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EDITORIALS: IS THERE A NEED FOR BLOOD SUBSTITUTES IN THE NEW MILLENNIUM AND WHAT SHOULD WE EXPECT IN THE WAY OF SAFETY AND EFFICACY?
Editorial by: Professor TMS Chang (Canada)
Editorial comments by:
(1) Professor A. Gerson Greenburg (USA);
(2) Professor Hui Sun Chen (PRChina);
(3)Professors Mario Feola & Jan Simoni (USA);
(4) Professor Eishun Tsuchida (Japan);
(5) Professor George Biro (Canada).
(6) Professor Chengmin Yang (PRChina)
(7) Dr. A. Alayash (USA)
(8) Professor Enrico Bucci (USA)
(9) Dr. C. Hsia (U.S.A)
EDITORIAL: IS THERE A NEED FOR BLOOD SUBSTITUTES IN THE NEW MILLENNIUM AND WHAT SHOULD WE EXPECT IN THE WAY OF SAFETY AND EFFICACY? (From: Artif. Cells, Blood Sub. Immob. Biotech 28 (1), i-vii.,2000) by T.M.S.Chang,O.C.,M.D.,C.M.,Ph.D.,FRCP[C], Director, Artificial Cells & Organs Research Centre, Professor of Physiology, Medicine & Biomedical Engineering, Faculty of Medicine, McGill University, Montreal, Quebec, Canada, H3G 1Y6. www.artcell.mcgill.ca
Sensitive screening tests for H.I.V., hepatitis viruses and other potential infective organisms have now resulted in much safer donor blood. This being the case, is there any need for blood substitutes in the new millennium? This question sounds somewhat similar to the comments made some 40 years ago regarding the first reports of "modern" approaches to blood substitutes (1-6). The result is that little or no effort was made to develop these blood substitutes. Thus, there was nothing to replace donor blood during the H.I.V. crisis in the 1980s and patients had to take their chances until many years later when sensitive screening tests become available. This was then followed by hepatitis C etc. Many groups started to catch-up on blood substitute research and development. Unfortunately, given such a complicated product as blood substitutes, it has been more than 10 years of intensive efforts and we still have nothing ready for routine clinical use. Had serious efforts been made to develop the modern concepts of blood substitutes 40 years ago, it would be likely that blood substitutes could have been ready for the H.I.V. crisis in the 1980s. In the new millennium is anyone willing to say that we no longer need blood substitutes and take the responsibility if something similar to H.I.V. or hepatitis C should unexpectedly come up? In addition, there is also the continuing need in emergency situations, peri-operative needs and also in less accessible regions.
Any blood substitutes must be safe and efficacious before they can be used clinically (7). However, safety and efficacy can have many interpretations. Should we demand that blood substitutes have to be equivalent to blood before they can be considered useful clinically? To answer this question, we only have to look at the most commonly used volume replacement solution in the form of Ringer-Lactate solution. It is nothing more than a solution with electrolytes and glucose in concentrations similar to that in the plasma. There is no plasma protein and no blood cell in the solution. It is not even equivalent to plasma. Yet it has been a well-established and effective solution for volume replacement in less severe blood loss and volume depletion. No one ever expects or asks for equivalency tests with blood or even with plasma. The present first generation blood substitute is nothing more than Ringer-Lactate solution with modified hemoglobin or fluorochemicals added as oxygen carrier and also for colloid osmotic pressure. They do not have clotting factors, antioxidant enzymes, white blood cells nor platelets. This being the case, can we expect this simple solution to have the same equivalency as blood or even as red blood cells? Any equivalency tests should only be done in those clinical conditions that require only volume replacement and oxygen carrier. In this regard, first generation blood substitutes like polyhemoglobin in Phase III clinical trials (8-11) and perfluorochemicals (12) are being tested in clinical trials for peri-operative surgery for hemodilution, in surgery with large volumes of blood loss and in other conditions requiring only oxygen carrier and volume replacement. In these clinical trials up to 20 to 23 units of glutaraldehyde crosslinked polyhemoglobin (8-10) have been infused (13).
What happens if one tries to test the safety and efficacy of first generation blood substitutes in conditions requiring more than volume replacement and oxygen carrier? For instance, in conditions with potentials for ischemia-reperfusion injuries including sustained ischemia in stroke, sustained severe hemorrhagic shock with intestinal ischemia, sustained cerebral ischemia and transplantation of donor organs. Unlike red blood cells, first generation blood substitutes contain no red blood cell antioxidant enzymes like catalase and superoxide dismutase. In the absence of these antioxidant enzymes, hemoglobin in the blood substitutes can break down more easily to release heme and iron in the presence of oxidants in ischemia-reperfusion and intensify injuries (14,15). In a 1998 editorial (16) I have emphasized that in these conditions, not only is first generation blood substitutes not efficacious, they are not safe to use. For these conditions, in addition to volume replacement and oxygen carrier, we need the addition of antioxidant enzymes to the blood substitutes. Thus, second generation blood substitutes that contain antioxidants as in the case of polyhemogloin-catalase-superoxide dismutase (14) are being developed.
First generation blood substitutes have a circulation half-time of only up to 24 hours and any equivalency test should only be within this time frame. Conditions requiring more than this time frame would require repeated infusion with blood substitutes or later replacement with donor blood. Studies are being actively carried out to increase the circulation time of blood substitutes. For example, lipid membrane encapsulated hemoglobin is being developed (17,18). The circulation half-time has been increased to more than 50 hours (17) and further increase are being studied. Nanotechnology and biodegradable polymer have been combined to form biodegradable hemoglobin nanocapsules and to increase their circulation time and to include multienzyme systems (19).
The beginning of this new millennium will be an exciting period for blood substitutes. First generation blood substitutes suitable as oxygen carrier and volume replacement are in the final stages of clinical trials. These first generation blood substitutes would have important potentials especially for peri-operative uses as in hemodilution and in surgery with extensive blood loss. For other conditions that require more than just oxygen carrier and volume replacement, new generations of blood substitutes are being actively developed (14-22). It is hoped that we have learned from the last millennium that we cannot wait for an emergency situation before starting to do catch-up research and development on available ideas of blood substitutes (23). They need to be actively developed before it is too late. After all, no one can be sure that there will not be another crisis similar to the H.I.V. crisis of the last millennium.
1. Chang, TMS (1957). Hemoglobin corpuscles. Report of a research, McGill University, 1-25, 1957. Medical Library, McIntyre Building, McGill University,1957 (reprinted in "30th Anniversary in Artificial Red Blood Cell Research" Biomaterials, Artificial Cells and Artificiall Organs 16:1-9, 1988 ).
2. Chang, TMS(1964) Semipermeable microcapsules. Science 146(3643):524-525
3. Bunn, HF, JH Jandl (1968). The renal handling of hemoglobin. Trans Assoc Am Physicians 81:147
4. Geyer, RP, RG Monroe, and K Taylor (1968). Survival of rats totally perfused with a fluorocarbon-detergent preparation. In Organ Perfusion and Preservation (eds. JC Norman, J Folkman, WG Hardison, LE Rudolf, FJ Veith), Appleton Century Crofts, New York, pp 85-96.
5. Chang, TMS (1971). Stabilization of enzyme by microencapsulation with a concentrated protein solution or by crosslinking with glutaraldehyde. Biochem Biophys. Res Com, 44:1531-1533.
6. Chang, TMS (1972). Artificial cells. Monograph. Charles C Thomas, Springfield, IL, 1972.
7. Frantantoni, JC(1991), Points to consider in the safety evaluation of hemoglobin based oxygen carriers. Transfusion. 31:(4)369-371
8. Gould, SA, LR Sehgal, HL Sehgal, R DeWoskin, GS Moss, The clinical development of human polymerized hemoglobin, in Blood Substitutes: Principles, Methods, Products and Clinical Trials. Vol.2 (Chang, TMS, ed.) vol 2 ,pp12-28 Basel Karger1998
9. Gould, SA, F.A Moore et al. Clinical Utility of Human polymerized hemoglobin as a Blood Stubstitute after Acute Trauma and Urgent Surgery. J. Trauma: Injury, Infection and Critical Care (1997) 43: 325-332
10. Pearce, LB, MS Gawryl(1998), Overview of preclinical and clinical efficacy of Biopures HBOCs in Blood Substitutes: Principles, Methods, Products and Clinical Trials. (Chang, TMS, ed.) vol 2, pp82-98 Basel Karger
11. Adamson, JG & C. Moore, Hemolink TM(1998) an o-Raffinose crosslinked hemoglobin-based oxygen carrier , in Blood Substitutes: Principles, Methods, Products and Clinical Trials (Chang, TMS, ed.) vol 2, pp62-79 Basel Karger
12. Keipert, PE (1998), "Perfluorochemical emulsions: future alternatives to transfusion,development,pool"in Blood Substitutes: Principles, Methods, Products and Clinical Trials. Vol.2 (Chang, TMS, ed.) pp101-121, Basel, Karger
13. Panel on Blood Substitutes, Annual Meeting, American Society for Artificial Internal Organs, 1999.
14. DAgnillo, F & TMS Chang (1998) Polyhemoglobin-superoxide dismutase. catalase as a blood substitute with antioxidant properties. Nature Biotechnology 16(7): 667-671
15. Alayash, AI, BA Brockner-Ryan, LL McLeod, DW Goldman & RE Cashon (1998), "Cell-free hemoglobin and tissue oxidants: probing the mechanisms of hemoglobin cytotoxicity", in Blood Substitutes: Principles, Methods, Products and Clinical Trials. Vol.2 (Chang, TMS, ed.) pp157-174, Basel Karger
16. Chang,TMS (1998). Editorial: Is there a role for first generation blood substitutes in the resuscitation of hemorrhagic shock? Artif Cells, Blood Sub Immob. Biotech, an international journal. 26(5-6);i-iii
17. Rudolph, AS, R Rabinovici and GZ Feuerstein (eds) (1997) Red Blood Cell Substitutes. Marcel Dekker, Inc., N.Y.
18. Tsuchida, E (editor) (1998). Blood Substitutes: Present and Future Perspectives. Elservier, Amsterdam.
19. Chang, TMS and WP Yu (1998) Nanoencapsulation of hemoglobin and red blood cell enzymes based on nanotechnology and biodegradable polymer. in Blood Substitutes: Principles, Methods, Products and Clinical Trials. Vol.2 (Chang, TMS, ed.) pp216-231, Basel Karger
20. Doherty, DH, MP Doyle, Curry SR, Vali RJ, TJ Fattor, JS Olson and DD Lemon (1998). Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin. Nature Biotechnology 16: 672-676
21. Chang, TMS (1997a) Monograph on "Blood Substitutes: Principles, Methods, Products and Clinical Trials" Vol. I, Basel Karger
22. Winslow, RM, KD Vandegriff, M. Intaglietta (eds) (1997) Blood Substitutes: industrial opportunities and medical challenges, Birkhauser, Boston, 1997.
23. Chang, TMS (1997). Editorial: An Urgent need to include blood substitute as a priority area in any national policies on blood supply. Artif. Cells, Blood Sub. Immob. Biotech, an international journal 25 (4), i-ii.
Editorial comments by A. Gerson Greenburg, M.D.,Ph.D., Professor of Surgery, Brown University School of Medicine, U.S.A. (From: Artif. Cells, Blood Sub. Immob. Biotech 28 (2),v-vi, 2000):
Dr Chang with the comfort of experience and the wisdom of age has raised most interesting and critical questions about the current status of blood substitutes(1). He has called upon the current active community of blood substitute scholars, many of who have dedicated much of their creative efforts the past score of years, to learn from the past. This call is well founded having been noted by Hegel, Santayna and Churchill in different context but relevant in principle.
I think there is resounding evidence to support the "need" of at least a red cell substitute at this time. Survival clearly depends on adequate oxygen delivery to tissues and a solution that carries and delivers oxygen has a place in the perioperative management of surgical patients today. Whether a fully functional "blood substitute" complete with immune and coagulation factors will ever be available remains, for now, an idealistic conjecture; ideal in principle perhaps, operationalizing it into practice may be more problematic.
Surely the blood supply is safer than it has ever been. Safe from the known risks to be sure. Yet we all know, and somewhat fear, that there may be lurking in that amorphous complex liquid other agents of concern. Can we ever be sure all of the risks of infectious complications of transfusion are eliminated? Can we ever be sure that non-infectious complications of blood and blood component transfusion will be eradicated? Granted a great deal is known about the complications of transfusion there remain unanswered questions relative to risk that concern the physician and lay public. Given a viable and safe alternative to traditional blood product transfusion, if only for the oxygen delivery component, known and unknown risk exposure will be minimized but not completely eliminated.
First generation substitutes have a place in the current therapeutic armamentarium if only as a bridge to more effective use of existing blood products. They may provide adequate perfusion or resuscitation to ischemic tissues until the site of hemorrhage is controlled. This increase in oxygen delivery is positive and clearly represents more than a crystalloid or colloid resuscitation fluid could accomplish. It may be that these properties will also apply to the concept of ischemic rescue and even reperfusion. Who is to say that the white cell elements of blood are idle passengers in these processes. Reperfusion is a complex physiologic process just now being unraveled. Oxygen is an element of the process to be sure and it serves both the positive and negative sides of the mechanism. The free radicals require oxygen for formation; the tissues require oxygen for basic function. Maintaining the balance, homeostasis, is the trick and what we have learned in the past few years is just how difficult this process really is.
As we enter the new millennium the call for newer and more sophisticated red cell and blood substitutes needs to be heard. The definitions of efficacy and safety singly and in combination need to be made explicit and criteria established to demonstrate when the criteria are met. The last decade of the 20th century has yielded remarkable progress in the field with obvious potential benefit to patients of the first generation products. Given we know more can be accomplished with the substitutes generally, as we integrate knowledge of the underlying pathophysiology of oxygen deprived states into their design, the new millennium presents an opportunity to move forward with developments that our scientific forefathers considered a dream.
1. Chang, TMS. Editorial: Is there a need for blood substitutes in the new millennium and what can we expect in the way of safety and efficacy? Artificial Cells, Blood Substitutes & Immobilization Biotechnology, an international journal. 28 (1) i-iv, 2000.
Editorial comments by Professor Hui Sun CHEN, People's Republic of China:(From: Artif. Cells, Blood Sub. Immob. Biotech 28 (2), vii, 2000)
On approach of the new millennium, some scholars have presented a
, that: "Screening tests are now so good that why do we
need blood substitutes? If we use them, they have to be equivalent to donor blood. There
is no need for further research on blood substitute." Firstly, is there really no
need for further research on blood substitute? We think that this conclusion should not be
made too early. Blood substitute, especially based on red blood cells, has much benefit
over donor blood in, for instance, storing for much longer period, having no immunity and
needing no cross match before transfusion. These means that blood substitute could be used
more conveniently, especially in disasters and war. Although the blood substitutes
presently being tested in clinical trials are a little far from perfect, new generations
of blood substitute are being developed, and with the beginning of this new millennium
some novel and more promising blood substitutes are likely to come out in the near future.
Editorial comments: by Professors Mario Feola and Jan Simoni, Department of Surgery, Texas Techn University Health Sciences Center, Lubbock, Texas 79430, USA(From: Artif. Cells, Blood Sub. Immob. Biotech 28 (2), vii-ix, 2000)
Hopefully, it will not take the next millennium to develop an effective blood substitute. The increasing demand for blood, together with a decreasing blood supply is of concern in the developed world. In fact, a shortage of 4 million units per year has been projected by the year 2030 . More importantly, however, is the concern about the safety of the blood supply in the undeveloped world, which represents most of humanity. In the U.S., the implementation of sensitive screening tests has reduced the risk of infectious disease transmission to 1: 60,000 blood transfusions for hepatitis B and 1: 500,000 for HIV, with intermediate transmission rates for hepatitis C and human T-cell leukemia virus . While the question of whether the blood can transmit Creutzfeldt-Jacob's disease, or its bovine variant, is yet to be answered, there is still a dramatic improvement in blood safety . On the other hand, it is reported that in India only 50% of the donated blood is screened, and 10% of the paid donors are HW-positive . Only 16 of the 35 member states of the Pan American Health Organization screen all their blood samples for hepatitis and the AIDS virus . In sub-Saharan Africa the situation is much worse, with no screening at all, and in many places no blood banks at all . This lack of safety of the blood supply ought to concern the World Health Organization and the international scientific community on an urgent basis.
An effective, safe and inexpensive blood substitute that could be stored at room temperature or in a regular refrigerator for a long period of time, and which could be administered without any need for blood typing and cross-matching, could save millions of people every year.
What can be expected in terms of safety and efficacy? The development of hemoglobin-based oxygen carriers has progressed significantly in the last 2 decades. The problems of high oxygen affinity and short half-life of hemoglobin in solution, which were uncovered in the 1970s, have been resolved in various ways. There are, however, newly discovered problems which need resolution. These are the vasoactivity, the pro-oxidant and the pro-inflammatory properties of hemoglobin in solution [7-11]. Products currently in various phases of clinical trials were developed before the recognition of these problems. Unlike intermolecularly crosslinked polyhemoglobin, intramolecularly crosslinked tetrameric hemoglobin has vasoactivity. Diaspirin intramolecularly cross-linked tetrameric hemoglobin has recently been withdrawn from clinical trials (3,12]. There is presently a disconnection between research laboratories that work toward better understanding and resolution of existing problems. It is reasonable to expect that the products, presently under clinical trials, will represent the "first family" of blood substitutes, with efforts now directed towards "new generations" of blood substitutes for other clinical applications.
Some research laboratories are already engaged in the development of new generations of blood substitutes. It has been reported that cross-linking of anti-oxidant enzymes with polyhemoglobin eliminates its pro-oxidative potential . Genetic alteration of the hemoglobin molecule can lessen the vasoconstrictor activity of tetrameric hemoglobin . We have also addressed these problems and have developed a novel hemoglobin chemical modification procedure. Our multi-functional cross-inking agents, used in hemoglobin intra- and intermolecular cross-linking and surface modification procedures, posses the desired chemical and pharmacological activities that suspend the pressor effect and suppress the hemoglobin pro-oxidative and pro-inflammatory potential. This blood substitute has been tested in both animals and humans [15-19].
EDITORIAL COMMENTS: NECESSITY OF BLOOD SUBSTITUTES IN THE NEW MILLENNIUM AND POINTS TO CONSIDER IN THE WAY OF SAFETY AND EFFICACY. by Professor Eishun Tsuchida, Professor, Department of Polymer Chemistry, Advanced Research Institute for Science and Technology,Waseda University, Tokyo 169-8555, Japan (From: Artif. Cells, Blood Sub. Immob. Biotech 28 (3), v-vii,2000):
The phase III clinical trial of resuscitation from traumatic hemorrhagic shock with the intramolecularly-crosslinked Hb revealed an unexpectedly higher mortality than resuscitation with saline, resulting in no further trials in 1998 . As had been often predicted by many researchers, this would be due to significant changes in blood circulation. Acellular Hb traps NO as an EDRF, induces vasoconstriction and hypertension, and causes blood flow redistribution. This has also been used as a vasopressor in critically ill patients or hemodialysis patients, and showed stable hemodynamics in clinical tests . However, the vasopressive property inversely induced negative effects. As a consequence, the recent trend in the development of acellular Hb-based blood substitutes is to increase the molecular dimension based on the physicochemical parameters : polymerization or polymer conjugation, and recombinant oligomeric Hb with low NO affinity, etc. Also, the cellular type of liposome-encapsulated Hb (Hb-vesicles, diameter: 200 - 250 nm) will be the most suitable to mimic the function of RBC [4-6].
The result of the phase II clinical trial of a perfluorocarbon emulsion for acute normovolemic hemodilution in orthopedic patients to avoid allogeneic transfusion was recently disclosed . There was no serious adverse event, and the safety of Perflubron was mostly confirmed. This is partly due to the safer protocol without a critical situation in comparison with the uncontrolled resuscitation from hemorrhagic shock. However, it should be considered if the controlled elective surgery is the primary indication for blood substitutes because autologous blood transfusion using EPO is possible as an alternative way. Other indications for which a blood substitute is strongly required are emergency situations such as hemorrhagic shock, cardiac and respiratory failures, and stroke. Historically, the development of transfusion and blood substitutes has been supported by the U.S. Department of Defense for combat casualties . On the other hand, the provision of blood substitutes was supposed to be required in Japan where people suffer from frequent natural disasters such as an earthquake.
Contrary to the present direction of the US Government, the Japanese Ministry of Health and Welfare strongly supports the artificial blood project as a part of the Research on Advanced Medical Technology as of four years ago based on the situation that Japan imports a huge amount of blood products and the tragedy of AIDS in hemophilia patients . This project is composed of three categories: 1) artificial red cells to carry oxygen, 2) artificial platelets to induce blood coagulation, and 3) artificial immune (immunoglobulin) to prevent infection. Among the three, the artificial red cell is the most advanced with Hb-vesicles and totally synthetic oxygen carriers using heme derivatives . Their sufficient oxygen transporting capability has been confirmed, and research is now concentrating on safety issues. The design and establishment of a pilot plant is underway in collaboration with the Ministry and companies. Since the Society of Blood Substitutes, Japan, was established in 1993, a symposium has been annually held with active interdisciplinary discussions. Guideline study groups for material properties, preclinical and clinical studies will be officially organized to prepare for clinical trials. Therefore, the answer to the question of Dr. Chang, Is there a need for blood substitutes? is indeed Yes! in Japan.
We have to keep it in mind that the lifetime of a blood substitute is much shorter than real blood in vivo. However, the characteristics of blood substitutes (long-term storage at room temperature, no blood type mismatching, no infectious virus, etc.) override the limitations of the blood transfusion system and contribute to benefiting clinical medicine. Research has been already started in the Middle and South American and Asian areas, thus the necessity and development of blood substitutes are becoming worldwide .
 Sloan, E.P., Koenigsberg, M., Gens, D., Cipolle, M., Runge, J., Malloy, M.N., and Rodman, Jr. G. Diaspirin crosslinked hemoglobin (DCLHb) in the treatment of severe traumatic hemorrhagic shock: a randomized controlled efficacy trial. JAMA 282: 1857-1864 (1999)
 Przyberski, R.J., Daily, E.K., Birnbaum, M.L. The pressor effect of hemoglobin - good or bad? In: Advances in blood substitutes: industrial opportunities and medical challenges (ed. by Winslow, Vandegriff, and Intaglietta) Chapter 5, pp.71-90, Birkhauser, Boston, 1997.  Intaglietta, M. Microcirculatory basis for the design of artificial blood. Microcirculation 6, 247-258, (1999).
 Tsuchida, E. ed. Blood substitutes: present and future perspective Elsevier, Amsterdam, 1998.
 Chang, T.M.S., ed. Blood substitutes: Principles, Methods, Products and Clinical Trials Karger, Basel, 1997.
 Rudolph, A.S., Rabinovici, R., Feuerstein, G.Z. (eds.) Red blood cell substitutes: basic principles and clinical applications Marcel Dekker, New York, 1998.
 Spahn, D.R., van Brempt, R., Theilmeier, G., Reibold, J., Welte, M., Heinzerling, H., Brick, K.M., Keipert, P.E., Messmer, K. Perflubron emulsion delays blood transfusion in othopedic surgery. Anesthesiology 91:1195-1208, (1999).
 Holmberg, J.A. Potential use of red blood substitutes within the military. In Red blood cell substitutes: basic principle and clinical applications (ed. by Rudolph, Rabinovici, Feuerstein) pp 17-27, Marcel Dekker, New York, 1998.
 Sekiguchi, S. Blood substitute research of the national health and welfare science. Artif. Blood 4, 85-89, 1996.
 Winslow, R.M. The role of blood substitutes in emerging healthcare systems. In Blood substitutes: present and future perspective (ed. by Tsuchida) pp. 15-32, Elsevier, Amsterdam, 1998.
EDITORIAL COMMENTS: THE USEFULNESS OF "RED CELL SUBSTITUTES" TO ENHANCE INTRA-OPERATIVE AUTOLOGOUS BLOOD DONATION IN CARDIAC SURGERY By George P. Biro, M.D., PhD.,Vice President, Medical Affairs, Hemosol, Inc.,Toronto, Ont., Canada, M9W 4Z4, and Professor, Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Adjunct Professor, Department of Physiology, Faculty of Medicine, University of Toronto. (From: Artif. Cells, Blood Sub. Immob. Biotech 28 (3), viii-xii, 2000):
Dr. T. M. S. Chang's recent Editorial, "Is there a need for blood substitutes in the new millenium and what should we expect in the way of safety and efficacy" makes the case for the need of these substances by reference to the blood-borne transmission of HIV and hepatitis viruses in the 1980's. After this tragic history, the introduction of more sensitive testing methods and stringent donor screening have rendered the transfusion of blood and blood products safer than ever before. The blood-borne transmission of the hepatitis and immunodeficiency viruses has been reduced from significant to very small probability (1), but the emergence of new threats in the future is possible. Complications not involving the transmission of infectious diseases also occur after transfusions of donor bank blood. In the United Kingdom, a voluntary survey of Serious Hazards Of Transfusion (S.H.O.T.) (2) has shown that, despite rigorous procedures, transfusion of the wrong unit, resulting in a serious or fatal reaction is the commonest hazard. Other complications also occur after transfusions of allogeneic blood (1). The immuno-modulatory effects of an allogeneic blood transfusion are believed to be associated with a higher frequency and severity of surgical infections, delayed wound healing and the recurrence or accelerated progression of malignant disease (3). These have a serious impact on the short- and longer-term recovery of surgical patients. If possible, these effects should be avoided.
Blood conservation methods (4) play a valuable role in contributing to safety, and sparing allogeneic blood use in particular surgical situations. One of these is the use of intra-operative autologous blood donation (IAD) used as an alternative to allogeneic blood transfusion. The benefits of blood conservation methods, including IAD blood transfusions in surgery, are the reduction of the risks and complications associated with allogeneic bank blood transfusions. "Red cell substitutes" may further enhance IAD and Dr. Chang has alluded to this potential.
Cardiac surgery with cardiopulmonary bypass is the commonest surgical procedure currently performed in North America, and it uses blood frequently (5). The frequency of blood transfusion in cardiac surgery ranges from 30% to 90% at various centres (6) and, on average, four to six allogeneic units are transfused, depending on the nature of the procedure and other associated risk factors (7). In some centres pre-operative autologous donation programs (PAD) make some of the patient's own blood available for peri-operative use, but these programs involve significant additional expense and require the patient to donate blood several weeks in advance of the planned date of surgery (4). As these units are stored in preservative solution for at least a week before their use, the blood has few functional platelets and the red cells exhibit the "storage lesion" due to depletion of ATP (8,9). The oxygen affinity of such red cells is suboptimal for oxygen supply to the tissues, and their survival in the circulation is shortened (10). The PAD program is limited to those candidates who have sufficiently large red cell mass to donate sufficient amount of blood and avoid the development of anemia after donation which may precipitate signs and symptoms of myocardial ischemia, particularly in candidates for coronary artery surgery, during activities of daily living (4).
An alternative to PAD is intra-operative autologous donation (IAD) which combines several advantages by providing the patient's own fresh, whole blood. This is the highest quality blood that can be procured, and cardiac surgery with cardiopulmonary bypass is the most favorable setting in which a large donation can be drawn rapidly without simultaneously diluting the blood greatly with the diluent solution used, and without inducing major hemodynamic instability by perturbing the blood volume. This "best" blood is available for needed transfusion during and immediately after the operation, avoiding allogeneic blood, returned to the patient to raise the hemoglobin and hematocrit after the bleeding is controlled. The following important advantages are derived from using the patient's own, fresh, whole blood. The units thus obtained are kept with the patient, and are used within about eight hours or less; this reduces or eliminates the small but significant possibility of transfusion errors and resultant potentially serious reactions. It does not cause allo-immunization to minor blood group antigens. It prevents the transmission of undetected infectious agents present in blood donors. This blood is fresh, having the full complement of the constituents of fresh blood, including fully functional platelets, clotting factors, as well as red cells of normal oxygen affinity and survival. It is not known whether a transfusion of such autologous blood would induce the immunomodulatory effects known to accompany allogeneic transfusions (3), but it is likely that such effects would be of lesser intensity, compared to those observed with allogeneic blood transfusions.
There are some potential risks offsetting the benefits of IAD blood use in the operative setting. Hemodynamic instability on drawing the donation is possible, but is avoidable or manageable in most cases. The possibility of bacterial contamination of these units, or administering one to the wrong recipient, exists, but is remote (4). The major limitation of obtaining a sufficiently large donation is the patient's red cell mass (principally determined by body size and hematocrit), and the possibility of reducing the hematocrit to a level which may induce a myocardial ischemic event (11,12). These are different from those that may occur in the case of PAD, since the blood is drawn as the patient is under intensive monitoring and management in the operating room, while being prepared for CABG surgery.
The use of "red cell substitutes" provides the best opportunity to enhance such IAD, by the replacement of the erythrocytic hemoglobin removed, with hemoglobin in the plasma phase, thereby maintaining a safe total hemoglobin level. For the duration of cardiopulmonary bypass many centers reduce the hemoglobin concentration to 60 g/L, and in the immediate post-operative period, under careful monitoring in the ICU, a hemoglobin of 70 g/L is considered safe.
The IAD procedure enhanced by the use of "red cell substitutes" would allow a significantly larger volume of IAD blood available for peri-operative use, and the benefits would be extended to those who could not donate sufficient blood without reducing their hemoglobin to levels at which myocardial ischemia is more likely. Avoidance or reduction of peri-operative allogeneic blood exposure, and its attendant risks, would clearly benefit most those who have a long life expectancy, those who have an expectation of a good surgical and cardiac outcome (i.e. improved exertion tolerance and ventricular function), and those who have none of the pre-operative conditions that predict a high risk for surgical complications (13) and excessive bleeding requiring many units of blood.
For the "low-risk" patients, who actually form the majority of those undergoing coronary artery bypass grafting (CABG) surgery, the benefits are substantial, but are difficult to fully quantify, since the avoidance of infections and other complications related to blood transfusion is difficult to detect. In addition, benefits would also accrue to the blood system as a whole, in that allogeneic blood and blood products (fresh frozen plasma, platelets) use would also be less in cardiac surgery, allowing better management of the supply of bank blood, preventing or reducing the frequency and severity of periodic shortages, and their downstream effects (eg. cancellation of elective surgical procedures, etc.). If half of the approximately 500,000 CABG operations, involving moderate blood loss would avoid two units of allogeneic blood transfused, one million units, or nearly 5-7% of bank blood used in North America, would be available to other uses.
[Acknowledgement: the author is grateful for the opportunity to discuss issues related to this matter with A. G. Greenburg MD, PhD, Surgeon-in-Chief, The Miriam Hospital, Providence, R.I. 02906, USA.]
1. Wolf , C.F.W. and J.P. Gold, Current Practice of Blood Transfusion in Cardiac Surgery. In: Blood Conservation in Cardiac Surgery, Krieger, K.H. and O.W. Isom (Eds) Springer-Verlag New York, Inc., New York, 1998, 3-27.
2. Williamson, L.M., S. Lowe et al. (1999). Serious hazards of transfusion (SHOT) initiative: analysis of the first two annual reports. BMJ 319:7201:16-19.
3. Blumberg, N. and J.M. Heal, Transfusion Immunomodulation. In: Scientific Basis of Transfusion Medicine, Anderson, K.C. and P.M. Ness (Eds) W.B. Saunders Company, Philadelphia, 2000, 427-443.
4. Blood Conservation in Cardiac Surgery, Krieger, K.H. and O.W. Isom (Eds) Springer-Verlag New York, Inc., New York, 1998.
5. Cosgrove, D.M., F.D. Loop et al. (1985). Determinants of Blood Utilization during Myocardial Revascularization. Ann Thorac Surg 40: 380-384.
6. Surgenor, D.M., E.L. Wallace et al. (1992). Red cell transfusions in coronary artery bypass surgery (DRGs 106 and 107). Transfusion 32: 458-464.
7. Surgenor, D.M., W.H. Churchill et al. (1996). Determinants of red cell, platelet, plasma, and cryoprecipitate transfusions during coronary artery bypass graft surgery: the Collaborative Hospital Transfusion Study. Transfusion 36: 521-532.
8. Valeri, C.R. (1970). Viability and Function of Preserved Red Cells. N Engl J Med 284: 81-86.
9. Sugerman, H.J., D.T. Davidson et al. (1970). The Basis of Defective Oxygen Delivery from Stored Blood. Surgery, Gynecology & Obstetrics 131: 733-741.
10. Valeri, C.R., D.A. Valeri et al. (1982). Viability and function of red blood cell concentrates stored at 4° C for 35 days in CPDA-1, CPDA-2, or CPDA-3. Transfusion 22: 210-216.
11. Hogue, C.W., Jr., L.T. Goodnough and T.G. Monk (1998). Perioperative myocardial ischemic episodes are related to hemacrit level in patients undergoing radical prostatectomy. Transfusion 38: 924-931.
12. Parsloe, M.R., R. Wyld et al. (1990). Silent myocardial ischaemia in a patient with mild anaemia before operation. Br J Anaesth 64: 634-637.
13. Hammermeister, K.E., C. Burchfiel et al. (1990). Identification of Patients at Greatest Risk for Developing Major Complications at Cardiac Surgery. Circ Suppl IV 82: 380-389.
COMMENTS ON IS THERE A NEED FOR BLOOD SUBSTITUTES IN THE NEW MILLENNIUM
AND WHAT CAN WE EXPECT IN THE WAY OF SAFETY AND EFFICACY? Chengmin
YANG, Professor, Chinese Academy of Medical Sciences
Peking Union Medical College, Former Director of Blood Transfusion, CAMS, Director, Chinese Red Cross National Blood Center, Deputy President , Chinese Society Council of Blood Transfusion
Committee Member, Committee of Technical Stardard, Ministry of Health of P.R.China
Unjust comment on blood substitutes is interfering with research and development of the blood product. I agree with your viewpoint about this problem . We should have an objective, historical, progressive attitude for new scientific discovery, new technique or new material. We should not look at things in a negative perspective. In the history of science and technology this situation has frequently been encountered. I hope that these unjust comments would not become a real obstacle for the advance of this field. As a person engaged in the blood science for over forty years, I have no reason to believe the problem of infection from blood transfusion will not occur in the future, even though we now have sensitive screening tests for HIV, HCV and others. Scientists have kept their eyes on the evolution of viruses and bacteria. Before 1980, because blood preparations entered into the clinics without strictly inactivating pathogenic components, it resulted in a widespread infection of HIV all over the world. The same case also took place in China. This is a disastrous retribution from nature. In the struggle for health and disease, mankind makes steadily progress, but at the same time, nature also undergoes constant change. Nowadays, HEV made an attack on mankind once more, who can predict what new infectious sources are not waiting for us, despite passing through tests against HIV, HCV, HBV and so on. FDA has worked out regulations for human factor-VIII blood preparation: it must be strictly inactivated for pathogenic organism before the product flows into the market. Some scientists carrying out research and development on blood substitute are shifted to the current demand for the product. Any new products are bound to have some imperfections at the beginning that needs to be improved. This cannot be taken as the reason to suppress their growth and further developments. As we know, when the first steam locomotive moved on the globe, it moved slower than horses. However with persistent efforts, modern high speed trains are now beyond comparison over the latter.
YANG, Professor, Chinese Academy of Medical Sciences
1. Chang, TMS. Editorial: Is there a need for blood substitutes in the new millennium and what can we expect in the way of safety and efficacy? Artificial Cells, Blood Substitutes & Immobilization Biotechnology, an international journal. 28 (1) i-iv, 2000
EDITORIAL COMMENTS: IS THERE A NEED FOR BLOOD SUBSTITUTES IN THE NEW MILLENIUM AND WHAT SHOULD WE EXPECT IN THE WAY OF SAFETY AND EFFICACY: A RESEARCH PRESPECTIVE.
by Abdu I. Alayash#, Ph.D. Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda Maryland 20892.(From: Artif. Cells, Blood Sub. Immob. Biotech 29 (1), 2001 in press)
Several editorial comments appeared in this journal in response to the question posed earlier by Dr Chang as to whether there is a need for blood substitutes (1). There was a general agreement among some of the leaders in the blood substitute community strongly endorsing the need for blood substitutes, reflecting the thinking of the community at large. As expected, there were some differences in how to demonstrate the safety and efficacy of these products. My response to Dr. Changs question will focus on the research aspects of blood substitutes carried out over the last decade, specifically some of the positive outcome (spin offs) of this research and why we still need to do more.
Nitric oxide (NO), crowned in 1992 as the molecule of the year (2), dramatically impacted the field of blood substitute research and continues to shape our understanding of human physiology in the new decade. This simple diatomic gas, produced by the vascular endothelium, regulates a host of biochemical reactions including vascular smooth muscle relaxation. It has also been implicated in the hypertensive response to the infusion of cell-free hemoglobin (Hb) in humans (3). The discovery that cell-free Hb can reach and rapidly react with NO, by the virtue of its small molecular size and high affinity towards NO, led many investigators to design larger conjugated, or polymerized proteins, thereby potentially attenuating its hypertensive effects. Unfortunately this did not fully materialize for the simple reason, that once NO is produced it can cover considerable distances in and around its vascular production sites in its short half-life (4). Importantly, recent animal studies and calculation of NO kinetics at the vascular wall argue against extravasation as a singular mechanism (5)(6). Hb is not subjected to intravascular flow dynamics as red blood cell. This flow dynamics can effectively decrease RBC consumption of NO by creating a RBC-free zone. In other words, small or large cell-free Hbs will undoubtedly be close enough to the sources of NO; thus a more effective method to prevent this reaction is clearly needed. One experimental approach is to re-design of the heme-binding pocket of human Hb by site-directed mutagenesis, to reduce its affinity towards NO. This was shown recently to lower blood pressure in animals (7)(8).
Another less appreciated role for NO in mammalian physiology is its antioxidant function. Removal of NO by Hb will undoubtedly tip the balance towards the formation of a number of harmful oxygen and nitrogen-based radicals (hydrogen peroxide (H2O2), lipid peroxide (LOOH), and peroxynitrite (ONOO-)) to the detriment of both Hb and the vasculature (4). The impact of these interactions on modulation of cell signaling pathways regulated by these reactive species is extremely important and as yet unexplored area of research (9). The presence of red cell enzymes (i.e., superoxide dimutase and catalase) in some stroma-free Hb preparations (10) or simply the cross-linking of these antioxidant enzymes to the Hb molecule (11) may provide a protective mechanism against Hb side reactions.
A novel product from the reaction between NO and Hb, involving S-nitrosation of cysteine-93 of the b-chain, has recently been identified (12). The mechanisms for the proposed role of the so-called SNO-Hb in regulating of blood flow are still controversial. Nevertheless, if proven, this will pose an additional challenge for the blood substitute developers in that a careful balance between Hbs own competing reactions i.e., nitrosylation (heme reactivity) versus S-nitrosation (b-globin reactivity) must be found in order to maintain hemodynamic stability.
Another proposed mechanism for the cause of hypertensive effects does not involve NO, but instead involves an autoregulatory constrictive response of the arterioles to excess supplies of oxygen (Hb is more efficient at delivery of oxygen than are red blood cells). In this hypothesis, cell-free Hb unloads its oxygen in small arterioles, triggering an autoregulatory response that leads to constriction and closure of capillary bed.
Regardless of the causes of vasoconstriction, unraveling the underlying mechanism(s) will surely lead not only to safer second-generation products, but a better understanding of oxygen and nitric oxide physiologies.
1. Chang TMS. Editorial: Is there a need for blood substitutes in the new millennium and what can we expect in way of safety and efficacy? Art Cells Blood Sub Immob Biotech 28:i-vii, 2000.
2. Editorial: NO, the molecule of the year, Science 254:1853,1992.
3. Gulati A, Barve A, and Sen PA. Pharmacology of hemoglobin therapeutics. J Lab Clin Med 133:112-119, 1999.
4. Alayash AI. Hemoglobin-based blood substitutes: oxygen carriers, pressor agents or oxidants? Nat Biotech 17:545-549, 1999.
5. Liu X, Miller MJS, Josh MS et al. Diffusion-limited reaction of free nitric oxide with eyrthrocytes. J Biol Chem 273:18709-18713, 1998.
6. Lio JC, Hein TW, Vaughn MW et al. Intravacular flow decreases erthyrocyte consumption of nitric oxide. Pro Natl Acad Sci USA 96:8757-8761, 1999.
7. Doherty DH, Doyle MP, Curry SR et al. Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin. Nat Biotech 16:672-676, 1998.
8. 8. Doyle MP, Apostol I, and. Kerwin B.A. Glutaraldehyde modification of recombinant human hemoglobin alters its hemodynamic properties. J Biol Chem, 274:2583-2591,1999.
9. Alayash AI, Patel RP and Cashon RE. Redox reactions of hemoglobin and myoglobin: biological and toxicological implications. Antiox Redox Signl (in press).
10. Privalle C, Talarico T, Keng T, et al. Pyridoxalated hemoglobin polyoxyethylene: a nitric oxide scavenger with antioxidant activity for the treatment of nitric oxide-induced shock. Free Rad Biol Med 28:1507-1517, 2000.
11. 11. DAgnillo F and Chang TMS. Polyhemoglobin-superoxide dismutase-catalase as a blood substitute with antioxidant properties. Nat Biotech 16:667-672, 1998.
Ji 12. L, Bonaventura J, Bonaventura C, et al. S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature 380:221-226, 1996.
#The opinions and assertions contained herein are the scientific views of the author and are not to be construed as the policy of the United States Food and Drug Administration.
HEMOGLOBIN BASED OXYGEN CARRIERS AT A
CROSS ROAD: THE OLD PARADIGMS MUST BE ABANDONED AND MUCH MORE BASIC SCIENCE
INVESTIGATION IS NECESSARY. Enrico
Professor, University of Maryland Medical School (
(Artificial Cells, Blood Substitutes & Immobilization Biotechnology, an international journal, for volume 29, 4th issue, 2001)
Research on blood substitutes started near the end of World War II when the military were impressed by the number of casualties with consequent need of blood transfusions. Unfortunately at that time this field was shunned by academia as an inferior kind of applied research, moreover marred by the sadness of war.
Thus the research was started by a group of courageous and dedicated pioneers who, challenged by the multidisciplinary nature of the endeavor and irrespective of their original background, had to improvise themselves simultaneously as chemists, protein chemists, physiologists, biochemists, surgeons, physicians, hematologists, blood bankers, hemoglobinologists. It was an overwhelming task, softened by the ideas that anything capable of exchanging molecular oxygen could be used as blood replacement. At that time the only available criterion for the viability of the various compounds was survival versus non-survival of infused animals. Also the conventional wisdom and present FDA requirements were and are that blood substitutes should have oxygen binding characteristics similar to those of blood.
Based on these criteria, very soon it appeared that the goal of obtaining a blood substitute was not easy. Thus the work was distributed into many different approaches including: intramolecularly crosslinked hemoglobins, polyhemoglobins, recombinant hemoglobins, conjugated hemoglobins, encapsulated hemoglobins.
Academia became interested, and private concerns started developing substitutes, lured by a potentially unlimited market. Thus, after 50 years of efforts full of ups and downs with academic laboratories starting and ending their research and private concerns oscillating between venture capital investments and bankruptcy, only three polyhemoglobins survive in the phase III clinical trails namely Polyheme (human Hb polymerized with glutaraldehyde, Northfield, Deerfield, IL), Biopure (bovine Hb polymerized with glutaraldehyde, Biopure, Boston, MA), Hemolink (raffinose polymerized human UB, Hemosol, Toronto, Canada). The clinical trials with Optro (a recombinant hemoglobin, Somatogen, Boulder,CO) and Hemassist (an intramolecularly crosslinked hemoglobin, Baxter,IL) were suspended. It may be noticed that all surviving products are from small private concerns. In fact, after an initial enthusiasm, Federal Agencies like NIH and DOD lost interest in supporting independent laboratories.
This is unfortunate, because the inevitable corporate policies of investments and confidentialities prevent small companies to develop new products and ideas. Their activity is locked into old technologies developed 15-30 years ago. The consequence is that at a recent FDA workshop in Bethesda, MD (Sept 1999) the representative of the three companies mentioned above confessed that the criterion for viability of their product is still the survival/not-survival of the prepositi (this time humans). They could not offer anything better to those who loudly objected.
These old ideas and technologies have been challenged. Recombinant hemoglobins are continuously produced (see in the web under Clara Fronticelli ,John S. Olson and Chen Ho), new chemistry is produced in the laboratory of this author (1,2) and of Dr. Thomas Chang (3), new encapsulations are proposed(4-6). Absorption of NO as cause of increasing mean arterial pressure (MAP), produced by most of the modified hemoglobins, is also very confusing. The old proposal that it is due to scavenging of NO by the cell free hemes is very reasonable and supported by experimental evidences. However it is not consistent with recent data obtained by Winslow (7) and Intaglietta (8). The last authors report that the size of the arterioles of rat mesentery is inversely proportional to the diameter of the infused hemoglobins. The cause of this correlation is not clear. The author of this editorial reports that tetrameric hemoglobins (that notoriously increase MAP) extravasate and appear in the lymph, even if they do not appear in the urine. Instead large polymeric hemoglobins do not extravasate and have little effect on MAP (9). Absorption of NO may not be the sole cause of the phenomenon. There are indications that there is a synergistic effect on oxygen diffusion from artificial capillaries when red cells are mixed with cell free hemoglobin (10-11). On this and other evidences Vandegriff and Winslow challenge the conventional wisdom that the substitutes should have characteristics similar to blood. It is proposed that high oxygen affinity compounds would be better carriers than low affinity ones (12).
It appears that cell free oxygen carriers, even those based on hemoglobin, are not blood substitutes whose characteristics should be compared to those of blood. They are new oxygen carrying compounds with their peculiar mechanism of oxygen transport in vivo. The negative experience of Baxter on trauma patients (13) testifies to the need of learning this new physiology before designing clinical trials. At the FDA workshop in Bethesda, the reported success of blood replacement in hemorrhagic patients, based on the empirical survival/non-survival approach, only shows that they can be beneficial, as expected. This is the crossroad mentioned in the title of this editorial. The old paradigms that cell free oxygen carriers must have > characteristics similar to blood are not tenable and, if enforced by either > FDA requirements or NIH study sessions, prevent developments. There is much need > to study in depth the rheology and viscosity effects in vivo of the > infused carriers, their extravasation properties and, most important, the > physiology and mechanism of cell free oxygen transport.1.
1. Bucci, E., Razynska, A., Kwansa, H., Matheson-Urbaitis, B., O'Hearne, M., Ulatowski, J. A. and Koehler, R. C. Production and Charateristics of an Infusible Oxygen-Carrying Fluid Based on Hemoglobin Intramolecularly Cross-Linked with Sebacic Acid. J. Lab. Clin. Med. 128, 146-153 (1996).
2. Razynska A., and E.Bucci Zero-link Polymerization: a New Class of Polymeric Hemoglobins, Blood Substitutes, Present and Future Perspectives. Pg 265-279, Elsevier Science S.A. 1998, (1999)
3. Chang TMS (1997) (monograph) Red blood cell substitutes: Principles, Methods, Products and Clinical Trials Vol I (Monograph) Karger/Landes Systems, Basel, Switzerland
4. Rudolph AS. Encapsulated hemoglobin: Current Issues and Future Goals. Artificial Cells, Blood Substitutes andImmobilization Biotechnology, An International Journal, 22,: 347-360, 1994.
5. Tsuchida E. Stabilized Hemoglobin Vesicles. Artificial Cells, Blood Substitutes and Immobilization Biotechnology, AnInternational Journal, 22: 467-479, 1994
6. Yu WP & Chang TMS Submicron Biodegradable Polymer Membrane Hemoglobin Nanocapsules as Potential Blood substitutes: A Preliminary Report. Artificial Cells, Blood Substitutes and Immobilization Biotechnology, An International Journal, 22: 889-894, 1994.
7. Rohlfs, R.J., Bruner, E., Chiu, A., Gonzales, A., Gonzales, M.L., Magde, D., M.D. Magde Jr., Vandegriff, K.D., Winslow, R.M. Arterial blood pressure response to cell-free hemoglobin solutions and the reaction with nitric oxide. J. Biol. Chem.1998;14: 351-358,
8. Sakai H., Hara H., Yuasa M., Tsai A.G., Takeoka S., Tsuchida E., Intaglietta M. Molecular dimension of Hb based O2 carriers determine constriction of resistance arteries and hypertension. A. J. Physiol Heart Circ. Physiol. 279:H908-15, 2000
9. Matheson, A. Razynska, H. Kwansa & E. Bucci. Appearance of dissociable and crosslinked hemoglobins in renal hilar lymph. J. Lab. Clin. Inv. 135:459-464 (2000)
10. Page T.C., Light W.R., Hellum J.D. Oxygen transport in 10 microns artificial capillaries. Adv. Exp. Med. Biol. 471:715-21, 1999
11. Page T.C., Light W.R., Hellum j.D. Prediction of microcirculatory oxygen transport by erythrocyte/hemoglobin solution mixtures. Microvasc. Res. 56:113-26, 1998.
12. Vandegriff, K.D and Winslow R.M. A theoretical analysis of oxygen transport; a new strategy for the design of hemoglobin-based red cell substitutes. Blood Substitute, Physiological Basis of Efficacy. (Winslow, R.M., Vandrgriff, K.D., and Intaglietta, M ed) Blood Substitutes. (1995) Birkhauser, Boston, MA.
13. Sloan E.P., Koenigsberg D., Gens M., Cipolle J., Runge J., M.N.R.G.Jr Mallory. Diaspirin crosslinked hemoglobin (DCLB) in the treatment of severe traumatic hemorrhagic shock.. J. Am. Med. Ass. 282:1857-64, 1999
14. Asano Y, Koehler RC, Ulatowski JA, Traystman RJ, Bucci E Effect of cross-linked hemoglobin transfusion on endothelial-dependent dilation in cat pial arterioles. Am J Physiol. 275:H1313-21 (1998).
15. Ulatowski JA, Asano Y, Koehler RC, Traystman RJ, Bucci E Sustained endothelial dependent dilation in pial arterioles after crosslinked hemoglobin transfusion.Artif Cells Blood Substit Immobil Biotechnol 25:115-20(1997);
16. Ulatowski JA, Bucci E, Razynska A, Traystman RJ, Koehler RCCerebral blood flow during hypoxic hypoxia with plasma-based hemoglobin at reduced hematocrit. Am J Physiol 274: H1933-42 (1998) .
17. Razynska A., and E.Bucci Zero-link Polymerization: a New Class of Polymeric Hemoglobins, Blood Substitutes, Present and Future Perspectives. Pg 265-279, Elsevier Science S.A. 1998, (1999)
EDITORIAL:IS THERE A NEED FOR BLOOD SUBSTITUTES IN THE NEW MILLENIUM, AND WHAT SHOULD WE EXPECT IN THE WAY OF SAFETY AND EFFICACY?A DEVELOPMENT PERSPECTIVE Carleton J C Hsia, Ph.D., President & CEO, SynZyme Technologies LLC, One Technology Drive, Suite E-309, Irvine, CA 92618 E-Mail: firstname.lastname@example.org in Artificial Cells, Blood Substitutes & Immobilization Biotechnology An international journal (for volume 29, 3rd issue, 2001)
One or more 1st generation hemoglobin (Hb) based blood substitute products with adequate safety and efficacy profile may soon be approved for use in surgery. However there are unsettling issues related to nitric oxide (NO) physiology and lack of anti-oxidant properties among these 1st generation products (1,2). From my perspective of developing a 1st generation and a 2nd generation blood substitute (3,4), I believe that we should continue to strive in the new millennium, for a product that outperforms whole blood, with respect of reduction of mortality and morbidity, in resuscitation scenarios where a blood substitute is critically needed. In these applications, an effective blood substitute needs antioxidant and anti-inflammatory properties to remedy the tissue ischemia, reperfusion and inflammatory injures that are common, for example, in severehemorrhagic shock and ischemic stroke.
A successful 2nd generation blood substitute should manifest the following properties:
1. Anti-oxidant activities: The 2nd
generation blood substitute needs to address the over-production of both superoxide (O2->) and hydrogen peroxide (H2O2)
during reperfusion of ischemic tissues. This
requirement has been met by either preparing an Hb co-polymer which incorporates both red
cell superoxide dismutase and catalase (5), or by the synthesis of a polynitroxylated
Hb-based blood substitute with the antioxidant enzyme-mimetic activities (6). The 2nd
generation blood substitute needs the capacity to remove the toxic oxylferryl ( FeIV=0
) intermediate (7). Free and covalently bound nitroxides in polynitroxyl Hb facilitate the
return of FeIV=0 to FeIII
with the release of oxygen; thus providing a catalase-mimetic activity (8,9). However,
even when resuscitation can be initiated within the 20 minute post-hemorrhagic shock
window, free radical injury can still occur from the over-production of peroxynitrite
(ONOO-). The 2nd
generation blood substitute needs to have the necessary antioxidant activity, to detoxify
ONOO- (10). In addition, the formation of the most injurious hydroxyl radical
(OH >) from H2O2 requires
catalysis by transition metal ions. Nitroxides prevent the redox cycling of these metal
2. Anti-inflammatory activities: Within the golden hour, free radical mediated injury can be reduced if treatment is initiated within 20 minutes of hemorrhagic shock. As the interval between the injury and treatment increases the shock becomes more refractory as resuscitation with shed blood 45 minutes post shock could not reduce mortality (12). An anti-inflammatory activity, derived in part from anti-oxidant activities, can be demonstrated in a 2nd generation blood substitute by its capability for in vivo inhibition of leukocyte/platelet aggregation and adhesion to microvessels (13). It is possible that a 2nd generation blood substitute with characteristics that include an inhibition of leukocyte/endothelium adhesion and a low P50 will facilitate capillary circulation and thereby reduce the severity of ischemia/reperfusion and inflammatory injury(14).
3. Vasodilation: A 2nd generation blood substitute can accomplish a beneficial reduction in Hb-associated vasoconstriction by incorporating a recombinant Hb molecule that is designed to reduce extravasation (e.g. recombinant Hb-dimer ) and to reduce intrinsic nitrosylation activity (heme reactivity) (15). Limiting Hb-mediated nitrosylation in the vascular space has enabled three 1st generation blood substitutes to advance to phase III clinical trials in controlled surgical patients (16). An alternative method to reduce Hb-associated vasoconstriction is to covalently attach NO analogues to Hb, such as b93 S-nitroso Hb (17) and polynitroxyl Hb (4). In a rat hemorrhagic shock model, resuscitation with polynitroxyl Hb compared with aaHb, did not produce pressor response, as a result, improved systemic hemodynamics, regional circulation, base deficit and survival time (18).
4. Oxygen delivery: The 2nd generation blood substitute, because its intrinsic short plasma half life, should complement, instead of replace red cells in the delivery of O2. This is best accomplished with a lower than physiological P50 for oxygen delivery to the hypoxic tissue(19). Dextran Hb with a P50=3~4 mmHg was shown be able to meet the resting O2 demand at red cells levels that was as low as 2% hematocrit or lower in the dog (20).
A number of 2nd generation blood substitutes have the potential to meet some or most of the above requirements. Their safety, cost and effectiveness to allogeneic blood transfusion will ultimately determine their viability as a 2nd generation blood substitute.
1. Chang TMS (2000) Editorial: Is there a need for blood substitutes in the new millennium and what can we expect in way of safety and efficacy? Art Cells Blood Sub Immob Biotech 28:i-vii.
2. Alayash AI (2000) Editorial: Is there a need for blood substitutes in the new millennium and what can we expect in way of safety and efficacy? From a research perspective. Artif. Cells, Blood Sub. Immob. Biotech 29 (1).
3. Hsia JC, (1989) US Patent No. 4,857,626, Pasteurizable, freeze-driable hemoglobin-based blood substitute.
4. Hsia JC, (1997) US Patent No. 5,591,710, Compositions and methods using nitroxides to avoid oxygen toxicity, particularly in stabilized, polymerized, conjugated or encapsulated hemoglobin used as a red cell substitute
5. D'Agnillo F and Chang TM (1998) Polyhemoglobin-superoxide dismutase-catalase as a blood substitute with antioxidant properties. Nat Biotechnol 16(7):667-71
6. Okayama N, Park JH, Coe L, Granger DN, Ma L, Hsia CJ and Alexander JS (1999) Polynitroxyl aa-hemoglobin (PNH) inhibits peroxide and superoxide-mediated neutrophil adherence to human endothelial cells. Free Radic Res 31(1):53-8.
7. McLeod LL, Alayash AI, (1999) Detection of a ferrylhemoglobin intermediate in an endothelial cell model after hypoxia-reoxygenation. Am J Physiol 277(1 Pt 2):H92-9
8. Krishna MC, Samuni A, Taira J,
Goldstein S, Mitchell JB and Russo A (1996) Stimulation by nitroxides of catalase-like
activity of hemeproteins. Kinetics and mechanism.
J Biol Chem 271(42):26018-25
9. Krishna MC , Ma L, Samuni A, Trimble CE, Mitchell JB, and Hsia CJC (1996), Superoxide Dismutase- and Catalase-Mimetic Activity of a Polynitroxyl Hemoglobin (PNH), Artif. Cells, Blood Sub. Immob. Biotech 24, 369
10. Faivre-Fiorina B, Caron A, Labrude P,
Vigneron C (1998) Erythrocyte, plasma and substitute hemoglobins facing physiological
oxidizing and reducing agents
Ann Biol Clin (Paris) 56(5): 545-56
11. Samuni A, Karmeli F, Moshen M and Rachmilewitz D (1999) Mechanisms underlying gastric anti-ulcerative activity of nitroxides in rats. Free Rad Res 30(2):133-40
12. Kentner R, Ma L, Behringer W, Safer P, Hsia CJC and Tisherman S (2000) Antioxidant polynitroxylated albumin (PNA) plus tempol for hemorrhagic shock(HS) in rats: improved survival with early treatment (submitted for publication)
13. Saetzler RK, Arfors KE, Tuma RF, Vasthare U, Ma L, Hsia CJ and Lehr HA (1999) Polynitroxylated hemoglobin-based oxygen carrier: inhibition of free radical-induced microcirculatory dysfunction. Free Radic Biol Med. 27(1-2):1-6
14. Patel PM, Harada H, Ma L, Trimble CE and Hsia CJC (1998) Polynitroxyl hemoglobin (PNH) reduces cerebral infraction following transient cerebral artery occlusion in the HRS rat, Shock, 9: 61
15. Eich RF, Li T, Lemon DD, Doherty DH, Curry SR, Aitken JF, Mathews AJ, Johnson KA, Smith RD, Phillips GN Jr. and Olson JS (1996) Mechanism of NO-induced oxidation of myoglobin and hemoglobin. Biochemistry 35(22):6976-83
16. Haney CR, Buehler PW and Gulati A.(2000) Purification and chemical modifications of hemoglobin in developing hemoglobin based oxygen carriers. Adv Drug Deliv Rev 28;40(3):153-69
17. Jia L, Bonaventura C, Bonaventura J, Stamler JS (1996) s-Nitrosohaemoglobin: a dynamic activity of blood involved control. Nature 380(6571):221-6
18. Buehler P, Ma L, Trimble CE, Hsia CJC and A. Gulati.(2000) Dextran conjugation of polynitroxylated aa-crosslinked hemodynamics and outcome of hemorrhaged rats. Shock, 13, 56
19. Winslow RM (2000) Blood substitutes: refocusing an elusive goal British J Heamatology 111: 1-11
20. Tam SC, Blumenstein J and Wong JT (1978) Blood replacement in dogs by dextran-hemoglobin. Can J Biochem 56(10)981-4