- Email: [email protected]
The multifactor nature of freezing injury
ABSTRACTS, suggests an increased permeability potassium.
17th
ANNUAL
to intracellular
51. Frozen Blood (Movie, 16 mm, colored, optical, 22 min). (1980) S. Sumida (Departmentof Cardiovascular Surgery and Medical Low Temperature Unit, National Fukuoka Central Hospital Jonai 2-2, Fukuoka, 810 Japan). In this motion picture, a new centrifuge “FBS” capable of deglycerolizing 4 units of thawed glycerolized red cells serially-intermittently in 4.5 min is introduced. This movie reports the 7-year, graft survivals of patients who underwent renal transplantation from living donors. About 25% or more patients received only thawed deglycerolized red cells to improve their anemia before surgery. Their graft survivals are the highest, compared to those of patients who received whole blood or no blood. We emphasize that thawed deglycerolized red cells do not jeopardize the graft survivals, but enhance them, as the thawed deglycerolized red cells include few viable leukocytes which allosensitize patients. No post-transfusion hepatitis has been seen for the past 7 years among the patients who received thawed red cells, indicating that the deglycerolization wash minimizes the amount of hepatitis virus to the noninfectious level. Cryomicroscopic observations of the freezing-thawing process of bull sperm, human red cells, rat islets of Langerhans, onion cells, animation of machanismus of freezing injuries, and resumption of rhythmic contraction and microcirculation of - 196°C freeze-preserved rat heart for 4 years when thawed is also shown. 52. A Method for Washing High Glycerol Freeze Reserved Red Blood Cells.* M. MINCHEFF, Tz.
TSVETKOV, CH. NICKOLOFF, AND K. KARAKOSTOFF(Institute of Haematology and Blood Transfusion, Central Problem Laboratory of Freeze Drying and Cryobiology Sofia, Bulgaria). Glycerolysed packed erythrocytes with a volume of 100 ml are frozen unprogrammed in a mechanical refrigerator at -20 or -80°C. The final glycerol concentration is 4.1-4.5 M. Glycerolisation is performed by the ordinary method of Meryman and Hornblower. Thawing is performed in a water bath at +4O”C. The packed erythrocytes are diluted with SOml 1.45 M solution of NaCl in the glass bottle or PVC- blood bag used as a container for the freeze preservation. Following 2 min of equilibration 250 ml of 6% HES in 0.145 M of NaCl solution are added. The final HES concentration in the suspension is 3.75% and the erythrocytes are sedimented in about 10 min. The supernatant is discarded and following another wash with 250 ml 6% HES in 0.145 M NaCl solution (sedimentation occurs in 6-8 min) the red blood cells are ready for transfusion. The results of 10 experiments show in vitro freeze thaw wash recovery to be
611
MEETING
98.1 -c 0.41%, total recovery 96 * 0.7%, therapeutic index 91.5 + 3%. 51Crrecovery in vivo is 94.88 ? 3.03%, 51Cr t,,, 31.6 2 1.9 days. There were no changes in ATPand 2,3,DPG erythrocytes’ content before and after freeze preservation. All the packed erythrocytes were frozen in 4 hr after blood donation. The supernatant haemoglobin in the final resuspending medium is 43 + 2.3 mg. The storage temperature for all the experiments is -80°C. The length of storage is 2 months. 53. Microwave Properties of Frozen and Thawed Blood Components. E. C. BURDETTEAND V. P.
POPOVIC (Engineering Experiment Station, Georgia Institute of Technology and Department of Physiology, Emory University, Atlanta, Georgia). Microwave thawing has been identified as a potentially attractive means for rapidly warming tissues or organs from cryogenic temperatures. Microwave radiation does not produce the injurious effects which result from conduction heating alone. Further, the volume to be warmed or thawed, the thawing rate, and the uniformity are more controllable using microwave radiation. However, microwave thawing of significant blood volumes is complicated. This is due to the difficulty in determining the temperature during thawing and to wide variations in electrical properties as thawing progresses. During several years of research in our laboratories, we have determined that the thawing method which has produced the most consistent results and highest recoveries of viable cells is microwave thawing using system designs based on measured electrical properties. These properties in fact are the key to the design of effective microwave thawing systems. Electrical properties of granulocytes, platelets, and erythrocytes have been measured at 2450 MHz as a function of temperature (-80 to +3O”C). Both DMSO and glycerol have been used as cryoprotectants and, through electrical property measurements, the effects of these cryoprotectants on phase+hange temperature and broadening of the phase transition region have been studied. In all cases, the dielectric constant and conductivity increase approximately IO-fold when the phase transition from frozen to thawed state occurs. However, the absolute values of these properties, rate of phase transition, and temperature of phase transition are all a function of blood component type and cryoprotectant type and concentration. Specific electrical property measurement results were used to optimize microwave thawing system design for maximum viable cell recovery. SYMPOSIUM-FUNDAMENTAL AND CELLULAR ASPECTS OF FREEZING AND THAWING 54. The Multifactor
Nature
of Freezing Injury.
JOHN
FARRANT (Clinical Research Centre, Watford Road, Harrow, United Kingdom, England).
612
ABSTRACTS, 17th ANNUAL
The search for mechanisms of freezing injury has been lengthy. Increased electrolyte concentration, excessive cell shrinkage, and recrystallization of intracellular ice are among the possibilities that have had and still have their proponents. An advance was made when a two-factor hypothesis was put forward attempting to separate the cause of the injuries seen following slow and rapid cooling. However, the rate of cooling is clearly not a primary variable and there is now much evidence that the injury observed to cells after thawing is brought about by a succession of timeand temperature-dependent stresses applied during the cooling, warming, and recovery phases. There is the added complication that many of these stresses are not independent of each other; exposure to one may affect the susceptibility of the cells to subsequent events. The net effect is that, whether cooling is slow or rapid, it is difficult to isolate the effect of individual stresses. It would seem that the study of complete freezing and warming procedures is a more fruitful approach than that of attempting to isolate single causal factors. One recently reintroduced procedure will be reviewed in which high survival is obtained using high concentrations of cryoprotective agents and high rates of cooling and warming. Cryoprotection. ALAN P. MACKENZIE (Center for Bioengineering, University of Washington, Seattle, Washington).
55. Macromolecular
More than one investigator has attempted to explain the cryoprotective action of substances such as polyvinylpyrrolidone (PVP) and hydroxyethylstarch (HES) on the basis of a supposed binding of the polymer to the plasma membrane. Others have thought that, despite their comparatively high molecular weights, PVP and HES might still somehow serve “colligatively” to prevent the concentration of, e.g., NaCl to damaging levels. It has been believed, on the one hand, that the macromolecules might, as they concentrate, attach to the surface of the plasma membrane with beneficial effect. It has been argued, in this connection, that the same substances could, in other respects, act only in direct proportion to their contributed mole fractions and that they could not, therefore, have any significant influence on effective salt concentrations developed during freezing. It has been argued, on the other hand, that the concentrating aqueous polymer serves to “buffer” the NaCl or other nonpermeating salt, though the physical chemical means by which this might have been done has not been apparent. The question of the latter mechanism would now seem to be resolved in more than one respect by some recent freeze/thaw studies on HES. Aqueous solutions containing NaCl and HES in varying concentrations and various w/w ratios have been subjected to differential thermal analysis and to the determination of electrical resistance during freezing and thawing. First- and second-order phase transition
MEETING
temperatures have been determined by each method and it has been concluded that the thermodynamic activity of the NaCl (aNBCI) is, in some way, prevented from rising in the presence of the concentrating HES as much as it would have done in its absence. Where the w/w ratio: HES:NaCl exceeds the value 9: 1, aNaCl does not reach that of crystalline salt, even at -30°C. The Endings furnish the first direct experimental evidence that the macromolecular HES is not “inert” in a physical-chemical sense during the freezing and thawing of its aqueous saline solutions, but that it may act in a manner very similar to that of glycerol and Me,SO. The action of some of the other common macromolecular cryoprotectants might well be explained in the same general way. 56. Fundamental and Cellular Aspects of Freezing and Thawing. PETERMAZUR (Biology Division,
Oak Ridge National Laboratories, Oak Ridge, Tennessee).* and Structural Injury. H. LE B. SKAER (ARC Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom, England).
57. Freezing
The aims and methodology of freezing to preserve structural detail and freezing to maintain viability show distinct differences which demonstrate the contrasting approaches adopted by the exponents of these two arts. In the former, the concern is with fidelity to the fully active, living cell at ambient, while the latter is preoccupied with the achievement of an essentially unphysiological condition of the cell which allows it to withstand the rigours of cooling and rewarming. An exposition on the contributions that can be made by studies of the structure, cytochemistry and electrolyte composition of cells in a condition as close as possible to the in vivo ideal, will be given in an attempt to highlight areas of common interest and possible cross-fertilization between these two fields. Some consideration will also be given to the rationale underlying the methodology employed during ultrarapid cooling and in particular to recent developments concerning the achievement of vitreous or near vitreous states. The dangers of excessive rewarming of quenched specimens have recently become clear and this problem, long an area of study in the freezing of tissue for the preservation of function, is ripe for the exploration of shared knowledge and ideas. SESSION *FUNDAMENTAL AND CELLULAR ASPECTS OF FREEZING AND THAWING 58. Determination Coefficients.
of Water and Solute Permeability
L. E. M&ANN, J.-M. TURC (Department of Pathology, Blood Transfusion Ser-
* Abstract not available.
17th
ANNUAL
to intracellular
51. Frozen Blood (Movie, 16 mm, colored, optical, 22 min). (1980) S. Sumida (Departmentof Cardiovascular Surgery and Medical Low Temperature Unit, National Fukuoka Central Hospital Jonai 2-2, Fukuoka, 810 Japan). In this motion picture, a new centrifuge “FBS” capable of deglycerolizing 4 units of thawed glycerolized red cells serially-intermittently in 4.5 min is introduced. This movie reports the 7-year, graft survivals of patients who underwent renal transplantation from living donors. About 25% or more patients received only thawed deglycerolized red cells to improve their anemia before surgery. Their graft survivals are the highest, compared to those of patients who received whole blood or no blood. We emphasize that thawed deglycerolized red cells do not jeopardize the graft survivals, but enhance them, as the thawed deglycerolized red cells include few viable leukocytes which allosensitize patients. No post-transfusion hepatitis has been seen for the past 7 years among the patients who received thawed red cells, indicating that the deglycerolization wash minimizes the amount of hepatitis virus to the noninfectious level. Cryomicroscopic observations of the freezing-thawing process of bull sperm, human red cells, rat islets of Langerhans, onion cells, animation of machanismus of freezing injuries, and resumption of rhythmic contraction and microcirculation of - 196°C freeze-preserved rat heart for 4 years when thawed is also shown. 52. A Method for Washing High Glycerol Freeze Reserved Red Blood Cells.* M. MINCHEFF, Tz.
TSVETKOV, CH. NICKOLOFF, AND K. KARAKOSTOFF(Institute of Haematology and Blood Transfusion, Central Problem Laboratory of Freeze Drying and Cryobiology Sofia, Bulgaria). Glycerolysed packed erythrocytes with a volume of 100 ml are frozen unprogrammed in a mechanical refrigerator at -20 or -80°C. The final glycerol concentration is 4.1-4.5 M. Glycerolisation is performed by the ordinary method of Meryman and Hornblower. Thawing is performed in a water bath at +4O”C. The packed erythrocytes are diluted with SOml 1.45 M solution of NaCl in the glass bottle or PVC- blood bag used as a container for the freeze preservation. Following 2 min of equilibration 250 ml of 6% HES in 0.145 M of NaCl solution are added. The final HES concentration in the suspension is 3.75% and the erythrocytes are sedimented in about 10 min. The supernatant is discarded and following another wash with 250 ml 6% HES in 0.145 M NaCl solution (sedimentation occurs in 6-8 min) the red blood cells are ready for transfusion. The results of 10 experiments show in vitro freeze thaw wash recovery to be
611
MEETING
98.1 -c 0.41%, total recovery 96 * 0.7%, therapeutic index 91.5 + 3%. 51Crrecovery in vivo is 94.88 ? 3.03%, 51Cr t,,, 31.6 2 1.9 days. There were no changes in ATPand 2,3,DPG erythrocytes’ content before and after freeze preservation. All the packed erythrocytes were frozen in 4 hr after blood donation. The supernatant haemoglobin in the final resuspending medium is 43 + 2.3 mg. The storage temperature for all the experiments is -80°C. The length of storage is 2 months. 53. Microwave Properties of Frozen and Thawed Blood Components. E. C. BURDETTEAND V. P.
POPOVIC (Engineering Experiment Station, Georgia Institute of Technology and Department of Physiology, Emory University, Atlanta, Georgia). Microwave thawing has been identified as a potentially attractive means for rapidly warming tissues or organs from cryogenic temperatures. Microwave radiation does not produce the injurious effects which result from conduction heating alone. Further, the volume to be warmed or thawed, the thawing rate, and the uniformity are more controllable using microwave radiation. However, microwave thawing of significant blood volumes is complicated. This is due to the difficulty in determining the temperature during thawing and to wide variations in electrical properties as thawing progresses. During several years of research in our laboratories, we have determined that the thawing method which has produced the most consistent results and highest recoveries of viable cells is microwave thawing using system designs based on measured electrical properties. These properties in fact are the key to the design of effective microwave thawing systems. Electrical properties of granulocytes, platelets, and erythrocytes have been measured at 2450 MHz as a function of temperature (-80 to +3O”C). Both DMSO and glycerol have been used as cryoprotectants and, through electrical property measurements, the effects of these cryoprotectants on phase+hange temperature and broadening of the phase transition region have been studied. In all cases, the dielectric constant and conductivity increase approximately IO-fold when the phase transition from frozen to thawed state occurs. However, the absolute values of these properties, rate of phase transition, and temperature of phase transition are all a function of blood component type and cryoprotectant type and concentration. Specific electrical property measurement results were used to optimize microwave thawing system design for maximum viable cell recovery. SYMPOSIUM-FUNDAMENTAL AND CELLULAR ASPECTS OF FREEZING AND THAWING 54. The Multifactor
Nature
of Freezing Injury.
JOHN
FARRANT (Clinical Research Centre, Watford Road, Harrow, United Kingdom, England).
612
ABSTRACTS, 17th ANNUAL
The search for mechanisms of freezing injury has been lengthy. Increased electrolyte concentration, excessive cell shrinkage, and recrystallization of intracellular ice are among the possibilities that have had and still have their proponents. An advance was made when a two-factor hypothesis was put forward attempting to separate the cause of the injuries seen following slow and rapid cooling. However, the rate of cooling is clearly not a primary variable and there is now much evidence that the injury observed to cells after thawing is brought about by a succession of timeand temperature-dependent stresses applied during the cooling, warming, and recovery phases. There is the added complication that many of these stresses are not independent of each other; exposure to one may affect the susceptibility of the cells to subsequent events. The net effect is that, whether cooling is slow or rapid, it is difficult to isolate the effect of individual stresses. It would seem that the study of complete freezing and warming procedures is a more fruitful approach than that of attempting to isolate single causal factors. One recently reintroduced procedure will be reviewed in which high survival is obtained using high concentrations of cryoprotective agents and high rates of cooling and warming. Cryoprotection. ALAN P. MACKENZIE (Center for Bioengineering, University of Washington, Seattle, Washington).
55. Macromolecular
More than one investigator has attempted to explain the cryoprotective action of substances such as polyvinylpyrrolidone (PVP) and hydroxyethylstarch (HES) on the basis of a supposed binding of the polymer to the plasma membrane. Others have thought that, despite their comparatively high molecular weights, PVP and HES might still somehow serve “colligatively” to prevent the concentration of, e.g., NaCl to damaging levels. It has been believed, on the one hand, that the macromolecules might, as they concentrate, attach to the surface of the plasma membrane with beneficial effect. It has been argued, in this connection, that the same substances could, in other respects, act only in direct proportion to their contributed mole fractions and that they could not, therefore, have any significant influence on effective salt concentrations developed during freezing. It has been argued, on the other hand, that the concentrating aqueous polymer serves to “buffer” the NaCl or other nonpermeating salt, though the physical chemical means by which this might have been done has not been apparent. The question of the latter mechanism would now seem to be resolved in more than one respect by some recent freeze/thaw studies on HES. Aqueous solutions containing NaCl and HES in varying concentrations and various w/w ratios have been subjected to differential thermal analysis and to the determination of electrical resistance during freezing and thawing. First- and second-order phase transition
MEETING
temperatures have been determined by each method and it has been concluded that the thermodynamic activity of the NaCl (aNBCI) is, in some way, prevented from rising in the presence of the concentrating HES as much as it would have done in its absence. Where the w/w ratio: HES:NaCl exceeds the value 9: 1, aNaCl does not reach that of crystalline salt, even at -30°C. The Endings furnish the first direct experimental evidence that the macromolecular HES is not “inert” in a physical-chemical sense during the freezing and thawing of its aqueous saline solutions, but that it may act in a manner very similar to that of glycerol and Me,SO. The action of some of the other common macromolecular cryoprotectants might well be explained in the same general way. 56. Fundamental and Cellular Aspects of Freezing and Thawing. PETERMAZUR (Biology Division,
Oak Ridge National Laboratories, Oak Ridge, Tennessee).* and Structural Injury. H. LE B. SKAER (ARC Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom, England).
57. Freezing
The aims and methodology of freezing to preserve structural detail and freezing to maintain viability show distinct differences which demonstrate the contrasting approaches adopted by the exponents of these two arts. In the former, the concern is with fidelity to the fully active, living cell at ambient, while the latter is preoccupied with the achievement of an essentially unphysiological condition of the cell which allows it to withstand the rigours of cooling and rewarming. An exposition on the contributions that can be made by studies of the structure, cytochemistry and electrolyte composition of cells in a condition as close as possible to the in vivo ideal, will be given in an attempt to highlight areas of common interest and possible cross-fertilization between these two fields. Some consideration will also be given to the rationale underlying the methodology employed during ultrarapid cooling and in particular to recent developments concerning the achievement of vitreous or near vitreous states. The dangers of excessive rewarming of quenched specimens have recently become clear and this problem, long an area of study in the freezing of tissue for the preservation of function, is ripe for the exploration of shared knowledge and ideas. SESSION *FUNDAMENTAL AND CELLULAR ASPECTS OF FREEZING AND THAWING 58. Determination Coefficients.
of Water and Solute Permeability
L. E. M&ANN, J.-M. TURC (Department of Pathology, Blood Transfusion Ser-
* Abstract not available.