Annals of the Royail College of Surgeons of England( (1976) vlc 581 ASPECTS OF TREATMENT*
Blood substitutes Witold Rudowski MD Hon.FRCS President, International Federation of Surgical Colleges Eva Kostrzewska M D Department of Surgery, Institute of Haematology, Warsaw
Summary With the development of modern methods of surgery, anaesthesia, and pre- and postoperative care the requirement for blood substitutes is continuously increasing. We present a review of the different blood substitutes which are already in clinical use or in an advanced stage of experimental investigation for possible practical administration. Our own clinical experience with dextrans and experimental studies on stroma-free haemoglobin and hydroxyethyl starch solutions are described.
Introduction Blood substitutes are fluids which, after infusion into the vascular bed, imitate the functions of blood in its haemodynamic action and in transport and exchange of oxygen. Their purpose is to ensure the adequate oxygenation and maintenance of the viability and metabolism of the cell. The recently available blood substitutes exert their action by replacing lost blood and maintaining the effective volume of circulating blood, providing adequate haemodynamic conditions and tissue perfusion. Theoretical and experimental investigations on 'ideal' blood substitutes which can transport and exchange oxygen are fairly advanced. Other functions of blood as a tissue participating in metabolic and immune processes can be replaced only to a very limited extentfor examole, the maintenance of electrolyte concentrations or buffering capacity. The requirements for blood substitutes are very wide and keep increasing owinz to many factors-for example, contraindications for transfusions of blood and blood derivatives in cases of hypovolaemia, nrsing incidence of injuries associated wTith induqstril development
and increase of traffic, as well as the extended range of indications for the use of plasma expanders in medical first aid, particularly in mass disasters. Not without significance also is the lower cost and wider availability of blood substitutes produced commercially. Blood substitutes differ in their biological action. Transport and exchange of oxygen are based on the possibility of chemical binding, using preparations of natural haemoglobin, or of physical solution of oxygen in neutral chemical compounds such as fluorocarbons, advantage being thus taken of the high solubility of gases in certain fluids. A sufficiently long maintenance of blood substitutes in the circulation is determined usually by the colloidoosmotic action of plasma expanders and their ability to bind water. Impairment of blood flow in the microcirculation after intravenous infusion of artificial blood substitutes is prevented by ensuring an adequate degree of dispersion and a similar viscosity of the infused solution to that of blood. Plasma expanders, such as dextrans, gelatin preparations, polyvinylpyrrolidone, and retently hydroxyethyl starch, have found a wide application in replacement of blood and plasma losses, in extracorporeal circulation, for perfusion of organs, and in syndromes of excessive blood concentration, but studies on agents capable of replacing blood cells are still in the experimental stage'. Apart from the potential therapeutic value of such an 'artificial blood', the importance of these studies lies in the possibility of gaining an insight into the physiology of the blood and the circulation in conditions which are as vet unattainableafter elimination of the blood cells or even of the whole blood from the animal organism.
*Fellows and Members intcrcsted in sutbmittinig articles for consideration with a view to publication in this series should first write to the Editor.
16
W'itold Rudoowski, aind Ewa Kostrzcwska
Fluorocarbon compounds Liquid fluorocarbons are neutral chemical compounds in which hydrogen ions have been replaced by fluorine. They are sufficiently good solvents for oxygen and carbon dioxide to mnaintain the respiration of animal organisms'-210 . Fluorocarbx)ns are colourless, odourless, non-inflammable fluids of high density, stable in very high temperatures, entering poorly into chemical reactions, and poorly soluble. Their boiling point is low, as are their surface tension and viscosity'1. They differ among themselves in their molecular structure, boiling point, an(l oxygen solubility, and the differences determine the variations in their biological action and in the time of their persistence in the organism2"2. For blood replacement and for systemic and organ perfusion fluorocarbons are used in the form of emuLsions: FX80, FC7,, FC,,, FC, 7 (perfluorotetrahydrofuran, perfluorotributylamine) and lately also perfluorodecaline and
perfluoromethyldecaline',5'8^'3' Fluiorocarbon alone injected intravenously causes death of the animal since it does not nmix with blood and produces pulmonary embolisnm, right ventricuilar failure, and asphyxia2"5. Sloviter et al.' showed that the pulmonarv changes result from the production of platelet and not fluorocarbon emboli. Fluorocarbon emuilsions are usually well tolerated, although at the beginning of perfusion they often cauise a fall in arterial blood pressure. The emulsifiers used include bovine albumin, lipids, and polyols for example, Plulronic F68, a mixture of polyoxypropylene and polyoxvethylene. The emulsions are produced by the use of mechanical energy or ultrasound, and selection of the right degree of dispersion is of great importance for ensuring easy flow throu,gh the capillary vascular system and for obtaining the nroper viscosity, gas diffusion capacity, and time of retention of the fluorocarbon in the circulation2. Larger particles have a better contact with the capillary walls, thus facilitating gas exchange'7, but the prom abilitv of embolism rises with increasing size of the particles. Larger particles are also much more rapidly taken up by the reticuloendothelial svstem, particularlv in the liver and spleen, where thev can be fouind many months
after their administration'9"'8. Emulsions whose particle diameter ranges from 0.2 to 5 ,um are used, tho¢se with the smallest particles, resembling colloids rather than emulsions, being regarded as the best. Their half-time in the circulation varies considerably, depending on the preparation, from about 2 days, with oxygen transport ability lasting about 20 min4, to 7 days in the case of emulsions with the smallest particles9. Pluronic F68 provides the correct colloidoosmotic pressure of the solution and maintenance of the effective volume of circulating blood2" 0"8 and improves blood flow20 by decreasing the viscosity of the preparation and by mobilizing blood cells sequestrated in the microcirculation . Its stable water coat protects the lipids and proteins in the plasma, cell membranes of erythrocytes, and platelets against denaturation during extracorporeal circulation'8'22. The molecular weight of Pluronic, used not only as an emulsifier but also as a plasma expander, is 820021. The use of emulsions makes it possible to incorporate electrolvtes into the blood substitute together with ntutrients and buffering systems"4. The final preparation usually contains 15-200/0 fluorocarbon and 2.5-1io% Pluronic, together with electrolytes, glucose, buffering systems, and agents improving blood flow, such as hvdroxyethvl starch2'22. 'Artificial blood' produced in this way, with fluorocarbon playing the role of the erythrocvtes and Plutronic, with a proper mixture of electrolytes, buffering substances, and other agents, forming the liquid phase of the emtulsion serving as plasma, has been used for perfusion of the kidneys, brain, stomach, and liver of experimental animals9" 12"1923, maintaining the functions of these organs for many minuites or even hours. After that time their functions cease gradually and structural chances appear12. The use of fluorocarbon for the partial replacement of lost blood3",5 or for complete exchange of blood in an animal5"5'22'24 has given good results in the hands of experienced investigators such as Clark, Sloviter, and Geyer, with suirvival of the animals nrovided, however, that the haematocrit could be maintained in the range of 6-7% and that a small residual voltume of plasma remained in the
Blood substitutes
circulation5. An interesting and as yet unexplained observation is the fact that the rate and efficiency of erythrocyte renewal are greater after total systemic perfusion to haematocrit value zero than after replacement of one-third of the blood volume2. The blood cells remaining after perfusion and over go9/0 of the blood cells produced during renewal are reticulocytes. The survival of animals depends mainly on the rapidity of erythrocyte renewal since plasma proteins are regenerated more effectively. Fluorocarbon should serve in its role until renewal of blood cells sets in. Because of that the animals after perfusion are kept in an atmosphere of oxygen, the concentration of which is gradually decreased"8. After the infusion of fluorocarbon emulsion histological changes are observed in the organs. The pulmonary changes include oedema, focal fibrosis, inflammation, sometimes emphysema (caused by pressure of fluorocarbon vapour), haemorrhages, and thromboembolic lesions in the pulmonary blood vessels"6. In the liver venous congestion is associated with vacuolization, necrosis, and focal degeneration'9'26. Similar changes occur in the spleen. Renal lesions take the form of haemorrhagic necrosis, acute focal necrosis of the tubules, congestion, and tubular degeneration26. These changes are not always the same and depend on the preparation used. Experimental investigations on the possibility of application of fluorocarbon emulsions indicate the need for the solution of several problems, such as fluorocarbon storage in the tisstues, the mode of its elimination from the organism (as yet no fluorinated metabolites or other substances have been found in urine and tissues), and the finding and matching of the particular fluorocarbon and surfactant which would ensure optimum conditions of diffusion in the vascular bed where gas exchange is taking place9. It has not yet been decided whether 'artificial blood' requires fluorocarbon for transport of carbon dioxide2. what is the best diameter of the particles and the best thickness and type of emulsifying layer, and what is the optimum concentration of fluorocarbon. There are still difficulties with the preparation of properly purified and nontoxic fluiorocarbon emulsions.
I I7
Stroma-free haemoglobin solutions The first experimental trials of the application of haemoglobin solutions as blood substitutes were conducted in the years I947-48 by Hamilton27. The preparations used by him were toxic and caused renal damage, haemoglobin was converted to methaemoglobin, and elements of the erythrocyte stroma, as demonstrated in later investigations, initiated intravascular clotting28"0. Fifteen years later, in I963, Rabiner and his colleagues in Chicago returned to the concept of haemoglobin utilization since large amounts of this substance are destroyed during the processing of blood for storage. It was assumed that haemoglobin would be particularly suitable for this aim because it is the natural compound for transporting and exchanging oxygen. Investigations on the preparation of stroma-free haemoglobin for intravenous infusion are now far advanced and the possibility of its practical use as a blood substitute appears much more likely3"-. In Poland investigations on stroma-free haemoglobin solutions have been conducted by Rudowski et al.84 Many investigations have been carried out on experimental animals to establish the effects of these solutions on vitally important organs, to study the problem of oxygen distribution in the organism during treatment with the solutions, and to test them as perfusion fluids for the extracorporeal storage of organs63. A haemoglobin solution is a colloidal solution with particles 6.5 X 5.5 X 5.o nm (65 X .95 X So A) in size and viscosity below that of blood5 possessing the ability to transport and exchange oxygen. The concentration of haemoglobin in the solution ranges from 3.5 to ..6 g/dl and its pH is about 7.0. Solutions of haemoglobin in saline contain no elements of erythrocyte stroma, while in Rabiner's preparations lipid remnants of the stroma are also absent31. At pH values below . and sodium chloride concentrations above I% the solutions are not stable; haemoglobin and haematin are precipitated and may give rise to side effects after infusion. Infusions of stroma-free haemoglobin solutions, even exchan0e transfusions with haematocrit falling to °3%, are well tolerated in experiments36. Fifteen minutes after the beginning of infu,sion haemoglobiniuria begins to
Witold Ruidowski anld Ewa Kostrzcwska
118 360' 8 a,
n
320 280 F
-
DL
-
n
40
240
200 160 120
80
40 0
FIG. I Free haemoglobin level in serum and urine after infusion of 500 ml of stroma-free
haemoglobin solution into healthy dogs (from Rudowski et al34). appear7 (Fig. i), indicating that the haemoglobin-binding capacity of the plasma proteins has been exceeded. In the first 2 h after infusion the plasma haemoglobin concentration falls steeply, 25% of the infused haemoglobin being excreted within 3 h, 6o% within 6 h, and another 7%/0 after i8 h36'37. After 24 h haemoglobin has disappeared from the urine of the experimental dogs and its concentration in the plasma is about 9.o ± 4.7 mg/ d134 (Fig. 2). Haemoglobin infusion restores normal oxygen consumption, improves oxygen supply to the tissues"6, and increases peripheral blood flow and oxygen transport capacity37. During perfusion of the lungs no vasoconstrictive action is observed in the lobar arteries and veins37. In vitro it has been demonstrated that haemoglobin solution can carry oxygen and maintain metabolic processes in the isolated myocardium of puppies in hypothermia"3. It has been shown that stroma-free haemoglobin solutions which decrease the blood clotting time31 have a reduced anticoagulant effect after filtration, depending on the rate of clearance of the stroma, and the
degree of prolongation of clotting time after filtration is inversely proportional to the diameter of pores in the filter used38. The mechanism of this anticoagulant activity is unknown. With the Polish preparation of stroma-free haemoglobin in investigations in vitro and after infusion of the preparation into dogs only slight abnormalities were found in the blood clotting system39. Histological examinations have failed to demonstrate nephrotoxic action or structural changes in the kidneys after administration of these preparations34'3a. Deposits of haemoglobin and change of colour were observed only in the kidneys and liver. No evidence of renal dysfunction has been demonstrated with turea and creatinine or para-aminohippurate clearance tests34'37. Infused and native haemoglobin could not be distinguished in spectrophotometric investigations. No toxic substances were found in the urine37. It seems that stroma-free haemoglobin solutions may become in future an effective blood substitute40. Their main disadvantage at present is the rapid disappearance of haemoglobin from the circtulation41. Subjects needing further inve-Itication are possible pyrogenic effects, duiration of storage, prolongation of biological life, and improvement of production efficiency.
A 8 140 ; 130 1 20 n 110 _
X100_ iz 90 _
80 _ 60 _ 50
40
-
30
-
20
-
10 _ 0O
I
' i
;I %i' .,
24h
4Oh
Tirn*
Free haemoglobin level in serum and urine after infusion of 500 ml of stroma-free haemoglobin solution into dogs after controlled haemorrhage of 500 ml (from Rudowski et al. 4).
FIG. 2
Blood substitutes
Hydroxyethyl starch A new plasma substitute is the hydroxyethylated derivative of starch (HES). It was introduiced by Wiedersheim in I95742 and prepared by Thompson et al. in I962 for clinical use4'. Preparations of HES are made from corn starch carrying the gene Wx containing the highly branched fraction amylopectin, and they are more stable in solution and show lower viscositv than common starch, which contains linear amylose and is easily decomposed in the organism. HES gives colloidal soltutions which are chemically neutral and non-toxic and. can be sterilized. The similarity of the highlv branched HES molecule to glycogen greatly increases its biological compatibility. Allergic reactions to HES are observed very
rarely' 5'44 HES has not been used extensively in clinical trials and the results of observations published aus yet are equivocal. It seems that the main problem preventing a more widespread use of the preparation in clinical practice is the fact that HES remains for a long time in the organism, and its metabolism in the organs has not yet been sufficiently elucidated. The haemodynamic effects of HES resemble those of dextran 70 preparations. and both these agents have a similar effect in improving blood flow95. HES is easier to prepare than dextran and it is also less expensive because the initial product can be produced in any desired amount. Many new paners on HES have appeared in recent years4848. In 1972 Irikura et a!. reported many investigations evaluating Hespander, a Japanese plasma exnander containing HES49-58. HES used for clinical purposes is a 6%/ solution of hvdroxyethyl starch without terato
Blood substitutes Witold Rudowski MD Hon.FRCS President, International Federation of Surgical Colleges Eva Kostrzewska M D Department of Surgery, Institute of Haematology, Warsaw
Summary With the development of modern methods of surgery, anaesthesia, and pre- and postoperative care the requirement for blood substitutes is continuously increasing. We present a review of the different blood substitutes which are already in clinical use or in an advanced stage of experimental investigation for possible practical administration. Our own clinical experience with dextrans and experimental studies on stroma-free haemoglobin and hydroxyethyl starch solutions are described.
Introduction Blood substitutes are fluids which, after infusion into the vascular bed, imitate the functions of blood in its haemodynamic action and in transport and exchange of oxygen. Their purpose is to ensure the adequate oxygenation and maintenance of the viability and metabolism of the cell. The recently available blood substitutes exert their action by replacing lost blood and maintaining the effective volume of circulating blood, providing adequate haemodynamic conditions and tissue perfusion. Theoretical and experimental investigations on 'ideal' blood substitutes which can transport and exchange oxygen are fairly advanced. Other functions of blood as a tissue participating in metabolic and immune processes can be replaced only to a very limited extentfor examole, the maintenance of electrolyte concentrations or buffering capacity. The requirements for blood substitutes are very wide and keep increasing owinz to many factors-for example, contraindications for transfusions of blood and blood derivatives in cases of hypovolaemia, nrsing incidence of injuries associated wTith induqstril development
and increase of traffic, as well as the extended range of indications for the use of plasma expanders in medical first aid, particularly in mass disasters. Not without significance also is the lower cost and wider availability of blood substitutes produced commercially. Blood substitutes differ in their biological action. Transport and exchange of oxygen are based on the possibility of chemical binding, using preparations of natural haemoglobin, or of physical solution of oxygen in neutral chemical compounds such as fluorocarbons, advantage being thus taken of the high solubility of gases in certain fluids. A sufficiently long maintenance of blood substitutes in the circulation is determined usually by the colloidoosmotic action of plasma expanders and their ability to bind water. Impairment of blood flow in the microcirculation after intravenous infusion of artificial blood substitutes is prevented by ensuring an adequate degree of dispersion and a similar viscosity of the infused solution to that of blood. Plasma expanders, such as dextrans, gelatin preparations, polyvinylpyrrolidone, and retently hydroxyethyl starch, have found a wide application in replacement of blood and plasma losses, in extracorporeal circulation, for perfusion of organs, and in syndromes of excessive blood concentration, but studies on agents capable of replacing blood cells are still in the experimental stage'. Apart from the potential therapeutic value of such an 'artificial blood', the importance of these studies lies in the possibility of gaining an insight into the physiology of the blood and the circulation in conditions which are as vet unattainableafter elimination of the blood cells or even of the whole blood from the animal organism.
*Fellows and Members intcrcsted in sutbmittinig articles for consideration with a view to publication in this series should first write to the Editor.
16
W'itold Rudoowski, aind Ewa Kostrzcwska
Fluorocarbon compounds Liquid fluorocarbons are neutral chemical compounds in which hydrogen ions have been replaced by fluorine. They are sufficiently good solvents for oxygen and carbon dioxide to mnaintain the respiration of animal organisms'-210 . Fluorocarbx)ns are colourless, odourless, non-inflammable fluids of high density, stable in very high temperatures, entering poorly into chemical reactions, and poorly soluble. Their boiling point is low, as are their surface tension and viscosity'1. They differ among themselves in their molecular structure, boiling point, an(l oxygen solubility, and the differences determine the variations in their biological action and in the time of their persistence in the organism2"2. For blood replacement and for systemic and organ perfusion fluorocarbons are used in the form of emuLsions: FX80, FC7,, FC,,, FC, 7 (perfluorotetrahydrofuran, perfluorotributylamine) and lately also perfluorodecaline and
perfluoromethyldecaline',5'8^'3' Fluiorocarbon alone injected intravenously causes death of the animal since it does not nmix with blood and produces pulmonary embolisnm, right ventricuilar failure, and asphyxia2"5. Sloviter et al.' showed that the pulmonarv changes result from the production of platelet and not fluorocarbon emboli. Fluorocarbon emuilsions are usually well tolerated, although at the beginning of perfusion they often cauise a fall in arterial blood pressure. The emulsifiers used include bovine albumin, lipids, and polyols for example, Plulronic F68, a mixture of polyoxypropylene and polyoxvethylene. The emulsions are produced by the use of mechanical energy or ultrasound, and selection of the right degree of dispersion is of great importance for ensuring easy flow throu,gh the capillary vascular system and for obtaining the nroper viscosity, gas diffusion capacity, and time of retention of the fluorocarbon in the circulation2. Larger particles have a better contact with the capillary walls, thus facilitating gas exchange'7, but the prom abilitv of embolism rises with increasing size of the particles. Larger particles are also much more rapidly taken up by the reticuloendothelial svstem, particularlv in the liver and spleen, where thev can be fouind many months
after their administration'9"'8. Emulsions whose particle diameter ranges from 0.2 to 5 ,um are used, tho¢se with the smallest particles, resembling colloids rather than emulsions, being regarded as the best. Their half-time in the circulation varies considerably, depending on the preparation, from about 2 days, with oxygen transport ability lasting about 20 min4, to 7 days in the case of emulsions with the smallest particles9. Pluronic F68 provides the correct colloidoosmotic pressure of the solution and maintenance of the effective volume of circulating blood2" 0"8 and improves blood flow20 by decreasing the viscosity of the preparation and by mobilizing blood cells sequestrated in the microcirculation . Its stable water coat protects the lipids and proteins in the plasma, cell membranes of erythrocytes, and platelets against denaturation during extracorporeal circulation'8'22. The molecular weight of Pluronic, used not only as an emulsifier but also as a plasma expander, is 820021. The use of emulsions makes it possible to incorporate electrolvtes into the blood substitute together with ntutrients and buffering systems"4. The final preparation usually contains 15-200/0 fluorocarbon and 2.5-1io% Pluronic, together with electrolytes, glucose, buffering systems, and agents improving blood flow, such as hvdroxyethvl starch2'22. 'Artificial blood' produced in this way, with fluorocarbon playing the role of the erythrocvtes and Plutronic, with a proper mixture of electrolytes, buffering substances, and other agents, forming the liquid phase of the emtulsion serving as plasma, has been used for perfusion of the kidneys, brain, stomach, and liver of experimental animals9" 12"1923, maintaining the functions of these organs for many minuites or even hours. After that time their functions cease gradually and structural chances appear12. The use of fluorocarbon for the partial replacement of lost blood3",5 or for complete exchange of blood in an animal5"5'22'24 has given good results in the hands of experienced investigators such as Clark, Sloviter, and Geyer, with suirvival of the animals nrovided, however, that the haematocrit could be maintained in the range of 6-7% and that a small residual voltume of plasma remained in the
Blood substitutes
circulation5. An interesting and as yet unexplained observation is the fact that the rate and efficiency of erythrocyte renewal are greater after total systemic perfusion to haematocrit value zero than after replacement of one-third of the blood volume2. The blood cells remaining after perfusion and over go9/0 of the blood cells produced during renewal are reticulocytes. The survival of animals depends mainly on the rapidity of erythrocyte renewal since plasma proteins are regenerated more effectively. Fluorocarbon should serve in its role until renewal of blood cells sets in. Because of that the animals after perfusion are kept in an atmosphere of oxygen, the concentration of which is gradually decreased"8. After the infusion of fluorocarbon emulsion histological changes are observed in the organs. The pulmonary changes include oedema, focal fibrosis, inflammation, sometimes emphysema (caused by pressure of fluorocarbon vapour), haemorrhages, and thromboembolic lesions in the pulmonary blood vessels"6. In the liver venous congestion is associated with vacuolization, necrosis, and focal degeneration'9'26. Similar changes occur in the spleen. Renal lesions take the form of haemorrhagic necrosis, acute focal necrosis of the tubules, congestion, and tubular degeneration26. These changes are not always the same and depend on the preparation used. Experimental investigations on the possibility of application of fluorocarbon emulsions indicate the need for the solution of several problems, such as fluorocarbon storage in the tisstues, the mode of its elimination from the organism (as yet no fluorinated metabolites or other substances have been found in urine and tissues), and the finding and matching of the particular fluorocarbon and surfactant which would ensure optimum conditions of diffusion in the vascular bed where gas exchange is taking place9. It has not yet been decided whether 'artificial blood' requires fluorocarbon for transport of carbon dioxide2. what is the best diameter of the particles and the best thickness and type of emulsifying layer, and what is the optimum concentration of fluorocarbon. There are still difficulties with the preparation of properly purified and nontoxic fluiorocarbon emulsions.
I I7
Stroma-free haemoglobin solutions The first experimental trials of the application of haemoglobin solutions as blood substitutes were conducted in the years I947-48 by Hamilton27. The preparations used by him were toxic and caused renal damage, haemoglobin was converted to methaemoglobin, and elements of the erythrocyte stroma, as demonstrated in later investigations, initiated intravascular clotting28"0. Fifteen years later, in I963, Rabiner and his colleagues in Chicago returned to the concept of haemoglobin utilization since large amounts of this substance are destroyed during the processing of blood for storage. It was assumed that haemoglobin would be particularly suitable for this aim because it is the natural compound for transporting and exchanging oxygen. Investigations on the preparation of stroma-free haemoglobin for intravenous infusion are now far advanced and the possibility of its practical use as a blood substitute appears much more likely3"-. In Poland investigations on stroma-free haemoglobin solutions have been conducted by Rudowski et al.84 Many investigations have been carried out on experimental animals to establish the effects of these solutions on vitally important organs, to study the problem of oxygen distribution in the organism during treatment with the solutions, and to test them as perfusion fluids for the extracorporeal storage of organs63. A haemoglobin solution is a colloidal solution with particles 6.5 X 5.5 X 5.o nm (65 X .95 X So A) in size and viscosity below that of blood5 possessing the ability to transport and exchange oxygen. The concentration of haemoglobin in the solution ranges from 3.5 to ..6 g/dl and its pH is about 7.0. Solutions of haemoglobin in saline contain no elements of erythrocyte stroma, while in Rabiner's preparations lipid remnants of the stroma are also absent31. At pH values below . and sodium chloride concentrations above I% the solutions are not stable; haemoglobin and haematin are precipitated and may give rise to side effects after infusion. Infusions of stroma-free haemoglobin solutions, even exchan0e transfusions with haematocrit falling to °3%, are well tolerated in experiments36. Fifteen minutes after the beginning of infu,sion haemoglobiniuria begins to
Witold Ruidowski anld Ewa Kostrzcwska
118 360' 8 a,
n
320 280 F
-
DL
-
n
40
240
200 160 120
80
40 0
FIG. I Free haemoglobin level in serum and urine after infusion of 500 ml of stroma-free
haemoglobin solution into healthy dogs (from Rudowski et al34). appear7 (Fig. i), indicating that the haemoglobin-binding capacity of the plasma proteins has been exceeded. In the first 2 h after infusion the plasma haemoglobin concentration falls steeply, 25% of the infused haemoglobin being excreted within 3 h, 6o% within 6 h, and another 7%/0 after i8 h36'37. After 24 h haemoglobin has disappeared from the urine of the experimental dogs and its concentration in the plasma is about 9.o ± 4.7 mg/ d134 (Fig. 2). Haemoglobin infusion restores normal oxygen consumption, improves oxygen supply to the tissues"6, and increases peripheral blood flow and oxygen transport capacity37. During perfusion of the lungs no vasoconstrictive action is observed in the lobar arteries and veins37. In vitro it has been demonstrated that haemoglobin solution can carry oxygen and maintain metabolic processes in the isolated myocardium of puppies in hypothermia"3. It has been shown that stroma-free haemoglobin solutions which decrease the blood clotting time31 have a reduced anticoagulant effect after filtration, depending on the rate of clearance of the stroma, and the
degree of prolongation of clotting time after filtration is inversely proportional to the diameter of pores in the filter used38. The mechanism of this anticoagulant activity is unknown. With the Polish preparation of stroma-free haemoglobin in investigations in vitro and after infusion of the preparation into dogs only slight abnormalities were found in the blood clotting system39. Histological examinations have failed to demonstrate nephrotoxic action or structural changes in the kidneys after administration of these preparations34'3a. Deposits of haemoglobin and change of colour were observed only in the kidneys and liver. No evidence of renal dysfunction has been demonstrated with turea and creatinine or para-aminohippurate clearance tests34'37. Infused and native haemoglobin could not be distinguished in spectrophotometric investigations. No toxic substances were found in the urine37. It seems that stroma-free haemoglobin solutions may become in future an effective blood substitute40. Their main disadvantage at present is the rapid disappearance of haemoglobin from the circtulation41. Subjects needing further inve-Itication are possible pyrogenic effects, duiration of storage, prolongation of biological life, and improvement of production efficiency.
A 8 140 ; 130 1 20 n 110 _
X100_ iz 90 _
80 _ 60 _ 50
40
-
30
-
20
-
10 _ 0O
I
' i
;I %i' .,
24h
4Oh
Tirn*
Free haemoglobin level in serum and urine after infusion of 500 ml of stroma-free haemoglobin solution into dogs after controlled haemorrhage of 500 ml (from Rudowski et al. 4).
FIG. 2
Blood substitutes
Hydroxyethyl starch A new plasma substitute is the hydroxyethylated derivative of starch (HES). It was introduiced by Wiedersheim in I95742 and prepared by Thompson et al. in I962 for clinical use4'. Preparations of HES are made from corn starch carrying the gene Wx containing the highly branched fraction amylopectin, and they are more stable in solution and show lower viscositv than common starch, which contains linear amylose and is easily decomposed in the organism. HES gives colloidal soltutions which are chemically neutral and non-toxic and. can be sterilized. The similarity of the highlv branched HES molecule to glycogen greatly increases its biological compatibility. Allergic reactions to HES are observed very
rarely' 5'44 HES has not been used extensively in clinical trials and the results of observations published aus yet are equivocal. It seems that the main problem preventing a more widespread use of the preparation in clinical practice is the fact that HES remains for a long time in the organism, and its metabolism in the organs has not yet been sufficiently elucidated. The haemodynamic effects of HES resemble those of dextran 70 preparations. and both these agents have a similar effect in improving blood flow95. HES is easier to prepare than dextran and it is also less expensive because the initial product can be produced in any desired amount. Many new paners on HES have appeared in recent years4848. In 1972 Irikura et a!. reported many investigations evaluating Hespander, a Japanese plasma exnander containing HES49-58. HES used for clinical purposes is a 6%/ solution of hvdroxyethyl starch without terato
