenon turns out to be something that can be repeated by other laboratories, it certainly casts some question about how much good we will ultimately do by infusing hemoglobin solutions. Perfluorocarbons have also been investigated. Much interest was stimulated by early pictures of a mouse that was submerged in a beaker of perfluorocarbon liquid that had been saturated with oxygen but was still awake and active. The mouse extracts the dissolved oxygen from the liquid the way a fish extracts dissolved oxygen from water. Perfluorocarbons do not bind oxygen selectively as hemoglobin or red cells do; fluorocarbons simply dissolve the oxygen. Perflubron is a second generation perfluorocarbon which carries more oxygen per amount of oxygen in the air than the first-generation compounds. It can dissolve more oxygen and can itself be more highly concentrated in an emulsion. The fluorocarbons are unusable with water, so, in order to infuse them, you have to put them into an emulsion form. Perfluorocarbons were developed in the United States in the mid-1960s and tested first clinically by the Japanese in the late 1970s. Then some clinical studies in the United States showed that there really was not any clinical benefit to severely anemic individuals, and a 1983 request to use this material as a blood substitute was not approved. However, in 1989 a perfluorocarbon was approved for selective use for perfusing angioplasty catheters. The market really did not support that material, and production was halted in about 1993. People have thought that the perfluorocarbons may be useful for perfusing ischemic tissues distal to an obstruction, since an emulsified particle of perfluorocarbon is much smaller than a red cell and so might be able to get to places that red cells could not. One of the problems with perfluorocarbons as a class is that it appears there does seem to be a somewhat dose-related thrombocytopenia that occurs. A research study by Bob Kaufman at Hemagen showed that a few days after the animal received the material, the platelet count went down from about 230 to about 120. This is a problem that is being looked at, although the clinical importance of this is something that is being debated. Demonstrating efficacy is certainly a challenge that people are looking at right now. A number of possible approaches are being considered. Obviously, you can show that these materials carry oxygen, or you can show that if you infuse hemoglobin, you increase the hemoglobin level, but that does not appear to be a very important end point. One can try to show that they are equivalent

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--> to red cells, but we have heard earlier that it is hard enough to show that red cells work. People are therefore looking for some sort of useful or practical benefit to an organ or to an organism. One possibility is showing that you can decrease allogeneic blood transfusions. Even though we know that the risk of allogeneic transfusion is very low, that might be something that could be considered of benefit and could support approval of such products. In order to make these demonstrations of efficacy a bit easier, people are looking at different levels of use, localized perfusion, hypovolemic shock, and perioperative hemodilution, trying to go one at a time rather than get approval for all indications at once. In conclusion, a red cell substitute appears feasible. There has been an enormous amount of research done in the past several years, and major advances have been made. However, progress has been slow. There have been toxicities, and there is still a lack of fundamental information. There was not the same enormous investment in all areas of basic research in the 1970s as there was in the molecular biology that spawned the biotech industry. When the AIDS crisis gave new stimulus to the development of blood substitutes in the 1980s, there was a large infusion of industrial activity. The military has been interested in red cell substitutes since the late 1970s or early 1980s and has been the mainstay of support for research. Nevertheless, it remains difficult to demonstrate efficacy. The nature of a successful product is going to depend upon the results of our future research, and it may well be that a successful product will be part of an overall approach to the avoidance of allogeneic transfusion.