Artificial Blood: What Is It? Will I Use It

موضوع: Artificial Blood: What Is It? Will I Use It 21/3/2007, 4:09 am

Since the 17th century, blood transfusions have been attempted to offset blood loss from trauma and childbirth, or as a therapeutic modality during leeching or bloodletting. Until the identification of isoagglutinating antibodies, however, transfusions were fraught with significant early complications. These early complications sparked interest in using hemoglobin as an oxygen carrier in plasma. Early trials of these solutions proved disastrous as well, with significant immediate complications resulting from infusions of stroma-free human hemoglobin solutions.1 These complications were most often acute renal failure thought to be the result of direct hemoglobin nephrotoxicity.2

History Of Artificial Blood
Development of a hemoglobin-based blood substitute was pursued vigorously by the military as a means to have an oxygen-carrying plasma expander available for battlefield use. Despite research throughout the Vietnam War, a clinically effective blood substitute was unable to be developed.

During this era of blood substitute research in the 1960s, Dr. Leland Clark began experimenting with a class of compounds known as perfluorocarbons. Oxygen has approximately 100 times greater solubility in perfluorocarbon solutions than in plasma. As a result, the amount of oxygen dissolved in plasma may be sufficient to sustain life, without the need for RBC-contained hemoglobin to provide additional oxygen. The hydrophobic nature of these compounds necessitated further development of perfluorocarbon emulsions prior to considering these compounds for use as a plasma oxygen carrier.

The use of Pluronic 64 as an emulsifying agent for perfluorocarbons enabled the production of Fluosol by the Green Cross Corporation of Japan. Clinical trials with this perfluorocarbon, however, were disappointing. Fluosol was present only in low concentrations in the emulsion, and Pluronic 64 caused rare but significant complications when the emulsion was infused intravenously. Further development of emulsion technologies resulted in the production of compounds which utilized smaller chain perfluorocarbon molecules to more effectively emulsify the perfluorocarbons, allowing higher concentrations of active agent in the emulsion and thus higher oxygen carrying capabilities. The improved stability of the newer emulsions are vastly superior to the first generations of perfluorocarbons; current emulsions can be stored at 4°C for extended periods of time (months) without appreciable degradation of activity.

Physiology Of Oxygen Transport
Normal oxygen transport is primarily a function of erythrocyte-contained hemoglobin. The heme-iron moiety of the hemoglobin molecule allows binding and release of oxygen, dependent upon the partial oxygen tension to which the hemoglobin is exposed. The tetrameric structure of the protein portion allows hemoglobin to bind four oxygen molecules within binding pockets in each protein subunit.

Modification of the ability of oxygen to bind to hemoglobin occurs naturally. Hemoglobin oxygen interactions result in structural conformational changes to facilitate loading and unloading of oxygen in the pulmonary circulation and peripheral tissues, respectively. The efficiency of oxygen binding and release can be altered by acid-base balance, the partial pressure of carbon dioxide, temperature, and 2,3-diphospho-glycerate. The resulting shift of the sigmoidal oxy-hemoglobin dissociation curve serves as a natural regulatory mechanism for the delivery of oxygen to tissues (Figure 1). In neutral pH, in the absence of any other modifying substances or conditions, the P50 of native hemoglobin outside of the RBC is 17 mmHg. Thus, the internal milieu of the RBC is critical to the effective delivery of oxygen to tissues.

Figure 1. Oxy-hemoglobin dissociation may be affected by several conditions, including acidosis (high CO2 levels), alkalosis (low CO2 levels), hyperthermia and hypothermia.

Artificial blood solutions based on hemoglobin function by oxygen delivery from plasma hemoglobin. Initial trials of free hemoglobin solutions demonstrated little benefit to patients with these unmodified hemoglobin molecules, most likely due to the high affinity of oxygen for the plasma hemoglobin.1, 3 Subsequent research has revealed several methodologies that are effective in altering the binding affinity of hemoglobin from oxygen in order to deliver oxygen to peripheral tissues. Ligands such as pyridoxyl groups, when bound to hemoglobin, alter the oxygen affinity, shifting the dissociation curve to the right. The decrease in oxygen affinity effected by these changes enables plasma hemoglobin to deliver oxygen to peripheral tissues.

Current Status Of Blood Substitutes
After many years of intensive research, blood substitute technology is finally reaching the point where safe, clinically effective solutions may become a reality. For perfluorocarbon emulsions, newer molecules, coupled with advances in emulsification technology, has produced solutions with great potential for clinical applications. Novel methods of crosslinking and chemical modification have made hemoglobin solutions a viable alternative as temporary oxygen carriers.

Perfluorocarbon Emulsions
After the initial excitement regarding Fluosol, subsequent small studies demonstrated no benefit from Fluosol infusions in patients with profound anemia.4,5 With colloid solutions as a comparator, Fluosol did not improve indirect measures of oxygenation. However, Fluosol continued to be available for infusion as an oxygen carrier during high-risk percutaneous transluminal angioplasty procedures until early 1993, when approval for this indication for the emulsion was rescinded by the Food and Drug Administration.

New emulsions have been developed which utilize emulsifying agents similar to the primary compound. In particular, perflubron (perfluorooctyl bromide) has been developed as a stable emulsion safe for intravenous infusion by the addition of small amounts of perfluorodecyl bromide as an emulsifying agent; the emulsion is then buffered with egg yolk phospholipids. The resulting emulsion has a calculated oxygen carrying capacity which is approximately three fold the amount of oxygen carrying capacity of the earlier Fluosol solutions.

Perflubron oxygen carrying capacity is directly related to the oxygen partial pressure (Figure 2). In this regard, perflubron oxygen delivery is predictable; direct diffusion of oxygen is the mechanism by which oxygen is off-loaded to peripheral tissues. Theoretically, oxygen delivered by diffusion may be more available, and more readily off-loaded from the bloodstream, than hemoglobin-delivered oxygen. However, no data have been produced which support this premise.

Figure 2. The oxygen content of perfluorocarbon emulsions obeys Henry's Law of partial pressures; the amount of oxygen dissolved in a perfluorocarbon solution is directly related to the partial pressure of oxygen to which the solution is exposed. Figure 3. A comparison of the amount of oxygen dissolved in normal plasma and two clinically achievable plasma concentrations of perflubron.

Benefits Of Perfluorocarbon Oxygen Transport
Transport of oxygen as soluble gas in plasma is radically different from hemoglobin-based oxygen transport. Although some oxygen is normally dissolved in plasma, the amount typically constitutes less than 1% of the total oxygen content in arterial blood, even with significant anemia. By contrast, administration of perflubron can increase dissolved oxygen to approximately 10-15% of the total arterial oxygen content, an increase from the norm of two to three fold, depending on the partial pressure of oxygen inspired (Figure 3).

There is evidence to suggest that diffusion of oxygen does occur, and increased tissue oxygenation is the result. Studies on solid tumor treatment with either chemotherapy or radiation therapy have demonstrated enhanced tumor kill ratios when animals are pretreated with perflubron. Diffusion of oxygen into the hypoxic core of these tumors, thus spurring these "dormant" hypoxic tumor cells to divide, results in greater sensitivity of these now dividing tumor cells to antimitotic agents, enhancing their effectiveness.6 This theory now awaits clinical trials to evaluate the efficacy of diffusion of oxygen into tissues.

Problems With Perfluorocarbons
Perfluorocarbons are inert biologically. The molecules are sequestered in the reticuloendothelial system, particularly in the Kupffer cells of the liver and macrophages, and subsequently released back into the plasma as a dissolved gas. The perfluorocarbon gas is then exhaled unchanged and non-----bolized via the lungs. While previous perfluorocarbons had a significant amount of retention in the reticuloendothelial system, current generation perfluorocarbons such as perflubron have a retention time of approximately one week. This allows effective elimination of perfluorocarbons from the liver and spleen without the potential for significant organ dysfunction.

However, despite the inert nature of perfluorocarbons, sequestration in the reticuloendothelial system may result in subtle consequences. Platelet count is known to decrease, presumably due to opsonization of platelets by the perfluorocarbon and subsequent sequestration and elimination by the reticuloendothelial system. Sufficient perfluoro-carbon may also overwhelm the reticuloendothelial system, resulting in potential infectious or other complications; however, this is only a theoretical concern, as no increase in infectious complications has been noted in early clinical trials.

The retention of perfluorocarbons does pose an additional problem with respect to dosage. Perfluorocarbons are relatively evanescent in the plasma, with a half life of approximately 3-4 hours in the plasma phase. The reticuloendothelial system, however, has an approximate 3-5 day retention phase prior to exhalation of the perfluorocarbon. Therefore, although extremely short-lived in the plasma phase, additional dosing of perfluorocarbons may not be possible for several half-lives of the tissue-reticuloendothelial terminal elimination, i.e., one to two weeks. Thus perfluorocarbons become a single dose drug, with limitation of dosage due to the capacity of the reticuloendothelial system to handle the plasma elimination phase. At present, this limitation of dosing is theoretical, as no clinical data exist to discern whether perfluorocarbon redosing results in serious adverse effects; future studies and newer generation emulsions will address this issue.

The dependence of perfluorocarbons on Henry's Law of partial pressures allows the potential for increased oxygen availability. This fact of oxygen delivery also limits the effective use of perfluorocarbons to situations when the partial pressure of oxygen is supranormal, i.e., when the partial alveolar oxygen tension approaches 400 mmHg or greater. This is impossible to attain without supplemental oxygen administration; an effective partial oxygen pressure may be impossible with any maneuvers at altitude. Even in the presence of supplemental oxygen and controlled ventilation, patients with significant pulmonary disease may be unable to reach partial pressures of oxygen to allow perfluorocarbon to function as an effective oxygen carrier.

Hemoglobin-Based Oxygen Carriers
Stroma free hemoglobin has been produced for some time, yet significant renal toxicity has heretofore prevented its widespread use. Hemoglobin is a tetrameric protein of approximately 64,000 daltons; outside of its red blood cell milieu, the hemoglobin molecule rapidly dissociates into dimers composed of an alpha and a beta subunit. In addition to rendering the hemoglobin non-functional, these dimers are then filtered by the kidney, and the interaction of these hemoglobin residua with minute amounts of cell wall pieces in the renal glomeruli results in rapid acute tubular necrosis and renal failure. Development of a suitable stroma free hemoglobin molecule therefore depends on the development of a stable, functional tetramer of hemoglobin which would not dissociate into dimers upon infusion. This problem has been solved in several novel ways.

Prevention of dissociation of the hemoglobin tetramer in plasma is accomplished by binding the hemoglobin protein subunits together to prevent dissociation. Binding of the hemoglobin tetramer has been approached both chemically and genetically. Chemical binding of the tetramer involves binding of the alpha subunits by a so-called bifunctional agent, such as diaspirin, which links the hemoglobin molecules and thus stabilizes them. These polyhemoglobins are now undergoing clinical trials as potential blood substitutes.

A second significant problem is the lack of 2,3-DPG associated with the stroma free hemoglobin; the resulting stroma free hemoglobin, although polymerized with bifunctional agents, will not be functional at physiologic levels of tissue oxygenation. The P50 of native stroma free hemoglobin in solution is approximately 17 mmHg. This has been overcome chemically by the binding of pyridoxal phosphate to the hemoglobin molecule. The resulting polymerized, pyridoxi-lated stroma free hemoglobin has a P50 of approximately 32 mmHg (as compared to native, RBC associated hemoglobin P50 of approximately 27 mmHg) (Figure 4). Therefore, chemically altered stroma free hemoglobin are functionally superior to native hemoglobin.

Figure 4. A comparison of the oxy-hemoglobin dissociation curves of native ("wild-type" or A1) hemoglobin contained with the normal red blood cell milieu ("RBC-Enclosed Native Hemoglobin"), native or "wild-type" hemoglobin after removal from a red blood cell ("Stroma Free Native Hemoglobin"), and typical hemoglobin based oxygen carrier solutions.

مشرف عدد الرسائل : 115 تاريخ التسجيل : 24/10/2006

Another approach to hemoglobin modification has been genetic engineering. The structure and amino acid sequence of wild-type hemoglobin is known. Therefore, by genetically altering the native hemoglobin by the addition of a single amino acid, it is possible to covalently bind two alpha subunits, thus preventing the dissociation of the hemoglobin tetramer. A single point mutation in the beta subunits produces a hemoglobin with a P50 of approximately 32 mmHg. Thus specific mutations in the hemoglobin molecule result in a functional, stable stroma free hemoglobin. Insertion of this engineered hemoglobin into E. coli plasmids results in the production of large quantities of hemoglobin.7 Purification of the hemoglobin would be similar to those processes used currently for other genetically engineered substances, such as insulin.

Xenograft material can also be used to produce stroma free hemoglobin. Bovine hemoglobin can be used after polymerization, as bovine hemoglobin does not require 2,3-DPG or other ligands to modify its oxy-hemoglobin dissociation.8 A ready supply of this hemoglobin is available, and chemical sterilization of this protein possible, although the prospect of zoonotic infection must be considered, particularly with concern for prion disease.

Benefits Of Hemoglobin-Based Oxygen Carriers
All blood substitutes utilizing chemical sterilization involve the reclamation of human blood cells from outdated red blood cell products. Questions regarding the ability to chemically sterilize these products sufficiently to avoid infectious disease transmission have been largely answered; however, production of this product involves a ready supply of outdated blood in a time when voluntary donations are decreasing. Genetically produced hemoglobin from E. coli does not suffer from supply problems associated with the use of polymerized human hemoglobin. The use of bovine hemoglobin should be in ready supply theoretically for the foreseeable future. These approaches may prove more effective in satisfying the predicted high demand for these products.

Use of these products is predicated on knowledge of the serum half-lives of these preparations. In general, poly-hemoglobin preparations will increase in plasma half-life as their size is enlarged; a limit to the size is the viscosity and oncotic effects of the larger hemoglobin molecules. Most preparations will be retained in the plasma for half-lives of 8-30 hours.

These hemoglobin products, however, do not require a supraphysiologic oxygen tension to be effective in delivering oxygen. Indeed, these compounds will most likely be more effective than native hemoglobin in delivering oxygen to tissues at physiologic arterial oxygen tensions (Figure 4). Thus hemoglobin-based oxygen carriers have an advantage over perfluorocarbons in this respect.

Hemoglobin-based oxygen carriers have some advantages over allogeneic red blood cell transfusions. The lack of iso-agglutinating antigens, due to the absence of a red cell membrane, obviates blood typing and screening and eliminates the most common morbidity and mortality of allogeneic and autologous transfusions, mismatching of blood units and the transfusion recipient. The lack of cross-matching requirements also allows virtually immediate availability of an oxygen carrier in critical periods of trauma or hemorrhage. However, there may be issues with administration of free hemoglobin in potentially septic situations.9

Disadvantages Of Hemoglobin-Based Oxygen Carriers
Plasma hemoglobin is not a true blood substitute; hemoglobin can replace only the oxygen transport capacity of whole blood, without the coagulation or immunologic aspects normally present in blood. While allogeneic blood may not supply these functions either, the plasma half-life of cross-matched allogeneic red blood cells is several fold greater than that of plasma hemoglobin. Thus, hemoglobin-based oxygen carriers will not replace blood, allogeneic whole blood, or allogeneic red blood cells completely. Thus use of these products may be limited to specific applications or in conjunction with specialized techniques, such as cardiopulmonary bypass with extracorporeal circulation or acute normovolemic hemodilution with harvesting of autologous whole blood for later reinfusion.

Free hemoglobin avidly binds nitric oxide. It is unknown whether this in vivo binding is of clinical significance, although binding of nitric oxide has been implicated as the cause of hypertension commonly seen with hemoglobin infusion. It remains to be determined what effects stroma free hemoglobin has on regional autoregulation of blood flow, and whether the hypertension associated with hemoglobin infusion has pathophysiologic consequences. At present, little data are available in large animal or clinical studies utilizing these compounds to elucidate the importance of this phenomenon.

----bolism of plasma free hemoglobin-based oxygen carriers is identical to native hemoglobin released as a red blood cell is destroyed. Bilirubin levels will rise as hemoglobin is ----bolized. Amylase levels also rise and some degree of lipase increase occurs; the pancreas appears to be the source of these increases in amylase and lipase, although no clinical evidence of pancreatitis has been documented in patients receiving hemoglobin-based oxygen carriers. The administration of these hemoglobin thus may cause significant alterations in laboratory values, potentially masking serious clinical consequences. Additionally, the consequences of ----bolism of hemoglobin-based oxygen carriers may be similar to those of multiple transfusions, namely hemosiderosis and chronic iron overload.

Clinical Utility Of Blood Substitutes
Current blood substitutes have been demonstrated to be safe when administered in small quantities to volunteers. Both perfluorocarbon and hemoglobin based oxygen carriers have undergone clinical trials designed to determine the safety of these compounds when given to otherwise healthy patients. These preliminary studies have shown that a clinical useful dose of a blood substitute can be infused to patients. However, further information regarding the effectiveness and clinical usefulness of these compounds is in short supply at present.

The short plasma half-life of these compounds limits the usefulness of blood substitutes to short periods of time. Ultimately, the blood substitute will be sequestered or ----bolized, and decreased oxygen carrying capacity will reappear as the plasma oxygen carrying capacity diminishes. Thus, if no longer acting agents are available, it is likely that these blood substitutes will merely delay an allogeneic transfusion, rather than avoiding exposure, when used in place of conventional allogeneic red blood cell transfusions.

In order to effectively use these compounds, special techniques should be considered. One technique which theoretically should optimize blood substitute utility is acute normovolemic hemodilution. Aggressive harvesting of potentially several units of autologous fresh whole blood is possible when the solution to replace the harvested blood is capable of transporting oxygen. Coupling of blood substitutes with acute normovolemic hemodilution has been successful in small clinical trials; whether this mode of using blood substitutes will result in substantial clinical and economic benefits await larger clinical trials.

Summary
Blood substitutes are currently undergoing preliminary clinical trials to determine their safety. Two distinctly different classes of oxygen carriers are being developed, each capable of transporting and delivering oxygen to peripheral tissues. The delivery of oxygen by these two methodologies may have both benefits and risks which are unique to its class. Early clinical trials have been promising; however, effective use of these blood substitutes may involve using them in conjunction with other techniques such as normovolemic hemodilution to effectively reduce or eliminate the need for transfusions in certain instances. However, this first generation of clinically safe blood substitutes will not replace allogeneic blood transfusions as a means of treating many types of anemia.