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Preservation of blood by freezing: A review
CRYOBIOLOGY Vol. I, No. 1, 1964
PRESERVATION
OF BLOOD
BY FREEZING:
A REVIEW*?
H. T. MERYMAN U. S. Naval Medical
Research
Institute,
Maryland
and subsequently extended to human erythrocytes? demonstrated that glycerin in concentrations between 10 and 15% permitted an excellent recovery of erythrocytes following slow freezing and thawing. This observation was actively pursued by Sloviter in the United States.l’ Exhaustive studies of the clinical merits of the technique were conducted, particularly by Chaplin and Mollison’ and by Hughes-Jones, Mollison, and Robinson” who reported excellent in viva survival of cells frozen and thawed in glycerin. From the theoretical as well as the practica1 point of view, one of the most important contributions was that of Lovelockl” who investigated the mechanism of glycerin protection, concluding that the concentration of electrolytes, particularly sodium chloride, which results from the freezing out of water was a major if not the most important cause of cell injury. In essence, Lovelock showed that cells exposed to a 0.8 M solution of sodium chloride become highly susceptible to thermal and osmotic shock and that this concentration of salt is produced when more than 80% of the water has been converted to ice, the result of freezing to below -3°C. Glycerin hydrogen bonds with water, tying up approximately 3 moles of water to each mole of glycerin and prevent’ing it from freezing. This glycerin-water complex appears to be an effective solvent for electrolyte so that the introduction of sufficient glycerin prevents the concentration of salt to a let.hal degree. In order to function as a protective agent, it is essential that the glycerin penetrate the red cell and it has been shown that bovine erythrocytes, which are relatively impervious to glycerin, are not protected by it. However, it is the penetration of the glycerin into the cell that has caused most of the difficulties surrounding its use. Although glycerin appears to penetrate red cells easily and rapidly, its removal has presented a substantial obstacle. When an erythrocyte equilibrated with a high concentration
The slowing of deleterious reactions through temperature reduction has long been recognized as an effective way of prolonging the storage of biological material. Storing whole blood at 4°C has extended its useful lifetime to 21 days. However, variations of supply and demand, particularly among the rare types, is such that even this extension in storage time is insufficient to prevent substantial loss of banked blood through outdating. Statistics gathered by the National Blood Council for the year 1956l’ indicate that of approximately 4.5 million units of blood drawn that year, only about 2.5 million were actually transfused, a large proportion presumably having been lost through outdating. an additional extension of Unfortunately, storage time through further reduction in temperature is not possible since the freezing of a red cell suspension to temperatures below -3°C results in the crystallization of sufficient water to cause virtually complete hemolysis. The first indication that this might not necessarily be an inevitable event was suggested by a report of Luyet in 1949” to the effect that a thin layer of red cells, sandwiched between two glass cover slips, frozen rapidly by immersion in liquid nitrogen and thawed rapidly in warm saline, showed the recovery of intact erythrocytes. The observation was not pursued further at the time. SLOW
Bethesda,
FREEZING
One of the most important milestones in cryobiology was the observation by the English investigators, Polge, Smith, and Parkes, of the protective effect of glycerin. Their work, originally conducted with bovine spermatozoal’ * Presented at the First Annual Meeting, Society for Cryobiology, August 24-26, 1964, Washington, D. C. t The opinions or assertions contained herein are the private ones of the author and are not to be construed as official or reflecting the view of the Navy Department or the Naval service at large. Bureau of Medicine and Surgery, Navy Department, Research Task MR 005.02-0001.07. 52
BLOOD PRESERVATION of glycerin is transferred to a glycerin-free medium, a momentary osmotic imbalance exists, since water enters the cell more rapidly than ,glycerin leaves. Many expedients have been used t,o remove g;lycerin after thawing without osmotic lysis. During the first several years of investigations, dialysis and sequential dilution were used. Both of these methods are time consuming. Chaplin and associates3 reported stepwise dilution in seven sequential cycles, requiring a period of 5 hours. Unfortunately, these laboratory procedures lent an aura of impracticality to the proposition that cells frozen in glycerin might be used for blood banking and, six years after the initial discovery of the protective effects of glycerin, interest in its pot,ential for transfusion had noticeably slackened. In the United States, in the meantime, it had been found that (sells could be glycerolized and deglycerolized in pint quantities under sterile conditions using the Cohn fractionator, which had been developed originally for the separation of plasma proteins on a continuous flow basis. Despite the expense and complexity of the apparatus and the relatively long processing time, 40 minutes to glycerolize and 60 minutes to deglgcerolize, the preparation of sterile pint units enabled for the first time extensive clinical experimentation. During the period 1954 to 1961, 2250 units of blood, frozen in glycerin and stored for varying periods of time were transfused at the Chelsea Naval Hospital in Boston.” This large mass of clinical experience, coupled with more recent studies of in vivo and in vitro cells frozen in glycerin’” fully supports the effectiveness and acceptability of this product for transfusion, regardless of the practical problems surrounding t,he post-t’haw washing procedure. In 1961, Lovelock and Bishopll reported the use of dimet.hyl sulfoxide (DMSO) as a prot,ective agent. Its outst,anding virt,ue was the fact that, unlike glycerin, it appeared to pass freely through cell membranes. DMSO appeared to give erythrocytes roughly the same protection from freezing as did glycerin in comparable concentrations, but t’he difficulties of removing the intracellular additive after thawing were greatly alleviated. Huggins” reported results comparable to those obtained with glycerin with the additional bonus of an ingenious washing technique. -4 phenomenon apparently involving the interaction of a y-globulin fraction
53
BY FREEZING
with the lipoprotein in cell membranes causes a reversible agglomeration of erythrocytes at salt concentrations lower than 0.02 M and at acid pH between 6.5 and 5.2. This agglomeration is sufficient to produce large clumps of cells which sediment rapidly in the absence of a centrifugal field. The cells redisperse on increasing the pH above 6.5. Utilizing three sedimentation and redilution sequences, Huggins’ reported reducing the DMSO concentration from over 30 g per cent to the order of 200 mg per cent, using 4 liters of wash solution. Using cells frozen with DMSO and washed by this procedure, Huggins reports normal survival following transfusion and good clinical effectiveness. Huggins has since applied this washing method to cells frozen in glycerin. The initial wash solution consists of 50% glucose which is designed to prevent osmot,ic hemolysis during t’he initial exposure of the cell to a glycerin-free medium. This is then followed by an 8% glucose, 1% fructose solution for the subsequent dilutions. He reports the washing of a unit of blood in 14 minutes using 6 liters of wash solution. The successful transfusion of over 200 units of blood frozen and thawed wibh glycerin additive and washed by cyto-agglomeration have been reported.’ Several of these transfusions have been multiple; no hemoglobinuria was observed. RAPID
FREEZING
In 1954, a report by Meryman and Kafig13 of the successful rapid freezing of bulk blood introduced a second radically different approach to blood freezing. Their technique consisted in spraying whole blood through a fine capillary onto the surface of liquid nitrogen. The lntle droplets thus formed floated for two to three seconds until they reached liquid nitrogen temperature, then sank to t,he bottom of the container. These frozen droplets were stored in liquid nitrogen and thawed by sifting into warm isotonic saline. Recoveries of the order of 97% were obtained when glucose was added to the whole blood at a final concentration of 7%. Two transfusions in humans were reported using cells tagged with CnSI. Twenty-four-hour survivals of 82 and 86% were reported. In contrast to the slow freezing technique, rapid freezing offered the hope of transfusing frozen and t,hawed cells without the necessity
54
H. T. MERYMAN
of washing them after thawing. The additive, glucose, was wholly familiar in clinical use and presumably acceptable in the concentration used. At that time there appeared to be no particular resistance to the presence of 2 to 3% hemolysis after thawing. Admittedly, at this stage the engineering obstacles were considerable. The spraying technique was clumsy and processing under sterile conditions had not been attempted. Nevertheless, in 1956 the Linde Division of Union Carbide undertook to engineer a device capable of freezing and thawing cells in pint quantities under sterile conditions. The devices constructed were bulky, complex, and expensive to operate, principally because of the need to clean and sterilize the assembly between each freezing run followed by retooling to operating temperature with considerable expenditure of liquid nitrogen:l* but, despite the engineering obstacles, the method did work. Another event of importance to the rapid freezing system was Strumia’s report’l that cells could be frozen in thin-walled, envelope-shaped, metal containers with an additive concentration of 5% glucose, 7.5% lactose, with recoveries almost as good as obtained by spraying. The appeal of a closed system which wholly avoided the complex problems of sterility with the spray method was irresistible, attention was immediately transferred to the closed container, and the prospects of achieving a practical, sterile, one-step rapid freeze process appeared bright indeed. This story might have had a considerably different ending had it not been for one critical error. The original in wivo studies reported by Meryman and Kafigl” had utilized a standard procedure by which erythrocytes were tagged with radioactive chromium prior to infusion. A 15-minute post-transfusion period was allowed for complete mixing of the infused sample, following which an initial aliquot of blood was removed and the radioactivity measured. This initial sample constituted the base line, or 100% sample, against which subsequent loss of radioactivity was compared. The validity of the method depended on the assumption that no celIs were lost between the moment of infusion and the 15-minute base line sample. Several years passed before it was fully appreciated that during this interval substantial hemolysis could and did occur for which glucose was pri-
marily responsible.” Glucose penetrates the red cell membrane rapidly to a concentration around 2.5%, then more slowly thereafter. Cells equilibrated with 7.5% glucose become hypertonic, particularly if permitted to equilibrate for periods in excess of 30 minutes. Such cells transferred suddenly to an isotonic medium such as the circulation, suffer considerable osmotic hemolysis with losses as high as 35% during the first few moments following infusion.’ Glucose was forthwith abandoned as an unusable additive and attention was transferred to other sugars notably lactose, which afforded protection during rapid freezing without penetrating the cell. When Strumia’s original envelope-shaped containers were scaled up to handle pint quantities of blood their dimensions became unwieldly and it was necessary to increase the cross section of the containers with a corresponding diminution in heat transfer rates. The reduction in freezing and thawing rates produced increased hemolysis and it was necessary to increase the additive concentration to offset this. A great deal of work was conducted with lactose-fortified blood, frozen and thawed by shaking in corrugated aluminum containers, but to maintain recovery in the upper 90 percentile range, lactose concentrations as high as 16% were needed. These investigations were climaxed by a report by Pert, Schork, and Moore” of the infusion into dogs of lactose concentrations comparable to those which a human would receive in several units of the currently contemplated product. Massive osmotic effects, particularly pulmonary, were observed whereupon lactose joined glucose in the purgatory of unacceptability. Previous investigations at the Linde Laboratories into the mechanism of additive protection had entailed the examination of a wide variety of compounds for their protective effects. One of these, the plasma expander polyvinyl-pyrrolidone (PVP), although not originally proposed as a protective additive, exhibited an unusual ability to protect cells from freezing injury. With the abandonment of the penetrating additives, (e.g., glucose) and of the nonpenetrating sugars, (e.g., lactose) PVP appeared to be the only additive capable of permitting good cell recovery in reasonable concentrations. Virtually all of the subsequent
BLOOD
PRESERVATION
efforts to develop the rapid-freezing process to a clinically acceptable level have relied on PVP as the protective additive. CONCLUSION
efforts to preserve human In summary, erythrocytes by freezing have proceeded along two separate lines : slow freezing with penetrating additives which must be removed by washing, and rapid freezing, using nonpenetrating additives hopefully present in low enough concentrations to permit transfusion without post-thaw processing. The slow freezing method has the advantages that the rates of freezing and thawing are not excessively demanding and that specialized equipment is not necessary for the purpose. Temperatures in the vicinity of -100°C or lower are advisable for very long term preservation without the loss of cells or impairment of their in viva survival. However, as HughesJones, Mollison, and Robinson” have shown, it appears possible to use storage temperatures as high as -20°C for periods of a few weeks or more without excessive loss. The concentration of glycerin currently in use ranges between 20 and 30% and it is absolutely essential that the cells be washed after thawing. The washing process alleviates the need for very high cell recoveries and low additive concentrations since both the additive and the products of hemolysis will be removed during the washing. The cells will also presumably be suspended in an isotonic medium at the end of washing so that no osmotic hemolysis should be expected on infusion. ill1 washing procedures currently in use require entering the blood container to introduce and remove wash solution. Public Health regulations limit the subsequent storage of blood after such a break in the closed system. The principal virtue of the rapid-freezing process lies in the prospect of avoiding the complex post-thaw washing procedure and entering the closed system. Since very rapid thawing is a necessary component of the rapid-freezing process, such blood should be available for transfusion within a very few minutes of its removal from storage. Specialized equipment is necessary in order to achjeve high rates of freezing and thawing. Storage temperatures for long term preservation are similar to those required for glycerinated cells, and should be below -100°C.
BY FREEZING
Xi
Exposure to temperatures above -70°C results in rapid injury and even brief breaks in storage temperature can be disastrous. The principal obstacles facing the rapidfreezing process are the limits of acceptability for additives and the products of hemolysis. Low additive concentration results in high freeze-thaw hemolysis, whereas very high recoveries are achieved only with high additive concentrations. Furthermore, excessive elevation of additive concentration disturbs the osmotic balance of the cell and results in additional infusion hemolysis. The utility of the rapid freeze process therefore depends on the concentration of additive and the total amount of hemolysis that is clinically acceptable for transfusion. This decision is also influenced by the extent to which the one-step procedure has special merit in special situations. In very recent months several modified processes have been reported. Lower concentrations of glycerin than those necessary for convent,ional slow freezing give effective protect,ion at higher rates of freezing. Pert” has reported such a process using an additive mixture of glycerin and sucrose, and Krijnen” has reported a glycerin-sorbital additive mixture, also used with rapid freezing. Both of these processes require that the cells be washed following thawing but the reduced concentration of additive reduces the time and the wash volume required. If any criticism is to be levied against the blood freezing research of the past 15 )-ears, it would be that the emphasis has largely been on! the empirical development of a process rather than on fundamental research into t,he mechanism of freezing injury and additive protection. However, in justification of this, it should be recognized that the goal has always appeared to be just around the corner and, in view of the urgency of developing frozen blood storage, its early achievement through empirical means appeared pragmatically correct. At t,he moment, from among the several methods under investigation it is probable that a usable and clinically acceptable technique can be developed. It is also apparent, however, that none of these methods approaches the ideal and that this ideal will not be attained without a greatly improved understanding of the mechanism of cell freezing through fundamental research.
H. T. MERYMAN
56 REFERENCES
1. Bloom, M. L., Rinfret, -4. P., Witebsky, E., Steinbern, H., Lawson, J., and Bow, T. M. Proceedings 8th Congress International Society Blood Transfusion, p. 443. S. Karger, Basel, 1962. 2. Chaplin, H., and Mollison, P. I,. I,ancet, 264: 215, 1953. 3. Chaplin, H., Crawford, H., Cutbush, M., and Mollison, P. L. Clin. Sci., 15: 27, 1956. 4. Haynes, L. L., Turville, W’. C., Sproul, M. T.. and Zemp, J. W. J. Trauma, 1: 5, 1962. 5. Huggins, C. E. Science, 139: 504, 1963. 6. Huggins, C. E. Transfusion, 3: 483, 1963. 7. Huggins, C. E. Paper delivered at 10th International Congress of Blood Transfusion, Stockholm, Sept. 1964. 8. Hughes-Jones, N. C., Mollison, P. L., and Robinson, M. A. Proc. Roy. Sot. (Biol.), 147: 476, 1957. 9. Krijnen, H. W., Paper delivered at 10th International Congress of Blood Transfusion, Stockholm, Sept. 1964. 10. Lovelock, J. E. Biochem. et Biophys. Acta, 11: 28,1953.
11. Lovelock, J. E., and Bishop, M. W. H. Nature (London), 183: 1394, 1959. 12. Luvet. B. J. Biodvnnmica, 6: 217, 1949. 13. M&man, H. T.,‘and Kafig, E. Proc. Sot. Exp. Biol Med., 90: 587,1955. 14. Nation’s Blood Transfusion Facilities and Serrices, Joint Blood Council, Washington, D. C., 1960. 15. Pert,, J. H. Paper delivered at 10th International Congress of Blood Transfusion, Stockholm, Sept. 1964. 16. Pert, J. H., Schork, P. K., and Moore, R. Proceedings 9th Congress International Society Blood Transfusion, S. Karger, Basel, 1963. 17. Polge, C., Smith, A. U., and Parkes, A. S. Nature (London), 164: 666,194Q. 18. Rinfret, A. P., and Doebbler, G. F. Biodynamica, 8: 181, 1969. 19. Sloviter, H. A.. Lancet, i: 823, 1951. 20. Smith, A. U. Lancet, ii: 910, 1950. 21. Strumia, M. M., Colwell, L. C., and Strumia, P. V. Science, 128: 1002, 1958. 22. Strumia, M. M. J. Lab. Clin. Med., 5G: 587, 1960. 23. Valeri, C. R., and Henderson, M. E. J. A. M. A., 188: 1125, 1964.
PRESERVATION
OF BLOOD
BY FREEZING:
A REVIEW*?
H. T. MERYMAN U. S. Naval Medical
Research
Institute,
Maryland
and subsequently extended to human erythrocytes? demonstrated that glycerin in concentrations between 10 and 15% permitted an excellent recovery of erythrocytes following slow freezing and thawing. This observation was actively pursued by Sloviter in the United States.l’ Exhaustive studies of the clinical merits of the technique were conducted, particularly by Chaplin and Mollison’ and by Hughes-Jones, Mollison, and Robinson” who reported excellent in viva survival of cells frozen and thawed in glycerin. From the theoretical as well as the practica1 point of view, one of the most important contributions was that of Lovelockl” who investigated the mechanism of glycerin protection, concluding that the concentration of electrolytes, particularly sodium chloride, which results from the freezing out of water was a major if not the most important cause of cell injury. In essence, Lovelock showed that cells exposed to a 0.8 M solution of sodium chloride become highly susceptible to thermal and osmotic shock and that this concentration of salt is produced when more than 80% of the water has been converted to ice, the result of freezing to below -3°C. Glycerin hydrogen bonds with water, tying up approximately 3 moles of water to each mole of glycerin and prevent’ing it from freezing. This glycerin-water complex appears to be an effective solvent for electrolyte so that the introduction of sufficient glycerin prevents the concentration of salt to a let.hal degree. In order to function as a protective agent, it is essential that the glycerin penetrate the red cell and it has been shown that bovine erythrocytes, which are relatively impervious to glycerin, are not protected by it. However, it is the penetration of the glycerin into the cell that has caused most of the difficulties surrounding its use. Although glycerin appears to penetrate red cells easily and rapidly, its removal has presented a substantial obstacle. When an erythrocyte equilibrated with a high concentration
The slowing of deleterious reactions through temperature reduction has long been recognized as an effective way of prolonging the storage of biological material. Storing whole blood at 4°C has extended its useful lifetime to 21 days. However, variations of supply and demand, particularly among the rare types, is such that even this extension in storage time is insufficient to prevent substantial loss of banked blood through outdating. Statistics gathered by the National Blood Council for the year 1956l’ indicate that of approximately 4.5 million units of blood drawn that year, only about 2.5 million were actually transfused, a large proportion presumably having been lost through outdating. an additional extension of Unfortunately, storage time through further reduction in temperature is not possible since the freezing of a red cell suspension to temperatures below -3°C results in the crystallization of sufficient water to cause virtually complete hemolysis. The first indication that this might not necessarily be an inevitable event was suggested by a report of Luyet in 1949” to the effect that a thin layer of red cells, sandwiched between two glass cover slips, frozen rapidly by immersion in liquid nitrogen and thawed rapidly in warm saline, showed the recovery of intact erythrocytes. The observation was not pursued further at the time. SLOW
Bethesda,
FREEZING
One of the most important milestones in cryobiology was the observation by the English investigators, Polge, Smith, and Parkes, of the protective effect of glycerin. Their work, originally conducted with bovine spermatozoal’ * Presented at the First Annual Meeting, Society for Cryobiology, August 24-26, 1964, Washington, D. C. t The opinions or assertions contained herein are the private ones of the author and are not to be construed as official or reflecting the view of the Navy Department or the Naval service at large. Bureau of Medicine and Surgery, Navy Department, Research Task MR 005.02-0001.07. 52
BLOOD PRESERVATION of glycerin is transferred to a glycerin-free medium, a momentary osmotic imbalance exists, since water enters the cell more rapidly than ,glycerin leaves. Many expedients have been used t,o remove g;lycerin after thawing without osmotic lysis. During the first several years of investigations, dialysis and sequential dilution were used. Both of these methods are time consuming. Chaplin and associates3 reported stepwise dilution in seven sequential cycles, requiring a period of 5 hours. Unfortunately, these laboratory procedures lent an aura of impracticality to the proposition that cells frozen in glycerin might be used for blood banking and, six years after the initial discovery of the protective effects of glycerin, interest in its pot,ential for transfusion had noticeably slackened. In the United States, in the meantime, it had been found that (sells could be glycerolized and deglycerolized in pint quantities under sterile conditions using the Cohn fractionator, which had been developed originally for the separation of plasma proteins on a continuous flow basis. Despite the expense and complexity of the apparatus and the relatively long processing time, 40 minutes to glycerolize and 60 minutes to deglgcerolize, the preparation of sterile pint units enabled for the first time extensive clinical experimentation. During the period 1954 to 1961, 2250 units of blood, frozen in glycerin and stored for varying periods of time were transfused at the Chelsea Naval Hospital in Boston.” This large mass of clinical experience, coupled with more recent studies of in vivo and in vitro cells frozen in glycerin’” fully supports the effectiveness and acceptability of this product for transfusion, regardless of the practical problems surrounding t,he post-t’haw washing procedure. In 1961, Lovelock and Bishopll reported the use of dimet.hyl sulfoxide (DMSO) as a prot,ective agent. Its outst,anding virt,ue was the fact that, unlike glycerin, it appeared to pass freely through cell membranes. DMSO appeared to give erythrocytes roughly the same protection from freezing as did glycerin in comparable concentrations, but t’he difficulties of removing the intracellular additive after thawing were greatly alleviated. Huggins” reported results comparable to those obtained with glycerin with the additional bonus of an ingenious washing technique. -4 phenomenon apparently involving the interaction of a y-globulin fraction
53
BY FREEZING
with the lipoprotein in cell membranes causes a reversible agglomeration of erythrocytes at salt concentrations lower than 0.02 M and at acid pH between 6.5 and 5.2. This agglomeration is sufficient to produce large clumps of cells which sediment rapidly in the absence of a centrifugal field. The cells redisperse on increasing the pH above 6.5. Utilizing three sedimentation and redilution sequences, Huggins’ reported reducing the DMSO concentration from over 30 g per cent to the order of 200 mg per cent, using 4 liters of wash solution. Using cells frozen with DMSO and washed by this procedure, Huggins reports normal survival following transfusion and good clinical effectiveness. Huggins has since applied this washing method to cells frozen in glycerin. The initial wash solution consists of 50% glucose which is designed to prevent osmot,ic hemolysis during t’he initial exposure of the cell to a glycerin-free medium. This is then followed by an 8% glucose, 1% fructose solution for the subsequent dilutions. He reports the washing of a unit of blood in 14 minutes using 6 liters of wash solution. The successful transfusion of over 200 units of blood frozen and thawed wibh glycerin additive and washed by cyto-agglomeration have been reported.’ Several of these transfusions have been multiple; no hemoglobinuria was observed. RAPID
FREEZING
In 1954, a report by Meryman and Kafig13 of the successful rapid freezing of bulk blood introduced a second radically different approach to blood freezing. Their technique consisted in spraying whole blood through a fine capillary onto the surface of liquid nitrogen. The lntle droplets thus formed floated for two to three seconds until they reached liquid nitrogen temperature, then sank to t,he bottom of the container. These frozen droplets were stored in liquid nitrogen and thawed by sifting into warm isotonic saline. Recoveries of the order of 97% were obtained when glucose was added to the whole blood at a final concentration of 7%. Two transfusions in humans were reported using cells tagged with CnSI. Twenty-four-hour survivals of 82 and 86% were reported. In contrast to the slow freezing technique, rapid freezing offered the hope of transfusing frozen and t,hawed cells without the necessity
54
H. T. MERYMAN
of washing them after thawing. The additive, glucose, was wholly familiar in clinical use and presumably acceptable in the concentration used. At that time there appeared to be no particular resistance to the presence of 2 to 3% hemolysis after thawing. Admittedly, at this stage the engineering obstacles were considerable. The spraying technique was clumsy and processing under sterile conditions had not been attempted. Nevertheless, in 1956 the Linde Division of Union Carbide undertook to engineer a device capable of freezing and thawing cells in pint quantities under sterile conditions. The devices constructed were bulky, complex, and expensive to operate, principally because of the need to clean and sterilize the assembly between each freezing run followed by retooling to operating temperature with considerable expenditure of liquid nitrogen:l* but, despite the engineering obstacles, the method did work. Another event of importance to the rapid freezing system was Strumia’s report’l that cells could be frozen in thin-walled, envelope-shaped, metal containers with an additive concentration of 5% glucose, 7.5% lactose, with recoveries almost as good as obtained by spraying. The appeal of a closed system which wholly avoided the complex problems of sterility with the spray method was irresistible, attention was immediately transferred to the closed container, and the prospects of achieving a practical, sterile, one-step rapid freeze process appeared bright indeed. This story might have had a considerably different ending had it not been for one critical error. The original in wivo studies reported by Meryman and Kafigl” had utilized a standard procedure by which erythrocytes were tagged with radioactive chromium prior to infusion. A 15-minute post-transfusion period was allowed for complete mixing of the infused sample, following which an initial aliquot of blood was removed and the radioactivity measured. This initial sample constituted the base line, or 100% sample, against which subsequent loss of radioactivity was compared. The validity of the method depended on the assumption that no celIs were lost between the moment of infusion and the 15-minute base line sample. Several years passed before it was fully appreciated that during this interval substantial hemolysis could and did occur for which glucose was pri-
marily responsible.” Glucose penetrates the red cell membrane rapidly to a concentration around 2.5%, then more slowly thereafter. Cells equilibrated with 7.5% glucose become hypertonic, particularly if permitted to equilibrate for periods in excess of 30 minutes. Such cells transferred suddenly to an isotonic medium such as the circulation, suffer considerable osmotic hemolysis with losses as high as 35% during the first few moments following infusion.’ Glucose was forthwith abandoned as an unusable additive and attention was transferred to other sugars notably lactose, which afforded protection during rapid freezing without penetrating the cell. When Strumia’s original envelope-shaped containers were scaled up to handle pint quantities of blood their dimensions became unwieldly and it was necessary to increase the cross section of the containers with a corresponding diminution in heat transfer rates. The reduction in freezing and thawing rates produced increased hemolysis and it was necessary to increase the additive concentration to offset this. A great deal of work was conducted with lactose-fortified blood, frozen and thawed by shaking in corrugated aluminum containers, but to maintain recovery in the upper 90 percentile range, lactose concentrations as high as 16% were needed. These investigations were climaxed by a report by Pert, Schork, and Moore” of the infusion into dogs of lactose concentrations comparable to those which a human would receive in several units of the currently contemplated product. Massive osmotic effects, particularly pulmonary, were observed whereupon lactose joined glucose in the purgatory of unacceptability. Previous investigations at the Linde Laboratories into the mechanism of additive protection had entailed the examination of a wide variety of compounds for their protective effects. One of these, the plasma expander polyvinyl-pyrrolidone (PVP), although not originally proposed as a protective additive, exhibited an unusual ability to protect cells from freezing injury. With the abandonment of the penetrating additives, (e.g., glucose) and of the nonpenetrating sugars, (e.g., lactose) PVP appeared to be the only additive capable of permitting good cell recovery in reasonable concentrations. Virtually all of the subsequent
BLOOD
PRESERVATION
efforts to develop the rapid-freezing process to a clinically acceptable level have relied on PVP as the protective additive. CONCLUSION
efforts to preserve human In summary, erythrocytes by freezing have proceeded along two separate lines : slow freezing with penetrating additives which must be removed by washing, and rapid freezing, using nonpenetrating additives hopefully present in low enough concentrations to permit transfusion without post-thaw processing. The slow freezing method has the advantages that the rates of freezing and thawing are not excessively demanding and that specialized equipment is not necessary for the purpose. Temperatures in the vicinity of -100°C or lower are advisable for very long term preservation without the loss of cells or impairment of their in viva survival. However, as HughesJones, Mollison, and Robinson” have shown, it appears possible to use storage temperatures as high as -20°C for periods of a few weeks or more without excessive loss. The concentration of glycerin currently in use ranges between 20 and 30% and it is absolutely essential that the cells be washed after thawing. The washing process alleviates the need for very high cell recoveries and low additive concentrations since both the additive and the products of hemolysis will be removed during the washing. The cells will also presumably be suspended in an isotonic medium at the end of washing so that no osmotic hemolysis should be expected on infusion. ill1 washing procedures currently in use require entering the blood container to introduce and remove wash solution. Public Health regulations limit the subsequent storage of blood after such a break in the closed system. The principal virtue of the rapid-freezing process lies in the prospect of avoiding the complex post-thaw washing procedure and entering the closed system. Since very rapid thawing is a necessary component of the rapid-freezing process, such blood should be available for transfusion within a very few minutes of its removal from storage. Specialized equipment is necessary in order to achjeve high rates of freezing and thawing. Storage temperatures for long term preservation are similar to those required for glycerinated cells, and should be below -100°C.
BY FREEZING
Xi
Exposure to temperatures above -70°C results in rapid injury and even brief breaks in storage temperature can be disastrous. The principal obstacles facing the rapidfreezing process are the limits of acceptability for additives and the products of hemolysis. Low additive concentration results in high freeze-thaw hemolysis, whereas very high recoveries are achieved only with high additive concentrations. Furthermore, excessive elevation of additive concentration disturbs the osmotic balance of the cell and results in additional infusion hemolysis. The utility of the rapid freeze process therefore depends on the concentration of additive and the total amount of hemolysis that is clinically acceptable for transfusion. This decision is also influenced by the extent to which the one-step procedure has special merit in special situations. In very recent months several modified processes have been reported. Lower concentrations of glycerin than those necessary for convent,ional slow freezing give effective protect,ion at higher rates of freezing. Pert” has reported such a process using an additive mixture of glycerin and sucrose, and Krijnen” has reported a glycerin-sorbital additive mixture, also used with rapid freezing. Both of these processes require that the cells be washed following thawing but the reduced concentration of additive reduces the time and the wash volume required. If any criticism is to be levied against the blood freezing research of the past 15 )-ears, it would be that the emphasis has largely been on! the empirical development of a process rather than on fundamental research into t,he mechanism of freezing injury and additive protection. However, in justification of this, it should be recognized that the goal has always appeared to be just around the corner and, in view of the urgency of developing frozen blood storage, its early achievement through empirical means appeared pragmatically correct. At t,he moment, from among the several methods under investigation it is probable that a usable and clinically acceptable technique can be developed. It is also apparent, however, that none of these methods approaches the ideal and that this ideal will not be attained without a greatly improved understanding of the mechanism of cell freezing through fundamental research.
H. T. MERYMAN
56 REFERENCES
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