Recent Advances in Haematology Renu Saxena, HP Pati, VP Choudhry
INDEX
×
Chapter Notes

Save Clear

Blood Substitutes1

Ramesh Kumar Arya
Key Words Transfusion • Transfusion alternatives • Red cell substitutes • Platelet substitutes  
INTRODUCTION
The term “Blood Substitutes” was first used for plasma expanders and later for blood components. Presently, it is applied to ex vivo produced therapeutic materials with potential to replace or reduce transfusion requirement of blood and blood components.1 Such materials that on therapeutic administration can perform function of the blood component, which is meant to be replaced, are referred to as “real blood substitutes”. Those that help to reduce its transfusion requirement by enhancing in vivo production of the desired component, such as therapeutic erythropoietin, G-CSF, thrombopoietin, interleukins, DDAVP, etc. are called “virtual blood substitutes”.2 The latter also includes management guidelines that help to rationalize and thus reduce the use of blood component in a given situation.1 In future, in vitro activated stem cells and gene therapy may become major players in this field.3
 
REAL BLOOD SUBSTITUTES
Among the real blood substitutes, major development has occurred for two components: (a) red blood cell substitutes, and (b) platelet substitutes.
 
Need for Development of Blood Substitutes
The following arguments appear convincing:
  1. With rapid advances in surgical sophistication and increasingly aggressive protocols of cancer management in the recent past, the transfusion requirements of both red cells and platelets have 2 outgrown human resource. Transfusion alternatives and blood substitutes are necessary to narrow the gap between demand and supply.4
  2. Risk of transmissible infection such as HIV, HVB, CMV, malaria, and syphilis, Variant Creutzfeldt Jacob Disease, etc. with blood transfusion has occupied front line attention.5 Although, mandatory regulations for pre-transfusion screening of allogenic blood have now reduced the panic, yet no component can be declared 100 per cent safe. Moreover, there is no guarantee that some new infectious agent will not emerge.6 Blood substitutes which can be subjected to vigorous in vitro sterilization can alleviate both the risks and costs in this regard.
  3. Sudden transfusion requirements triggered by massive blood loss in accidents, natural disasters, conflicts and war cannot be entirely met with allogenic blood transfusion. Off-the-shelf available blood substitutes can be life saving.7
  4. Immunogenic risks of allogenic transfusion like incompatibility reactions; graft-versus-host reaction, allergic and febrile reactions and transfusion related lung injury, though presently much reduced, couldn’t be completely ignored. Success to enzymatically scrub or cloak red cell membranes of blood group antigens is still elusive.8 The cost of donor blood collection, transport, storage in cold, compatibility testing, processing for leucocyte depletion, irradiation and other manipulations is enormous. Blood substitutes that are devoid of immune identity markers and thus universally acceptable help to cut across all these problems.
  5. Limited shelf life of allogenic components resulting in wastage of beyond-the-expiry-date unused packs are a serious economic handicap. Blood substitutes on the other hand, show promise of much longer shelf life, even for several years when packed in lyophilized state.
  6. It is also important to realize that some people on account of their religious belief (Jehovah’s witnesses) are prohibited from taking blood from other persons.1 Blood substitutes are the only alternatives.
Efforts toward development of a desired blood substitute have , therefore, been directed to produce a product that (a) is available as non-toxic, stable, pre-packed material that can be easily stored and transported, has long shelf life and can be easily reconstituted for immediate administration (b) should withstand vigorous sterilization against viruses and bacteria (c) is completely non-immunogenic and universally acceptable without compatibility testing (d) is cost effective for mass pharmaceutical production and above all (e) should be bio-effective with efficacious performance of the functions of the blood component that it is meant to substitute.
 
DEVELOPMENT OF RED CELL SUBSTITUTES
3Although efforts have been made to develop blood substitutes for all the cellular elements, yet it is the red blood cell substitutes that have attracted most of the researches and resources. By far the largest transfusion requirement pertains to red blood cells all over the world. According to the estimates of pharmaceutical industry, nearly 12-20 billion dollar market exists for a red cell substitute.
As a result of massive research and competition in pharmaceutical industry, three categories of red cell substitutes have emerged:
  • Cell-free haemoglobin solutions
  • Encapsulated haemoglobins
  • Perfluorochemical emulsions.
 
Cell-Free Haemoglobin Solutions
The most important function of red blood cells is to carry oxygen and carbon dioxide by virtue of its haemoglobin content. Therefore, in search of an alternative to red blood cell transfusion, RBC free haemoglobin solution has rightly attracted the attention. However, the following major problems were encountered in its use and have demanded solutions:
  1. When outside the RBC, the normal tetrameric molecular configuration of haemoglobin molecule undergoes rapid fragmentation into dimeric form, which is imbued with the following untoward effects:
    1. Haemoglobin dimers are easily filtered through renal glomeruli and precipitated in proximal convoluted tubules causing serious renal damage.1,9,10 Acute renal failure as hallmark of intravascular haemolysis is well known.
    2. Dimeric fragments have increased ability to transmigrate endothelial cell barriers and act as endothelial cell toxin. Iron moiety of haemoglobin molecule provides excellent nidus for increased free oxygen radicals.9
    3. Dimeric fragments rapidly bind nitric oxide and thus result in significantly increased vasoconstriction causing hypertension and oesophageal spasm. Scavenging of nitric oxide also wipes away its protective action on endothelial cells from the damaging effect of platelet and white cell binding.11
  2. Outside the red cell milieu, free haemoglobin looses its natural buffering environment of 2,3-diphosphoglyceraldehyde (2,3-DPG). This adversely affects the binding and release of oxygen by the free haemoglobin molecule. Normally, in the oxygen dissociation curve that is depicted by sigmoid pattern, 50 per cent oxygen saturation (P50) of RBC enclosed haemoglobin is at approximately 26 torr. Once removed outside the erythrocyte, free 4 haemoglobin has much greater affinity for oxygen with the oxygen dissociation curve moving considerably to the left with P50 at around 15-16 torr,12 meaning thereby, that free haemoglobin does not release oxygen as easily as when it is inside the RBC. When free haemoglobin solution is utilized for exchange transfusion in animals, the mixed venous oxygen saturation that reflects tissue oxygenation may drop drastically, though haemoglobin itself is well saturated.12
  3. Intra-erythrocytic environment provides for the important reductive enzymes and mechanisms which combat the very oxidative nature of oxyhaemoglobin.
  4. RBCs contain catalase, superoxide dismutase and other enzymes that are significantly important in counteracting tissue injury caused by free oxygen radicals.10 It is well known that ischaemia consequent to lack of tissue oxygen supply (such as in haemorrhagic shock, stroke and other causes of inadequate circulation) leads to production of hypoxanthin. When the ischaemic tissue is re-perfused with oxygen, xanthin oxidase converts hypoxanthin to superoxide that results in formation of free oxygen radicals. Red cell enzymes, quite importantly, help to prevent this. Superoxide dismutase converts superoxide to hydrogen peroxide and catalase breaks up hydrogen peroxide to water and oxygen. Ex-RBC free haemoglobin devoid of these red cell enzymes is, therefore, incompetent to prevent “re-perfusion tissue injury”.13
The above mentioned problems in using cell-free haemoglobin as red blood cell substitute have engaged researchers for many years. To overcome these difficulties, three strategies have emerged leading to development of the following products
  1. Modified haemoglobins
    • Polymerized (cross-linked) haemoglobins (or Polyhaemoglobins)
      • i. Inter-molecularly cross-linked haemoglobins
      • ii. Intra-molecularly cross-linked haemoglobins
    • Conjugated haemoglobins
  2. Recombinant haemoglobins
  3. Encapsulated haemoglobins (artificial RBC)
  4. Perfluorochemical emulsions.
 
CROSS-LINKED HAEMOGLOBINS (MODIFIED HAEMOGLOBINS)
Taking advantage of the existence of many amino-groups (rich in lysin residues) on the surface of haemoglobin molecule, it has been possible to cross-link several haemoglobin molecules (inter-molecular cross-linkage) to create haemoglobin molecule of larger size and higher5 molecular weight. Such polymerized haemoglobins (polyhaemoglobins) show two important advantages:
  1. These do not break into dimeric fragments in circulation and are thus devoid of renal toxicity and endothelial cell damage. Nitric oxide scavenging is also eliminated which obviates the risk of hypertension and oesophageal spasm.1
  2. Change in surface configuration of polymerized haemoglobins renders them unrecognizable by the host immune system. Thus these polyhaemoglobins are non-immunogenic and universally acceptable for transfusion without cross-matching.14
 
Inter-Molecularly Cross-Linked Haemoglobins
First attempts for inter-molecular cross-linking were made with bifunctional “diacid” reagent-diaspirin,15 but later replaced by gluteraldehyde.16
Northfields Lab’s “ polyHaeme” and Biopure’s “Haemopure” utilized gluteraldehyde for cross-linking human and bovine source haemoglobin respectively. Addition of 2,3-DPG analogue–pyridoxal phosphate to cross-linked haemoglobin improved their P50.17 Other cross-linkers are being developed, some of which have the dual function of cross-linking and acting as 2,3-DPG analogues.17 One such approach presently in clinical trials involves the use of a dialdehyde prepared from oxidizing a sugar molecule to form open ring raffinose. Hemosol’s “Hemolink” utilizes this approach. “O-raffinose” polymerized haemoglobin shows good P50 without addition of 2,3-DPG.18
 
Intra-Molecularly Cross-Linked Haemoglobins
Cross-linkers mentioned above have been used for both inter-molecular and intra-molecular polymerization. Specifically, intra-molecular cross-linking of 2-beta subunits of haemoglobin molecule is brought about with a bifunctional agent, 2-nor-formopyridoxal 5-phosphate which is also a 2,3-DPG analogue.17 Another 2,3-DPG-pocket modifier, bis (3,5-dibromosalicyl) fumerate has been successfully used for intra-molecular cross-linkage of 2-alpha subunits of haemoglobin molecule. Intramolecular cross-linking has been successful in preventing dimer formation and shifts the oxygen dissociation curve to the right, thus improving P50 to facilitate oxygen release. Many other bi-functional 2,3-DPG-pocket modifiers are also in the pipeline.10
 
Conjugated Haemoglobins
Conjugated haemoglobins are obtained by cross-linking soluble haemoglobin polymers to other large inert molecule. Apart from the usual advantages of cross-linked polyhaemoglobins, conjugated haemoglobins show improved circulation time (T/2 bio-availability) after infusion.4
6 Apex biochemical’s and Ajinomoto of Japan have experimented with conjugation of polyoxyethelene to human pyridoxilated polyhaemoglobin17 and Enzon Inc. has used polyethylene glycol to conjugate bovine haemoglobin.
 
RECOMBINANT HAEMOGLOBINS
With advances in bioengineering technology, recombinant haemoglobin has been successfully produced in E. coli. 19Somatogen who have pioneered this technology claim that their recombinant haemoglobin “Optro” in which fusion of 2-alpha subunits has also been achieved, retains its tetrameric structure without breaking up into dimers, thus eliminating the renal toxicity and reducing the other toxic effects caused by dimeric fragments.20,21 Further modifications have resulted in achieving improved P50 and a second generation tailor-made recombinant haemoglobin has been created in which the receptor site for nitric oxide has also been blocked.22
 
Usage and Limitations of Modified Haemoglobins
Since modified haemoglobins produced by cross-link polymerization, conjugation and recombinant technology are resistant to fragmentation into dimers, renal toxicity and nitric oxide scavenging has been considerably controlled and eliminated. Additional linkage with 2,3-DPG pocket modifiers to cross-linked haemoglobins has improved their P50 for oxygen delivery. Careful filtration to completely remove all cell membrane fragments and stromal elements has almost completely eliminated the dangers of complement activation and endothelial cell micro-vascular toxicity. The risk of re-perfusion tissue injury that had remained unsolved with first generation modified haemoglobins has also been diminished by successful addition of superoxide dismutase and catalase enzymes to cross-linked haemoglobins.23,24
All modified haemoglobins can be subjected to rigorous sterilization against transmissible infections. These can be conveniently stored and transported. Devoid of immunogenicity, the modified haemoglobins can be used as universal therapeutic agents without risks of incompatibility. These can be easily administered by paramedicals, like any other pharmaceutical intravenous fluid.
Therefore as oxygen carriers, modified haemoglobins have come a long way to be accepted for human use. Clinical trials (see update below) for some of these products are in phase III, with applications already with Regulatory State Health Authorities for approval.
However, a serious limitation that has, so far, defined all efforts is their short bio-availability (25-30 hours) after infusion.25 Compared with RBC-enclosed haemoglobin that circulates for nearly 3 months, bioavailability of modified haemoglobins for 30 +/– hours is only meager. Therefore, it is suitable only for short-term or emergency blood 7 replacement or as bridge transfusion until compatible allogenic blood becomes available. Some of these conditions, in which modified haemoglobins have found effective application include cardio-pulmonary bypass surgery, haemorrhagic shock, and emergency resuscitation in natural disasters, accidents and conflicts/war casualties.7,25 Medical uses include cardioplegia, balloon angioplasty, thrombosis and embolism to improve distal tissue oxygenation where RBC cannot reach. In addition, modified haemoglobins have been found to be beneficial in normo-volumic exchange transfusion and in auto-transfusion.4
 
Clinical Trials Update of Modified Haemoglobins
Several clinical trials with modified haemoglobins as red cell substitutes have been conducted. The results reported until August 2001 have been reviewed by Chang26 and are summarized here.
  1. Northfield Laboratories, in 1998 reported the results of their prospective randomized trial comparing therapeutic benefit of gluteraldehyde cross-linked human haemoglobin “Polyhaeme” with allogenic red cell transfusion in the treatment of haemorrhagic shock.27 In 1999, they reported a randomized trial of polyhaeme in acute trauma and in emergency surgery. Polyhaeme was shown to maintain satisfactory total haemoglobin concentration and helped to reduce the need for donor blood transfusion to the extent of nearly fifty per cent. Now well-into Phase III clinical trial, they have reported no side-effects after having infused 10,000 ml of their product. FDA approval is awaited. (www.northfieldlabs.com/polyhaeme.htm).
  2. Biopure reported in 1999 that their gluteraldehyde cross-linked bovine polyhaemoglobin “haemopure” has been infused in large amounts and repeatedly. It has shown no side effects with infusion amounts up to 10,000 ml in human subjects. In a single blind multi-centric study in the year 2000, involving 72 patients requiring aortic surgery, their product HBOC-201 could help to reduce allogenic blood transfusion requirements significantly.28 It has been accorded FDA approval for routine use in veterinary medicine for treatment of canine anaemia.29 For human use, approval has been given in South Africa selectively for surgical patients with acute anaemia. In June 2000, Biopure have reported having safely and successfully used their bovine polyhaemoglobin in a patient with auto-immune haemolytic anaemia.30 Recently, (August 2001) they have described findings about its safety and efficacy in pivotal phase III clinical trials (http://www.biopure.com)
  3. Hemosol’s o-raffinose cross-linked polyhaemoglobin “hemolink” has completed phase III clinical trials in coronary artery bypass 8surgery (CABG) in Canada and UK and is awaiting regulatory approval for its routine use. Their recent analysis has confirmed that it was safe in cardiac surgery patients and that no clinically significant side effects were observed.31 Hemosol have also completed their phase II clinical trials for using “hemolink” in orthopaedic surgery and acute anaemia.26
  4. Enzon have reported on their phase II clinical trials with polyethyleneglycol (PEG) conjugated bovine haemoglobin for sensitizing tumours to chemotherapy in cancer patients.25
  5. Apex Bioscience (Ajinomoto) is currently involved in clinical trials using polyethylene human pyridoxolated (PHP) conjugated haemoglobin for scavenging nitric oxide in septic shock and a few other applications.18
  6. Baxter had produced intra-molecularly alpha-cross-linked human haemoglobin “Hemassist” by using bis- (dibromosalicyl) fumerate and were vigorously persuing FDA approval in 1998, when they suddenly discontinued due to safety concerns. Later, joined by Somatogen they have now been involved in developing recombinant haemoglobin, with high P50. They have also succeeded in creating a second generation recombinant haemoglobin in which the receptor for nitric oxide has been successfully blocked, thereby, counteracting vaso-constrictive effects when infused into animals.22
 
ENCAPSULATED HAEMOGLOBINS (ARTIFICIAL RBC)
First attempt to encapsulate free haemoglobin with artificial membranes mimicking RBC was reported in 1957.32 The membrane enclosed haemoglobin exists in tetrameric state and exhibits oxygen dissociation properties similar to natural RBC-contained haemoglobin. It remains protected from breakdown into dimeric fragments.32 Also, 2,3-DPG could later be added into the membrane enclosure, in addition to other enzymes like carbonic anhydrase and catalase. The encapsulated catalase could be successfully used for antioxidant effect against hydrogen peroxide in experimental animals.33 These contents could be membrane enclosed in 1-5 μm diameter spherules mimicking red blood cells. But the major problem encountered was their rapid removal from circulation by reticulo-endothelial cells allowing circulation time of only a few hours. An observation that removal of sialic acid from the enclosing membrane material further enhanced their disappearance from circulation proved to be of significant value. Preparations of artificial cells with modification of surface properties including addition of sialic acid rich polysaccharides and negative surface charge has helped to improve circulation time. Yet, it was not enough for practical application. Further attempts to enhance circulation time saw the development of smaller 0.2 μm diameter lipid membrane enclosed 9 spherules. Several workers have since carried out extensive research and have developed bilayer lipid membrane material including addition of sialic acid analogues and appropriate modification of surface charge.34 This has resulted in improving the circulation time of such artificial RBC to more than 24 hours.34
Extensive further work is on-going. For example:
  1. Rapid progress has been made to solve the problem of methaemoglobin by using artificial reductive systems and by addition of methaemoglobin reductase enzyme to the haemoglobin containing lipid spherules.33
  2. Studies for interaction with human complement by using in vitro screening methods have almost been completed.26
  3. Lipid human haemoglobin vesicles have successfully been used in treatment of massive haemorrhagic shock in experimental animals.35
  4. No adverse changes have been observed in histology of brain, heart, kidneys and lungs in experimental animals, even on repeated infusions.35
  5. Large scale commercial production appears quite feasible and it is likely that clinical testing will be carried out soon.34
Along with development of bilayer lipid membrane material, efforts have also succeeded to develop biocompatible and biodegradable polymer membrane material to prepare liposome enclosed haemoglobin. The biodegradable polymers like polyactide and polyglycolide provide greater strength and more porosity to the enclosing membranes, thus making the spherules permeable for glucose entry from outside plasma and for exit of deleterious metabolic products from inside the microspherules.36 Lately, by using nano-technology, it has now been possible to create nano-capsules of 80-200 nanometres in diameter with biodegradable membrane material to function as artificial RBC.37 Further, it has been possible to increase the content of haemoglobin in these nano-capsules. Superoxide dismutase and catalase can also be added in addition to methaemoglobin reductase system with promising results of conversion of methaemoglobin to haemoglobin in in vitro studies.37 Such nano-capsules have been successfully infused into animals to the extent of one-third of their blood volume without any deleterious effect.37 Attempts to increase their circulation time are in active pursuit.
 
PERFLUOROCHEMICALS
Perflurochemicals (PFC) are fluorinated (hallogenated) hydrocarbons. The very concept of their use as red cell substitutes is based on the fact that these chemicals have exceptionally high solubility for nonpolar gasses, and are one of the most stable compounds.38 Another unique property of these compounds relevant to their oxygen carrying ability is that10 they dissolve and release oxygen (and also carbon dioxide) along simple partial pressure gradient for diffusion.39
 
Physico-Chemical Properties of Perfluorochemicals39,40
To fully understand the importance of perfluorochemicals as red blood cell substitutes it is important to describe their relevant physico-chemical and bio-effective properties. This is followed by an account of their development as oxygen carrying blood substitutes for human use.
  1. PFCs are hydrocarbons that are produced by substituting single hydrogen of each carbon atom of the benzene ring with fluoride radical in the selected hydrocarbon base.
  2. PFCs are extremely stable compounds in which non-polar gasses (such as respiratory gasses) are highly soluble. Although much of the research on these compounds has been aimed at oxygen transport, but these can also carry other gasses in solution, such as nitrogen, carbon dioxide, xenon, argon, hydrogen and helium.
  3. Oxygen solubility in PFC is dependent on its equilibrium with atmospheric partial pressure of oxygen. In fact, this is the single important determinant factor for the amount of oxygen dissolved. Gasses move in and out of this liquid against partial pressure gradient for simple diffusion. Unlike the haemoglobin molecule, oxygen is not chemically bound to PFC. Instead it is simply held in solution. In pure form, PFC can carry 40-60 volumes per cent oxygen at 100 per cent partial pressure of oxygen at sea level. This reflects into PFC capacity of carrying 21 volumes per cent of oxygen in blood when it is in equilibrium with 100 per cent oxygen in the inspired air. However, if the gas pressure is increased, theoretically there is no limit to gas solubility. This fact may assume unique application in undersea medicine.
  4. Pure PFC exists as liquid oil. Because of this, the following two points are important for its biological use.
    1. Being an oil, it is completely immiscible with blood. This means that PFC cannot be administered into the bloodstream in pure form. Therefore a stable non-toxic emulsion must be created to take advantage of its immense oxygen carrying capacity. When emulsions are made, a significant amount of dilution takes place that limits its oxygen transport ability depending upon the proportion of PFC that can be driven into the emulsion
    2. Depending upon the volatility of the particular hydrocarbon chain, pure PFC may have tendency to vaporize at body temperature. Therefore, for biological use, it is important to select the parent hydrocarbon chain that is not volatile at 37°C. At the same time, it should not have such a high boiling point 11that it does not vaporize at all and thus create problems of clearance from the body.
 
Physico-Chemical Properties of PFC in Relevance to Bio-Application as Blood Substitute38-42
  1. Since it is necessary to emulsify pure PFC, search for suitable emulsifiers has been important. The earlier used emulsifying agent-Pluronic F68 used in “Flusol-DA-20 per cent” (see et seq) was found to activate complement and was hepatotoxic. Although further investigations have led to safer emulsifiers, but search is on going.
  2. A significantly important physical property of PFC is that the amount of oxygen dissolved in this liquid is chiefly determined by the atmospheric pressure of oxygen with which it is in equilibrium. Based upon this property, the following considerations are important in relevance to its bio-use as oxygen carrier.
    1. To increase the oxygen carrying capacity of PFC, it is important that the patients breathe a high inspired oxygen concentration. Therefore, the patients in haemorrhagic shock and those with poor lung perfusion may not be able to make full use of oxygen carrying capacity of PFC. On the other hand, however, in circumstances such as cardiopulmonary bypass, the membrane oxygenator might well provide the perfect way to utilize the enhanced oxygen carrying transport capabilities of PFC.
    2. Since oxygen in PFC is not chemically bound and its release is determined simply on flow across a diffusion pressure gradient, all the oxygen dissolved in PFC is potentially available for metabolic use. Similarly, other respiratory gasses such as nitrogen and carbon dioxide from tissues to the lungs can be completely removed from circulation in one pass, if there is no partial pressure of these gasses in the lungs.
    3. After being cleared from circulation by reticulo-endothelial cells, pure PFC is transiently lodged in the liver, but without any hepato-toxicity or release of hepatic enzymes. From the liver, it is slowly carried back to the lungs where it vaporizes and is exhaled as colourless and odourless vapors. There is no hepatic metabolism or renal clearance. A small proportion (nearly 10%) may also be transpired through the skin.
 
DEVELOPMENT OF PERFLUOROCHEMICALS FOR THERAPEUTIC USE
The first generation perfluorochemical was developed by Green Cross Corporation, Japan. Their product “Fluosol-DA-20 per cent” was a 20 per cent w/v emulsion of two chains of fluorocarbons (7 parts of12 perfluorodecalin and 3 parts of perfluorotripropylamine). It was emulsified in Pluronic F68, into which only 10 per cent of pure PFC could be driven, the rest of 90 per cent being emulsifier.43 Because of the low concentration of pure PFC in “Fluosol-DA-20 per cent” emulsion, its oxygen dissolving capacity was limited. It could not carry more than 1 volume per cent of oxygen. Moreover, the emulsifying agent (Pluronic F68) also showed considerable hepatotoxicity, inhibition of leucocytes and complement activation38 The total administrable dose of “Fluosol-DA-20 per cent” was, therefore, limited to less than 500 ml. Moreover, limitations of “Fluosol-DA-20 per cent” also included the need for stem emulsion to be stored in frozen state and the need to thaw and reconstitute before use; the whole exercise is taking about an hour.38
Because of these reasons, its general use as oxygen carrying blood substitute could not be supported. However, it was found effective in patients undergoing balloon angioplasty in as much as it could supply oxygen efficiently to the tissue beyond the inflated balloon catheter.44 Patients who received “Fluosol-DA-20 per cent” had fewer myocardial infarctions and had much better outcome than those who did not receive this infusion.45 FDA approval for “Fluosol-DA-20 per cent” was, thus, accorded for its use in this limited indication. The other first generation perfluorochemicals, Emulsion II and Perflubron (from China and Russia respectively), and Oxypherol (from Green Cross) were developed but could not be promoted.
The second generation perfluorocarbons such as “Oxygent” of Alliance Pharmaceuticals (58 w/v perfluorodecyl bromide) with egg yolk lecithin as surfactant46 and “Oxyfluor” of Hemogen (76% w/v perfluorodichloroctane) with sunflower triglycerides and egg yolk lecithin as emulsifier have shown greater promise. The higher concentrations of pure PFC that can be driven into these emulsions, have made it possible for these products to carry significantly larger amounts of oxygen, provided the patient breathes 100 per cent oxygen in inspired air.47 With change in emulsifier (from Pluronic F68 to egg yolk lecithin), hepato-toxicity and complement activation have nearly been eliminated when used in controlled doses. These also showed improved stability characteristics, hence longer shelf life and better bio-compatible and excretion properties.
In phase II clinical trials, “oxygent” when used in the dose of 0.9 g/Kg has been able to help avoid the need of up to 2 units of blood in surgical patients. The present emphasis is, therefore, to study the use of PFC in surgical patients with the objective of reducing the need for some amount of blood during surgery.48 Moreover, with the help of PFC infusion, surgery could be initiated at lower haematocrit, thereby reducing intra-operative blood loss.45 Its use in autologous transfusion has also been appreciated.48
13Among the other applications of second generation PFC, the following are significant:
  1. The use of PFC in lower dose levels is logical in patients with thrombosis, atherosclerosis and other causes of vascular obstruction, wherein the small particle size of emulsion has the advantage of finding its way past the area of vascular occlusion.39 This together with increased oxygen pressure in the inspired air may help to alleviate the ischaemic damage in the affected tissue beyond the occluded blood vessel.
  2. PFC enhances oxygen solubility in plasma by 25-100 folds. Oxygen cannot normally be carried in plasma since plasma is a very polar fluid. With PFC present, any oxygen molecule that is released by haemoglobin can travel down the column of PFC and follow a diffusion gradient rapidly to its target mitochondria.39
  3. Presently, air embolism is a serious problem in several surgical situations, particularly in neurosurgery. The almost universal problem of neuro-psychiatric dysfunction after cardio-pulmonary bypass is believed largely to be due to micro-air-embolism. Near elimination of air embolism has been achieved in a number of animal studies in cardio-pulmonary bypass surgery with addition of PFC to the bypass prime.39,49
  4. Several small animal studies have shown PFC to be excellent in treatment of decompression sickness. It is, though, not clear whether direct bubble absorption occurs through enhanced nitrogen solubility in PFC (nitrogen is 1000-10000 times more soluble in PFC) or actually unloading and quick perfusion of oxygen is responsible.41
  5. PFC has also been investigated for its use as adjunct to chemotherapy and radiotherapy for solid tumours. Like free haemoglobin preparations, it increases the oxygen content in the center of the tumour and thereby enhances its susceptibility to chemotherapy and radiotherapy.42
  6. By virtue of the small size of emulsion particles, PFC is also carried in lymphatics and, therefore, significant tissue oxygenation can occur through trickle flow. This should be an important aspect of its use in acute angina, stroke and transient ischaemic attacks.40
  7. The pure perfluorocarbons (without emulsifier) are being investigated for use as (i) respiratory tract infiltrates through liquid ventilation medium for both adult and paediatric respiratory distress syndrome39,46,50 (ii) as priming fluid for cardiopulmonary bypass machines39 (iii) as perfusate for isolated organs39,51 and (iv) in surgical tools in ophthalmology.52
  8. Radio-opaque and acoustic impedance properties of some of the perfluorochemicals (Perflubron) have been utilized for contrast 14enhancement for X-rays in gastro-enterology and bronchoscopy and as target specific contrast agent in imaging technology.53
Thus the promise of perfluorochemicals beyond their use as blood substitutes, in several other applications is also encouraging and exciting.
The greatest merit of perfluorochemicals is that these are synthetic agents which are accessible for mass production by chemical means without dependence upon donor blood or other biological sources. Simply, being chemical compounds, these can be subjected to intense sterilization, can be stored and transported without cold, are non-immunogenic; thus universally acceptable and can be safely infused like other pharmaceutical fluids.
However, significant restrictions for use of PFC today, concern
  1. limitation of administrable dose to restrict hepato-toxicity and complement activation
  2. retention in reticuloendothelial tissues and delayed clearance
  3. need for the patient to breathe 100 per cent oxygen in inspired air.
 
PLATELET SUBSTITUTES
 
Need to Develop Platelet Substitutes
Like in case of red blood cell substitutes, it is important to consider the reasons that have directed the efforts and research for developing alternatives to platelet transfusions. These can be summarized as follows:
  1. Demand on platelet transfusion has been mounting rapidly not only for its use in non-immune thrombocytopenia and platelet functional disorders, now diagnosed more often, but more so because, availability of platelet transfusion has allowed development of increasingly intense cancer chemotherapy protocols. Platelet transfusions have also made extracorporeal bypass surgery a safer procedure. Therefore, there is universal and ever increasing gap between demand and availability of platelet supply.
  2. Shelf life of stored platelets is too short (5 days) that restricts its easy availability off-the-shelf and makes wastage difficult to control.
  3. Since platelet concentrates are stored at room temperature, the risk of introducing infection into stored platelets is not unrealistic.
  4. Donor collection of platelets through apheresis requires special settings that are not within the means of routine health care establishments. Maintenance of donor panels and procedural management for donor apheresis for platelet collection has constantly expanded the costs.
  5. Development of anti-platelet antibodies to major histocompatibility (MHC)-class-I antigens and platelet antigens consequent to repeated platelet transfusions is an important event that keeps on making ever increasing demands on platelet supply.
15As mentioned earlier, development efforts have been directed to produce a product that (a) should be available as non-toxic, stable, pre-packed material that can be easily stored and transported, has long shelf life and can be easily reconstituted for immediate administration (b) should withstand vigorous sterilization against viruses and bacteria (c) should be completely non-immunogenic and universally acceptable (d) should be cost effective for mass pharmaceutical production and above all (e) should be bio-effective with efficacious haemostatic performance.
 
Development of Platelet Substitues
Although this chapter concerns the “real blood substitutes”, yet it is not out of order to mention that serious efforts have also been made for helping to reduce demands for platelet transfusion with implementation of rational guidelines for its use. And, research in photodynamic treatment of platelets in additive solutions with psoralens54 and ultraviolet irradiation has also helped to eliminate transfusion transmitted infections, febrile reactions and transfusion associated graft versus host rejection, thereby reducing wastage1,4 and controlling avoidable increased demands.
 
REAL PLATELET SUBSTITUTES
The following three products have shown good promise
  • Lyophilized platelets
  • Infusible platelet membranes
  • Fibrinogen coated micro-spheres (artificial platelets)
 
Lyophilized Platelets
In search for alternatives to fresh platelets for transfusion, three modifications, that could be derived from pooled out-dated platelets were developed and tried. These included:
  1. Thawable frozen platelets
  2. Sonicated platelets
  3. Freeze-dried or lyophilized platelets
Of these only the lyophilized platelets showed promise in being as effective as stored fresh platelets in tests of haemostasis in vitro and to provide effective haemostasis to thrombocytopenic animals in vivo.55,56 Additionally, these could stand intense sterilization, important to alleviate the risk of transmissible infections.
However, lyophilized platelets still remain immunogenic and can stimulate antibody response almost similar to stored fresh platelets. Clinical trials also remain to be completed.
 
Infusible Platelet Membranes
Platelet membranes that can be infused as platelet substitute are derived by fragmentation of both fresh as well as out-dated platelets, in the16 process of preparing freeze-dried preparations. These products have been shown to promote haemostasis in animal studies and in phase II clinical trials.57
Infusible platelet membranes can be subjected to intense sterilization procedures and are, therefore, safer in respect of donor transmissible infections. There is considerably reduced expression of major histocompatibility (MHC) class I antigens and therefore these are less immunogenic.57
In addition, since infusible platelet membranes can be prepared from out-dated platelets, these can be more easily accessible for mass procurement .
 
Fibrinogen Coated Microspheres
It has been possible to create microspheres of human albumin coated with human fibrinogen in an approach to produce synthetic platelet substitute (synthocytes).58 In both animal and human studies, such microspheres have been found to show remarkable haemostatic qualities.58 However, their ability to promote platelet plug formation involves interaction with recipient’s own platelets, though this can occur quite effectively even when the host platelet count is considerably low.59
No immediate toxicity has been noticed in rodents and primates, but studies about their safety and overall efficiency in human subjects are still underway.
This product can be intensely sterilized for safety against transmissible infections. Since, fibrinogen coated microspheres are independent of platelet source and are devoid of platelet or MHC class-I antigens, these are entirely non-immunogenic in terms of production of antibodies to platelet antigens.
Alternative forms of synthetic platelet substitutes involving liposomes and other carriers containing platelet receptors have also been evolved.60 These still remain in pre-clinical trial stages.
Efficiency of all the three above mentioned platelet substitutes, that is lyophilized platelets, infusible platelet membranes and fibrinogen coated microspheres still remains to be studied in prophylaxis of bleeding in severely thrombocytopenic patients. More immediate application of these products especially fibrinogen coated microspheres may be in improving haemostasis where the patient’s own platelet count is moderately reduced. Their application as adjunct to therapy where patients have become refractory to platelet transfusions through allo-immunization is also of considerable value.
 
SUMMARY
The search for “blood substitutes” as alternatives to donor derived blood component transfusion was chiefly driven by safety concerns in terms of donor transmissible viral infections and the ever increasing 17 gap between demand and supply. Ready-to-use availability of efficacious, stable, non-toxic, safe, and pharmaceutically producible products have been the goals of intense research and development. Convenient storage, easy transport, longer shelf life and more importantly universal acceptability without need for cross matching have been the development goals.
Massive efforts and resources have been expended on red cell substitutes. Three strategies have been persued. (a) modified (cross-linked, conjugated and recombinant) cell-free haemoglobin (b) encapsulated haemoglobin (c) perfluorochemical (inert respiratory gas-dissolving) emulsions. All have come a long way. There were initial concerns about ex-RBC free haemoglobin in circulation in respect to renal toxicity, vasoactivity (due to nitric oxide scavenging), inadequate oxygen dissociation (due to lack of 2,3-DPG), methaemoglobin reduction and free oxygen radical toxicity (due to absence of reduction systems) and hydrogen peroxide accumulation (due to lack of catalase and superoxide dismutase enzymes). Similarly, there were concerns about perfluorochemicals in respect of endotoxic toxicity and complement activation. Most of these problems have now been resolved. Nano-technology based haemoglobin containing nano-capsules (artificial RBC) to which most of the native RBC enzymes can be added appear immensely promising. However, very short bio-availability of all these red cell substitutes remains a serious limiting factor. Presently, their use is thus largely restricted to emergency resuscitation in post-traumatic/haemorrhagic shock, peri-surgical haemodilution, acute surgical anaemia, cardio-pulmonary bypass surgery, cardioplegia and balloon angioplasty in as much as that requirement of donor blood transfusion can be considerably reduced and that these substitutes act as bridge transfusion, until safe allogenic blood is available. Some of these products have already completed Phase III clinical trials and await regulatory approval for marketing.
Platelet substitutes include (a) lyophilized platelets (b) infusible platelet membranes and (c) fibrinogen coated micro-spheres (artificial platelets). Though these products largely meet the required quality characteristics and are shown to be efficacious in animal settings, yet their functionality depends upon the availability of at least a small number of functional host platelets. Some of these products are in phase II clinical trials, some in pre-clinical stage of development.
Apart from the above-mentioned blood substitutes to be used as replacement alternatives, several pharmaceutical products and biomaterials (like haemopoietic growth factors) have been developed, which promote in vivo production of blood cells. These along with rigorous implementation of regulatory guidelines to rationalize the use of blood components render considerable help in avoiding blood transfusion.
REFERENCES
  1. 18 Chang TMS. Blood Substitutes: Principles, methods, products and clinical trials. Vol. I. Karger-Landes system, Basel and Austin, 1998.
  1. Prowse CV. Alternatives to standard blood transfusion. Availability and promise. Transfusion Medicine 1999; 9: 287-89.
  1. Brenner MK, Heslop HE, Rooney CM. Gene and cell transfer for specific immunotherapy. Vox Sang 1998; 74: 87-90.
  1. Chang TMS. Red cell substitutes. Best practices and research. Clinical Hematol 2000; 13: 651-68.
  1. Saegusa A. UK plays safe on risks from blood products (ed). Nature 1998; 392-93.
  1. Voak D, Caffer EA, Barbara JAJ, Pollock A, Scott M, Contreras MC. Affordable safety for blood supply in developed and developing countries. Transfusion Medicine 1998; 8: 73-76.
  1. Bowersox JC, Hess JR. Trauma and Military applications of blood substitutes. Artificial cells, blood substitutes and immobilization. Biotechnology. An International Journal. 1994; 22: 145-59.
  1. Kruskall MS, AuBuchnon JP. Making Landsteiner’s discovery superfluous. Safety and economic implications of universal group (O group) red blood cell supply. Transfusion Science 1997; 18: 613-20.
  1. Fratatoni JC. Points to consider in the safety evaluation of haemoglobin oxygen carriers. Transfusion 1991; 31: 369-71.
  1. Winslow RM. Blood substitutes- a moving target. Nature Medicine 1995; 1: 1212- 15.
  1. Chang TMS. Modified hemoglobin blood substitutes. Present status and future perspectives. Biotech Annual Rev 1998; 4: 75-122.
  1. Spiess BD. Oxygen carriers. What is behind us and what is ahead? Transfusion Medicine 1999; 1: 27-33.
  1. Jill EO, Shirley LM. Haemoglobin based red cell substitutes-current status. Vox Sang 1995; 69: 302-08.
  1. Klein HG. Oxygen carriers and transfusion medicine. Artificial cells, blood substitutes and immobilization. Biotechnology. An International journal 1994; 22: 123-35.
  1. Walder JA, Zang RH, Walder RY, Steel JM, Koltz. , Diaspirin that cross-link alpha chains of haemoglobin with bis (3,5 dibromosalicyl) succinate and bis (3,5 dibromosalicyl) fumerate. Biochemistry 1979; 18: 4265-70.
  1. Payne JW. Glutalardehyde cross-linked protein to form soluble molecular weight markers. Biochem J 1973; 135: 866-73.
  1. Iwashita Y. Relationship between chemical properties of pyridoxylated hemoglobin-polyoxyethylene. Biomaterials, Artificial cells and Artificial Organs. 1992; 20: 299-308.
  1. Adamson JG, Moore C. Hemolink, an O-raffinose cross-linked haemoglobin based oxygen carrier. In: Chang TMS (ed). Blood Substitutes: Principles, Methods, Products and Clinical Trials. Karger Basel, Switzerland Vol. II 1998; 62-79.
  1. Kumar R. Recombinant haemoglobin as blood substitute; a biotechnology perspective. Proc Soc Exp Biol Med 1996; 208: 160-68.
  1. Shoemaker S, Gerber M, Evans G, Paik I, Scoggin C (Somatogen Co, USA). , Initial clinical experience with a rationally designed genetically engineered human haemoglobin. Artificial Cells, Blood Substitutes and Immobilization Biotechnology—An International Journal 1994; 22: 456-65.
  1. 19 Zuckerman SH, Doyle MP, Gorczynski R, Rosenthal GJ. Pre-clinical biology of recombinant haemoglobin rHB1. 1. Artificial Cells, Blood Substitutes and Immobilization. Biotechnology—An International Journal 1998; 26: 231-57.
  1. Lemon DD, Doharty DH, Curry SR, Mathew AJ, Doyle MP, Fattor TJ, et al. Control of nitric oxide scavenging activity of haemoglobin. Artificial Cells, Blood Substitutes and Immobilization. Biotechnology—An International Journal 1996; 24: 378-87.
  1. Quebec EA, Chang TMS. Superoxide dismutase and catalase cross-linked polyhemoglobin reduces methemoglobin formation in vitro. Artificial Cells, Blood Substitutes and Immobilization. Biotechnology—An International Journal 1995; 25: 693-706.
  1. Razak S, D’ Agnillo F, Chang TMS. Cross-linked hemoglobin-superoxide dismutase-catalase scavenges free oxygen radicals in rat model of intestinal ischemia reperfusion injury. Artificial Cells, Blood Substitutes and Immobilization Biotechnology 1997; 25: 181-294.
  1. Carmichael FJ. Recent developments in haemoglobin based oxygen carriers. An update on clinical trials. Transfus Apheresis Sci 2001; 24: 17-21.
  1. Chang TMS. Artificial Cell Biotechnology for Medical Applications (ISBP Award Lecture). Blood Purif 2000; 18: 91-96. (updated Sept. 2001-http://www.artcell.mcgill.ca/)
  1. Gould GA, Moore FA. The first randomized trial of human polymerized hemoglobin as a blood substitute in acute trauma and emergency surgery. J Am Coll Surg 1998; 187: 113-22.
  1. Jacobs EE. Use of hemoglobin based oxygen carrier (HBOC-201), When blood is not available or not acceptable. Transfusion 2000; 40:41S.
  1. LaMuraglia GM, O’Hara PJ, Baker WH, Nasblund TC, Norris EJ, Li J, Vandermeersch E. The reduction of allogenic transfusion requirement in aortic Surgery with haemoglobin based solution. J Vascular Surg 2000; 31: 299-08.
  1. Sprung J, Kindscher JD, Wahr JA, Levi JH, Monk TG, Moritz MW, et al. The use of bovine hemoglobin glutamer-250 (Hemopure) in surgical patients: Result of multi-center, randomized single blind trial. Anesth Analg 2002; 94: 799-808.
  1. Carmichael FJL. A phase I study of oxidized raffinose cross-linked human hemoglobin. Crit Care Med 2000; 28: 2283-92.
  1. Chang TMS. Hemoglobin corpuscles. Report of a research project for BSc Honours Physiology, Medical Library. McGill University, 1957, pp 1-25 (also Reprinted in J Biomaterials, Artificial Cells and Artificial Organs 1988; 16: 1-9.
  1. Rudolph AS. Encapsulated hemoglobin. Current issues and future goals. Artificial Cells, Blood Substitutes and Immobilization Biotechnology—An International Journal 1994; 22: 347-60.
  1. Chang TMS, Ponzbasky MJ. Semipermeable microcapsules containing catalase for enzyme replacement in acatalasaemia mice. Nature 1968; 218 (5138): 42-45.
  1. Tsuchida R (ed). Present and future perspectives of blood substitutes. Elsevier, Lausanne, Switzerland, 1998.
  1. Yu WP, Chang TMS. Submicron biodegradable polymer membrane nanocapsules as potential blood substitutes:Preparation and characterization. Artificial Cells, Blood Substitutes and Immobilization Biotechnology - An International Journal 1996; 24: 169-84.
  1. Chang TMS. Modified hemoglobin based blood substitutes. Cross-linked, recombinant and encapsulated hemoglobin. Vox Sang 1998; 74: 233-41.
  1. 20 Lowe KC. Perfluorochemical respiratory gas carriers. Applications in medicine and biotechnology. Science Prog 1997; 80: 169-93.
  1. Lowe KC. Perfluorinated blood substitutes and artificial oxygen carriers. Blood Reviews 1999; 13: 171-84.
  1. Riess JG. Fluorocarbon based oxygen delivery. Basic principles and product development. In Chang TMS (ed). Blood Substitutes:Principles, Methods, Products and Clinical trials. Vol II. Karger-Landes Systems. Basel, Austin 1998.
  1. Lowe KC. Fluorocarbon emulsions as blood substitutes. In Tsuchida E (ed). >Present and Future perspectives of Blood Substitutes. Lausanne-Elsevier 1998; 327-38.
  1. Riess JG, Krafft MP. Fluorinated materials for in vivo oxygen transport (blood substitutes). Diagnosis and drug delivery. Biomaterials 1998; 19: 1529-39.
  1. Mitsuno T, Ohyanagi . Present status of clinical studies of Flusol-DA (20%) in Japan. In Tremper KK. Blood Substitutes. Perfluorochemical Oxygen Transport. Little Brown and Co, Boston, 1985; 169-84.
  1. Kerins DM. Role of Perfluorocarbon-Fluosol DA in coronary angioplasty Am J Med Sci 1994; 307: 218-21.
  1. Keipert PE. Perfluorochemical emulsions. Future alternatives to transfusion. In Chang TMS (Ed). Blood Substitutes: Principles, Methods, Products and Clinical trials. Vol II. Karger-Landes Systems, Basel, Austin 1998.
  1. Riess J. Fluorocarbon based in vivo oxygen transport and delivery systems. Vox Sang 1991; 61: 225-39.
  1. Goodin TH, Grossbard EB, Kaufman XJ, Richard JJ, Kolata RJ, Allen JS, et al. A perfluorochemical emulsion for pre-hospital resuscitation of experimental hemorrhagic shock. A prospective randomized controlled study. Crit Care Med 1994; 22: 680-89.
  1. Waher JA, Tronwborst , Spence RK. A pilot study of efficiency of oxygen carrying emulsion oxygent TM in patients undergoing surgical blood loss. Anesthesiology 1994; 80: 397.
  1. Spiess BD, Cochran RP. Perfluorocarbon emulsions and cardiopulmonary bypass: A technique for future. J Thoracic Vasc Anaesth 1996; 10: 83-90.
  1. Hischl RB, Pranikoff T, Wise C. Initial experience with partial liquid ventilation in adult patients with acute respiratory distress syndrome. JAMA 1996; 275: 383-89.
  1. Geywe RP, Monrose RG, Taylor K. Survival of rats totally perfused with fluorocarbon-detergent preparation. In Norman Jc, Folkman J, Haridson WG, Rudolf LE and Veith FJ. Organ Perfusion and Preservation. Appleton Century Crofts, New York 1968; 85-96.
  1. Peyman GA, Schulman JA, Sullivan B. Perfluorocarbon liquids in Ophthalmology. Surv Ophthalmol 1995; 39: 375-95.
  1. Mattrey RF. The potential role of perflurochemicals (PFCs) in diagnostic imaging . Artificial Cells, Blood Substitutes and Immobilization Biotechnology–An International Journal 1994; 22: 295-13.
  1. Corash L. Inactivation of viruses, bacteria, protozoa and leukocytes in platelet concentrates:Current research perspectives. Transfusion. Med Review 1999; 14: 18-30.
  1. Blajchman MA, Lee DH. The thrombocytopenic rabbit bleeding time model to evaluate the in vivo hemostatic properties of platelets and platelet substitutes. Transfusion Med Review 1997; 12: 95-105.
  1. Lee DH, Blajchman MA. Novel platelet products and substitutes. Transfusion Med Review 1998; 13: 175-87.
  1. 21 Chao FC, Kim BK, Honraniech AM. Infusible platelet membrane microcapsules: A potential transfusion substitute for platelets. Transfusion 1996; 36: 536-42.
  1. Levi M, Friedrich PW, Middleton S, de Groot PG, Wu YP, Harris R, et al. Fibrinogen coated albumin microcapsules reduce bleeding in severely thrombocytopenic rabbits. Nature Med 1999; 5: 107-11.
  1. Yen R, Yobra L. World patent on protein particles for therapeutic and diagnostic use. WHO 96/3 9128.
  1. Alvig A. Potential for synthetic phospholipids as partial platelet substitutes Transfusion 1998; 38: 997-98.