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Hemolysis of human erythrocytes in hypertonic sodium chloride solutions at subphysiological temperatures
ABSTRACTS,
13TH
NaCl concentration of 12 wt%, while for cells saline the frozen in 1.85 M glycerol in buffered LTm corresponds to 3.0 wt% NaCI. When the NaCl concentration is expressed on a molal basis, the correlation is improved. (2) There is some correlation between LT, and the fraction of water remaining unfrozen at that temperature; 50% hemolysis occurs when about 90% of the water has been converted to ice. ’ Predoctoral Investigator supported by NIH Grant No. GM01972. ‘Visiting Investigator from Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan. 3 Operated by Energy Research and Development Administration under contract with the Union Carbide Corporation. The United States Government’s right to retain a nonexclusive, royalty-free license in and to copyright covering this abstract is acknowledged. 24. Water Transport in a Cluster of Closely Packed Erythrocytes during Cooling at Subzero E. G. CRATemperatures. R. L. LEVIN, VALHO, AND C. E. HUGGINS. (Cryogenic Engineering Laboratory, Massachusetts Institute of Technology and Blood Bank and Transfusion Services, Massachusetts General Hospital, Boston, Massachusetts 02114). The ideal, hydrated, nondilute multicomponent electrolyte solution model of the red bIood cell (RBC) intracellular solution developed by Levin et al. ( 1) has been applied to the case of water transport in a cluster of closely packed erythrocytes during cooling at constant rates at subzero temperatures. The results indicate that the volume flux of water out of the interior cells of a cluster will lag behind the volume flux of water out of the exterior cells. At any given time (temperature) during a cooling process the amount of water retained within the interior cells will therefore exceed the amount of water retained within the exterior cells. Only for very slow cooling or warming rates, for which the water volume fluxes are small, will the cells of a cluster be able to shrink or swell almost uniformly. At fast cooling or warming rates, the exterior cells will shrink or swell to a much greater degree than the interior cells. Our findings also indicate that, on an overall percentage basis, the amount of water retained within a cluster of closely packed cells of a given type will exceed the amount of water retained within a single, isolated cell of the same type at any given time (temperature) during a cooling process. The probability of intracellular ice nucleation at the low temperatures for a given cooling rate will therefore be greater in a cluster of
ANNUAL
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651
closely packed cells than in a single, isolated cell. Hence, the transition cooling rate for a cluster of closely packed cells of a given type will be much lower than the transition cooling rate of a singb, isolated cell of the same type. (For RBCs our model predicts that BrcR = -5O”C/min for a 1% cell cluster whereas B TCR = -1500”C/min for a single RBC ) . Consequently, the survival signature for an aggregate of cells should peak at a cooling rate which is less than the corresponding optimal value for a single, isolated cell. The behavior has been observed by McGrath et al. (2) who found for HeLa S-3 cells that the optimal cooling rate for survival is approximately SO”C/min for single cells but approximately 8”C/min for packed cells. 1. Levin, R. L., Cravalho, E. G., and Huggins, C. E. The effect of hydration on the water content of human erythrocytes. Manuscript submitted. 2. McGrath, J. J., Cravalho, E. G., and Huggins, C. E. An experimental comparison of intracellular ice formation and freeze-thaw survival of HeLa S-3 Cells. Cryobiology 12, 520-550 ( 1975).
of the Post25. Effect of Initial Cell Concentration Thaw Hemolysis of Frozen Erythrocytes. T. NEI. (The Institute of Low Temperature Science, Hakkaido University, Sapporo, Japan) . It was reported by Nei in 1968 and by Rapatz et al. in 1973 that post-thaw hemolysis of erythrocyte suspensions, frozen under various conditions, decreased as the cell concentration decreased. The present study was carried out to elucidate the mechanism of this phenomenon. Human red blood cells, suspended in saline with or without cryoprotective substances, were frozen at various cooling rates. The extent of hemolysis was measured spectrophotometrically after thawing. When cell suspensions were frozen slowly, the cells were initially confined in unfrozen narrow channels according to the growth of the surronnding ice and subsequently became highly concentrated. It is assumed that mechanical cell damage due to such intense concentration becomes greater in concentrated cell suspensions. In rapid freezing, however, cells were evenly scattered in the frozen state. The effect of cell concentration on post-thaw hemolysis appears to be due to different mechanisms in slow- and fastfrozen erythrocytes. 26. Hemolysis of Human Erythrocytes in Hypertonic Sodium Chloride Solutions at Subphysiological Temperatures. J. J. MCGRATH, E.
G.
CRAVALHO,
AND
C.
E.
HUGGINS,
652
ABSTRACTS,
13TH
(Cryogenic Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Surgery, Harvard Medical School, Low Temperature Surgical Unit and Blood Bank Transfusion Service, Massachusetts General Hopital, Boston, Massachusetts 02114). An optical system in communication with a thermally controlled environment has been developed and used to determine hemolysis as a function of time for human erythrocytes in hypertonic solutions at various subphysiological temperatures. This technique yields real-time hemolysis data without centrifugation, thereby providing access to short exposure time data and avoiding centrifugation artifacts. Comparison of hemolysis data obtained from this system with data obtained from the standard cyanmethemoglobin calorimetric method yields excellent agreement. Transient hemolysis data of human erythrocytes in hypertonic sodium chloride solutions between 1.0 and 4.0 M are compared with existing data of SoderStrom and Zade-Oppen at ambient temperatures and of Farrant and Woolgar at 0°C. This technique has revealed that hemolysis kinetics in hypertonic sodium chloride solutions occurs in two distinct modes characterized by a rapid reaction rate at short exposure times followed by transition to a reduced rate at long exposure times. The implications of these results are presented in terms of the cryobiological application of modeling freeze-thaw damage at suboptimal cooling rates. Supported in part by the National Heart and Lung Institute, Grant No. HL-14322. 27. Osmotic Augmentation of Thermal Shock in Human Erythrocytes. R. J, WILLIAMS AND H. T. MERYMAN. (American National Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Maryland 20014 ) . Human erythrocytes exposed to hypertonic saline solutions of greater than 1200 mosM show a resistance to volume loss and, when cooled to 4”C, undergo a hemolysis called thermal shock. We have examined the effect of high concentrations of extracellular solute on thermal shock and found that higher concentrations not only increase the severity of injury but displace the onset of injury to higher temperatures. An analogous interaction between osmotic and thermal stress has been shown in cells from plants which are subject to chilling injury above freezing. This chilling injury is thought to result from a lipid transition in the membrane. We have proposed that in these plant cells the pbsmolysis which produces reduced
ANNUAL
MEETING
volume also reduces cell membrane area. When membrane lipids have been compressed to their minimum area, two effects occur. The lipids resist further compression, producing a resistance to plasmolysis, and the strength of intermolecular (dispersion) forces increases, producing a higher lipid transition temperature. Human red cells are known to experience negligible loss in membrane area during hyptertonic exposure; nonetheless they show a disruptive change in permeability which occurs at higher temperatures in higher concentrations of extracellular solute. The osmotic disequilibrium across the cell membrane at high concentrations implies the storage of potential energy by the cell in some other form, the nature of which is now under investigation. 28. The Use of Permeability Coefficients in Predicting the Osmotic Response of Human Red Cells during the Removal of Intracellular Glycerol. PETER MAZUR AND R. H. MILLER. (Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830). Although some cells survive freezing in the absence of intracellular additive, others may require its permeation for optimum survival. Moreover, even when an additive has not permeated prior to freezing, the development of high solute concentrations and the possible alterations of membranes during freezing may drive additive into the cell. Once additive is in a cell, it can cause serious osmotic problems during thawing and during its subsequent removal. Knowledge of the permeability coefficients of the solute permits one in theory to calculate the changes in volume that a cell will be subjected to when attempts are made to remove protective additives by dilution. One should in theory be able to determine rapidly by computer modeling those dilution procedures which will minimize osmotic damage. TO test whether this expectation is met in practice, we made use of recent determinations of the permeability coefficients of human red cells in glycerol (Cryobiology, in press). The coefficients were used to calculate the cell volume changes when cells which had been fully equilibrated with 2 M glycerol were subjected to two sorts of dilution procedures: One procedure involved stepwise dilution with various volumes of isotonic phosphate-buffered saline (PBS) at various time intervals. The other procedure involved transferring the cells to a nonpenetrating solute (1.0 M sucrose) for various times followed by abrupt 20x dilution with PBS. In the red cell, hemolysis occurs when the volume exceeds rather precisely determinable values, and from these detemrinations we were able to calculate the amount of
13TH
NaCl concentration of 12 wt%, while for cells saline the frozen in 1.85 M glycerol in buffered LTm corresponds to 3.0 wt% NaCI. When the NaCl concentration is expressed on a molal basis, the correlation is improved. (2) There is some correlation between LT, and the fraction of water remaining unfrozen at that temperature; 50% hemolysis occurs when about 90% of the water has been converted to ice. ’ Predoctoral Investigator supported by NIH Grant No. GM01972. ‘Visiting Investigator from Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan. 3 Operated by Energy Research and Development Administration under contract with the Union Carbide Corporation. The United States Government’s right to retain a nonexclusive, royalty-free license in and to copyright covering this abstract is acknowledged. 24. Water Transport in a Cluster of Closely Packed Erythrocytes during Cooling at Subzero E. G. CRATemperatures. R. L. LEVIN, VALHO, AND C. E. HUGGINS. (Cryogenic Engineering Laboratory, Massachusetts Institute of Technology and Blood Bank and Transfusion Services, Massachusetts General Hospital, Boston, Massachusetts 02114). The ideal, hydrated, nondilute multicomponent electrolyte solution model of the red bIood cell (RBC) intracellular solution developed by Levin et al. ( 1) has been applied to the case of water transport in a cluster of closely packed erythrocytes during cooling at constant rates at subzero temperatures. The results indicate that the volume flux of water out of the interior cells of a cluster will lag behind the volume flux of water out of the exterior cells. At any given time (temperature) during a cooling process the amount of water retained within the interior cells will therefore exceed the amount of water retained within the exterior cells. Only for very slow cooling or warming rates, for which the water volume fluxes are small, will the cells of a cluster be able to shrink or swell almost uniformly. At fast cooling or warming rates, the exterior cells will shrink or swell to a much greater degree than the interior cells. Our findings also indicate that, on an overall percentage basis, the amount of water retained within a cluster of closely packed cells of a given type will exceed the amount of water retained within a single, isolated cell of the same type at any given time (temperature) during a cooling process. The probability of intracellular ice nucleation at the low temperatures for a given cooling rate will therefore be greater in a cluster of
ANNUAL
MEETING
651
closely packed cells than in a single, isolated cell. Hence, the transition cooling rate for a cluster of closely packed cells of a given type will be much lower than the transition cooling rate of a singb, isolated cell of the same type. (For RBCs our model predicts that BrcR = -5O”C/min for a 1% cell cluster whereas B TCR = -1500”C/min for a single RBC ) . Consequently, the survival signature for an aggregate of cells should peak at a cooling rate which is less than the corresponding optimal value for a single, isolated cell. The behavior has been observed by McGrath et al. (2) who found for HeLa S-3 cells that the optimal cooling rate for survival is approximately SO”C/min for single cells but approximately 8”C/min for packed cells. 1. Levin, R. L., Cravalho, E. G., and Huggins, C. E. The effect of hydration on the water content of human erythrocytes. Manuscript submitted. 2. McGrath, J. J., Cravalho, E. G., and Huggins, C. E. An experimental comparison of intracellular ice formation and freeze-thaw survival of HeLa S-3 Cells. Cryobiology 12, 520-550 ( 1975).
of the Post25. Effect of Initial Cell Concentration Thaw Hemolysis of Frozen Erythrocytes. T. NEI. (The Institute of Low Temperature Science, Hakkaido University, Sapporo, Japan) . It was reported by Nei in 1968 and by Rapatz et al. in 1973 that post-thaw hemolysis of erythrocyte suspensions, frozen under various conditions, decreased as the cell concentration decreased. The present study was carried out to elucidate the mechanism of this phenomenon. Human red blood cells, suspended in saline with or without cryoprotective substances, were frozen at various cooling rates. The extent of hemolysis was measured spectrophotometrically after thawing. When cell suspensions were frozen slowly, the cells were initially confined in unfrozen narrow channels according to the growth of the surronnding ice and subsequently became highly concentrated. It is assumed that mechanical cell damage due to such intense concentration becomes greater in concentrated cell suspensions. In rapid freezing, however, cells were evenly scattered in the frozen state. The effect of cell concentration on post-thaw hemolysis appears to be due to different mechanisms in slow- and fastfrozen erythrocytes. 26. Hemolysis of Human Erythrocytes in Hypertonic Sodium Chloride Solutions at Subphysiological Temperatures. J. J. MCGRATH, E.
G.
CRAVALHO,
AND
C.
E.
HUGGINS,
652
ABSTRACTS,
13TH
(Cryogenic Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Surgery, Harvard Medical School, Low Temperature Surgical Unit and Blood Bank Transfusion Service, Massachusetts General Hopital, Boston, Massachusetts 02114). An optical system in communication with a thermally controlled environment has been developed and used to determine hemolysis as a function of time for human erythrocytes in hypertonic solutions at various subphysiological temperatures. This technique yields real-time hemolysis data without centrifugation, thereby providing access to short exposure time data and avoiding centrifugation artifacts. Comparison of hemolysis data obtained from this system with data obtained from the standard cyanmethemoglobin calorimetric method yields excellent agreement. Transient hemolysis data of human erythrocytes in hypertonic sodium chloride solutions between 1.0 and 4.0 M are compared with existing data of SoderStrom and Zade-Oppen at ambient temperatures and of Farrant and Woolgar at 0°C. This technique has revealed that hemolysis kinetics in hypertonic sodium chloride solutions occurs in two distinct modes characterized by a rapid reaction rate at short exposure times followed by transition to a reduced rate at long exposure times. The implications of these results are presented in terms of the cryobiological application of modeling freeze-thaw damage at suboptimal cooling rates. Supported in part by the National Heart and Lung Institute, Grant No. HL-14322. 27. Osmotic Augmentation of Thermal Shock in Human Erythrocytes. R. J, WILLIAMS AND H. T. MERYMAN. (American National Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Maryland 20014 ) . Human erythrocytes exposed to hypertonic saline solutions of greater than 1200 mosM show a resistance to volume loss and, when cooled to 4”C, undergo a hemolysis called thermal shock. We have examined the effect of high concentrations of extracellular solute on thermal shock and found that higher concentrations not only increase the severity of injury but displace the onset of injury to higher temperatures. An analogous interaction between osmotic and thermal stress has been shown in cells from plants which are subject to chilling injury above freezing. This chilling injury is thought to result from a lipid transition in the membrane. We have proposed that in these plant cells the pbsmolysis which produces reduced
ANNUAL
MEETING
volume also reduces cell membrane area. When membrane lipids have been compressed to their minimum area, two effects occur. The lipids resist further compression, producing a resistance to plasmolysis, and the strength of intermolecular (dispersion) forces increases, producing a higher lipid transition temperature. Human red cells are known to experience negligible loss in membrane area during hyptertonic exposure; nonetheless they show a disruptive change in permeability which occurs at higher temperatures in higher concentrations of extracellular solute. The osmotic disequilibrium across the cell membrane at high concentrations implies the storage of potential energy by the cell in some other form, the nature of which is now under investigation. 28. The Use of Permeability Coefficients in Predicting the Osmotic Response of Human Red Cells during the Removal of Intracellular Glycerol. PETER MAZUR AND R. H. MILLER. (Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830). Although some cells survive freezing in the absence of intracellular additive, others may require its permeation for optimum survival. Moreover, even when an additive has not permeated prior to freezing, the development of high solute concentrations and the possible alterations of membranes during freezing may drive additive into the cell. Once additive is in a cell, it can cause serious osmotic problems during thawing and during its subsequent removal. Knowledge of the permeability coefficients of the solute permits one in theory to calculate the changes in volume that a cell will be subjected to when attempts are made to remove protective additives by dilution. One should in theory be able to determine rapidly by computer modeling those dilution procedures which will minimize osmotic damage. TO test whether this expectation is met in practice, we made use of recent determinations of the permeability coefficients of human red cells in glycerol (Cryobiology, in press). The coefficients were used to calculate the cell volume changes when cells which had been fully equilibrated with 2 M glycerol were subjected to two sorts of dilution procedures: One procedure involved stepwise dilution with various volumes of isotonic phosphate-buffered saline (PBS) at various time intervals. The other procedure involved transferring the cells to a nonpenetrating solute (1.0 M sucrose) for various times followed by abrupt 20x dilution with PBS. In the red cell, hemolysis occurs when the volume exceeds rather precisely determinable values, and from these detemrinations we were able to calculate the amount of