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Thermal shock hemolysis in isotonic solution
ABSTRACTS,
17th ANNUAL
erably reduced when the tissue was heated to only 37°C under conditions of whole body hypothermia. However, significant differences in blood flow existed between the hypothermic body and the normothermic (or hyperthermic) uterine horn. This suggests the possibility that normally toxic high doses of chemotherapeutic agents could be infused locally in the region to be warmed while the body is maintained in hypothermia, thus increasing the effectiveness of the drug. Cytotoxicity may possibly be further increased by warming the selected region to 42-43°C.
MEETING
609
concentration would be influenced by the relative volumes of intra- and extracellular solutions, i.e., the hematocrit. 47. Cell Packing and the Recovery of Human Erythrocytes Frozen and Thawed in the Presence of Glycerol. D. E. PEGGAND M. P. DIAPER (MRC
Medical Cryobiology Group, University Department of Surgery, Cambridge, United Kingdom, England).
Further studies have been carried out to determine the importance of the cell-packing effect in red cells frozen in the presence of glycerol, and to elucidate its 46. The Effect of Variations in Hematocrit on Red mechanism. A significant error in the measurement of Cell Lysis Following Freezing and Thawing. haemolysis in high-haematocrit samples, both by ourH. T. MERYMAN, M. ST. J. DOUGLAS,J. BROSS, selves and some other workers, will be described: AND W. SOMMERS,(Cryobiology Laboratory, when this error is avoided the magnitude of the effect ARC Blood Services Labs, 9312 Old Georgeis seen to be somewhat less than had previously been town Road, Bethesda, Maryland). supposed. The present experiments were carried out There is a difference of opinion in the literature as to with human red blood cells at packed cell volumes of whether or not the freeze-thaw hemolysis of red cells 10, 40, and 80%; cooling rates of approximately 0.5, is influenced by the hematocrit. We have examined 5.0, and SO.O”C/min; and warming rates of 5 and this question using both unprotected cells and ZOO”C/min.Cells were suspended in 0, 1, 2, or 3 M glycerolized cells. The hematocrits used ranged from 2 glycerol, and rewarming was commenced as soon as to 97%, cooling rates ranged from 0.6”C/min to -60°C was reached. In the absence of glycerol, O.Ol”C/min, all samples were seeded at less than 1°C haemolysis was virtually complete under all conditions below the melting point, the warming rate was always studied, and no effect of cell packing could be deidentical to the cooling rate. Unprotected cells frozen tected. A reduction in recovery with increasing to -6°C under these circumstances suffer between 40 haematocrit was observed with red cells suspended in and 60% hemolysis. At cooling rates of 0.3”C/min or 1 M glycerol at all the cooling and warming rates less, no significant effect of hematocrit was evident. At studied, but the extent of the effect varied: it was higher cooling rates, a slight hematocrit effect was maximal after rapid cooling, irrespective of warming seen with low hematocrits producing around 10% less rate. In 2 M glycerol the packing effect was prohemolysis than high. At these higher rates, samples nounced under some conditions (e.g., rapid cooling, cooled under identical circumstances do not actually slow warming), absent under others (rapid cooling, cool at the same rate because of differences in the rapid warming), and intermediate in extent at the other amount of latent heat of fusion evolved from samples cooling and warming rates. In 3 M glycerol, the pheof differing hematocrit, a factor that is exaggerated if nomenon was pronounced only with rapid cooling and the samples are not first seeded and allowed to equili- slow warming-fortuitously the very conditions brate. The hematocrit effect seen at higher cooling selected for our earlier experiments. It seems probable velocities may therefore reflect compliance stress dif- that the principal mechanism of the effect is that close ferences associated with high rates of cell volume packing inhibits dehydration during cooling, thus change rather than being directly dependent on hemat- making intracellular freezing more likely either during ocrit. When glycerol, DMSO, ethanol, or methanol cooling or during subsequent rewarming; however, was added to the suspension, a definite hematocrit ef- there may also be some augmentation of solution effect developed with the magnitude of the effect in- fects. With adequate concentrations of glycerol and creasing with the cryoportectant concentration at least careful selection of appropriate cooling and warming to 1 M. These observations are not consistent with the rates, the cell packing effect can be avoided. hypothesis that a reduction in ice channel size can cause hemolysis but rather imply that the influence of 48. Thermal Shock Hemolysis in Isotonic Solution. T. hematocrit on hemolysis is dependent on the presence TAKAHASHI AND R.J. WILLIAMS (Cryobiology of a penetrating cryoprotectant. As an alternative hyLaboratory, ARC Blood Services Labs, 9312 pothesis we suggest that the cryoprotectant enters the Old Georgetown Road, Bethesda, Maryland). cell during freezing, resulting in both a decrease in cell dehydration and an increase in the concentration of Thermal shock hemolysis has been studied in extracellular, nonpenetrating solutes. Both the extent hypertonically stressed human red cells. Thermal of glycerol influx and the degree of extracellular solute shock hemolysis in isotonic solution was reported by SESSION
5-ERYTHROCYTE
PRESERVATION
610
ABSTRACTS,
17th ANNUAL
Lovelock, who found that cells treated by bacteriotoxin were susceptible to thermal shock. We found several other compounds that can make human red cells susceptible to thermal shock in isotonic phosphate-buffered saline. Hypertonic thermal shock and isotonic thermal shock were compared with respect to morphological changes, the effect of cooling rate, and of critical temperature. The results were as follows: (a) Human red cells treated with chlorpromazine hemolyzed drastically in isotonic solution when they were cooled from 37 to 0°C. (b) Triton X-100 could cause thermal shock hemolysis, but SDS or Tween 80 could not. (c) Phospholipase C (Clostridium perfringens) could cause thermal shock hemolysis but phopholipase AZ could not. (d) Vinblastine could cause thermal shock hemolysis, but cholchicine could not. (e) Chemicals which made cells susceptible to isotonic thermal shock changed the cell shape from biconcave to cup shape or spherocyte. (f) Light-scattering studies showed that hemolysis began when cells were cooled down to a temperature between 15 and 10°C. (g) Light microscope observation showed that stomatocytic or spherocytic cells treated with chlorpromazine lost hemoglobin without any change of shape when they were cooled below 10°C. In contrast, hypertonically stressed biconcave cells became spherocytes when undergoing thermal shock. (h) During hypertonic thermal shock using either NaCl or sucrose, rapid cooling caused more hemolysis than slow cooling. The influence of cooling rate was independent of the duration of suspension in the hypertonic solution prior to cooling. In isotonic thermal shock, the effect of cooling rate was less. No clear pattern emerges from these observations and the mechanism of sensitization to thermal shock remains obscure. 49. Unusual Freeze Fracture Pattern of Unprotected Red Cells. E. D. ALLEN, L. WEATHERBEE,AND
P. A. PERMOAD (Veterans Administration Medical Center, 2215 Fuller Road, Ann Arbor, Michigan). We have examined the ultrastructure of red cells using the freeze fracture technique. To gain a better understanding of the cell damage which might occur during freezing, we examined cells frozen in the absence of cryoprotective agents. Cells were suspended in saline, placed in Hemoflex bags (30-ml final volume), and frozen by agitation in liquid nitrogen. Identical units when thawed indicate that greater than 95% of the cells are damaged (saline stability test following thaw). Replicas prepared from frozen units indicate an unusual appearance of the half-membrane faces. The PF half-membrane face (face closest to protoplast) appears to possess large deposits of material scattered over its surface. In contrast, the EF face (halfmembrane face closest to extracellular region) has gaps or holes scattered over its surface. Etching the
MEETING
fractured surfaces indicates that the deposits are not caused by irregular cell shapes or by ice crystals penetrating the cells. Etching appears to expose the external surface of the cell (ES surface) and to indicate that the deposits are below the external surface of the cell but above the PF face. If so, these deposits may represent the partial passage of cellular contents (hemoglobin) through the cell membrane or the redistribution of membrane components. The smooth appearance of the deposits suggest that they are not hemoglobin. 50. The Effect of Citrate on Red Cell Volume and on Freeze-Thaw Hemolysis. H. T. MERYMAN
AND M. ST. J. DOUGLAS(Cryobiology Laboratory, ARC Blood Services Labs, 9312 Old Georgetown Road, Bethesda, Maryland) Red cells frozen and thawed for clinical use undergo progressive hemolysis following deglycerolization with the supernatant hemoglobin concentration roughly doubling each 24 hr. This progressive lysis can be totally prevented by the addition of sodium citrate at a concenration of 20 mh4 or greater. Red cells suspended in buffered isotonic NaCl either unprotected or protected with glycerol and frozen to a temperature producing about 50% hemolysis, will suffer 15 to 30% less hemolysis when isotonic buffered sodium citrate is substituted for the sodium chloride. Studies of the volume distribution of red cells frozen and thawed in sodium chloride show a substantial population of swollen cells following washing to remove ghosts. After freezing in sodium citrate, no swollen cells are evident. The shape of the volume distribution curve is virtually identical to that of control cells in NaCl but is shifted slightly in the direction of smaller volume. Fresh red cells suspended in isotonic citrate solution are progressively reduced in volume, approaching equilibrium in a matter of hours. On resuspension in isotonic NaC 1, the mean cell volume returns to normal in about 24 hr. These observations suggest that, following freezing and thawing in NaCl, there are four categories of cells: those that are unaffected; those that have experienced some influx of extracellular solute and are swollen but remain intact and at less than lytic volume; those that have suffered too much solute influx and lyse hypotonically on thawing; and perhaps a group with irreversible membrane damage. On this basis, it can be hypothesized that citrate in some way alters the permeability of the cell membrane, increasing its defense against solute influx. As a result, the influx of extracellular solute is postponed to higher osmolalities and freezing injury becomes an all-ornone phenomenon with cells either unaffected or lysed. The ability of citrate to prevent continued swelling and lysis of deglycerolized red cells indicates that it can also reverse the increased membrane permeability that permits this colloid osmotic lysis. The shrinking of cells suspended in isotonic citrate also
17th ANNUAL
erably reduced when the tissue was heated to only 37°C under conditions of whole body hypothermia. However, significant differences in blood flow existed between the hypothermic body and the normothermic (or hyperthermic) uterine horn. This suggests the possibility that normally toxic high doses of chemotherapeutic agents could be infused locally in the region to be warmed while the body is maintained in hypothermia, thus increasing the effectiveness of the drug. Cytotoxicity may possibly be further increased by warming the selected region to 42-43°C.
MEETING
609
concentration would be influenced by the relative volumes of intra- and extracellular solutions, i.e., the hematocrit. 47. Cell Packing and the Recovery of Human Erythrocytes Frozen and Thawed in the Presence of Glycerol. D. E. PEGGAND M. P. DIAPER (MRC
Medical Cryobiology Group, University Department of Surgery, Cambridge, United Kingdom, England).
Further studies have been carried out to determine the importance of the cell-packing effect in red cells frozen in the presence of glycerol, and to elucidate its 46. The Effect of Variations in Hematocrit on Red mechanism. A significant error in the measurement of Cell Lysis Following Freezing and Thawing. haemolysis in high-haematocrit samples, both by ourH. T. MERYMAN, M. ST. J. DOUGLAS,J. BROSS, selves and some other workers, will be described: AND W. SOMMERS,(Cryobiology Laboratory, when this error is avoided the magnitude of the effect ARC Blood Services Labs, 9312 Old Georgeis seen to be somewhat less than had previously been town Road, Bethesda, Maryland). supposed. The present experiments were carried out There is a difference of opinion in the literature as to with human red blood cells at packed cell volumes of whether or not the freeze-thaw hemolysis of red cells 10, 40, and 80%; cooling rates of approximately 0.5, is influenced by the hematocrit. We have examined 5.0, and SO.O”C/min; and warming rates of 5 and this question using both unprotected cells and ZOO”C/min.Cells were suspended in 0, 1, 2, or 3 M glycerolized cells. The hematocrits used ranged from 2 glycerol, and rewarming was commenced as soon as to 97%, cooling rates ranged from 0.6”C/min to -60°C was reached. In the absence of glycerol, O.Ol”C/min, all samples were seeded at less than 1°C haemolysis was virtually complete under all conditions below the melting point, the warming rate was always studied, and no effect of cell packing could be deidentical to the cooling rate. Unprotected cells frozen tected. A reduction in recovery with increasing to -6°C under these circumstances suffer between 40 haematocrit was observed with red cells suspended in and 60% hemolysis. At cooling rates of 0.3”C/min or 1 M glycerol at all the cooling and warming rates less, no significant effect of hematocrit was evident. At studied, but the extent of the effect varied: it was higher cooling rates, a slight hematocrit effect was maximal after rapid cooling, irrespective of warming seen with low hematocrits producing around 10% less rate. In 2 M glycerol the packing effect was prohemolysis than high. At these higher rates, samples nounced under some conditions (e.g., rapid cooling, cooled under identical circumstances do not actually slow warming), absent under others (rapid cooling, cool at the same rate because of differences in the rapid warming), and intermediate in extent at the other amount of latent heat of fusion evolved from samples cooling and warming rates. In 3 M glycerol, the pheof differing hematocrit, a factor that is exaggerated if nomenon was pronounced only with rapid cooling and the samples are not first seeded and allowed to equili- slow warming-fortuitously the very conditions brate. The hematocrit effect seen at higher cooling selected for our earlier experiments. It seems probable velocities may therefore reflect compliance stress dif- that the principal mechanism of the effect is that close ferences associated with high rates of cell volume packing inhibits dehydration during cooling, thus change rather than being directly dependent on hemat- making intracellular freezing more likely either during ocrit. When glycerol, DMSO, ethanol, or methanol cooling or during subsequent rewarming; however, was added to the suspension, a definite hematocrit ef- there may also be some augmentation of solution effect developed with the magnitude of the effect in- fects. With adequate concentrations of glycerol and creasing with the cryoportectant concentration at least careful selection of appropriate cooling and warming to 1 M. These observations are not consistent with the rates, the cell packing effect can be avoided. hypothesis that a reduction in ice channel size can cause hemolysis but rather imply that the influence of 48. Thermal Shock Hemolysis in Isotonic Solution. T. hematocrit on hemolysis is dependent on the presence TAKAHASHI AND R.J. WILLIAMS (Cryobiology of a penetrating cryoprotectant. As an alternative hyLaboratory, ARC Blood Services Labs, 9312 pothesis we suggest that the cryoprotectant enters the Old Georgetown Road, Bethesda, Maryland). cell during freezing, resulting in both a decrease in cell dehydration and an increase in the concentration of Thermal shock hemolysis has been studied in extracellular, nonpenetrating solutes. Both the extent hypertonically stressed human red cells. Thermal of glycerol influx and the degree of extracellular solute shock hemolysis in isotonic solution was reported by SESSION
5-ERYTHROCYTE
PRESERVATION
610
ABSTRACTS,
17th ANNUAL
Lovelock, who found that cells treated by bacteriotoxin were susceptible to thermal shock. We found several other compounds that can make human red cells susceptible to thermal shock in isotonic phosphate-buffered saline. Hypertonic thermal shock and isotonic thermal shock were compared with respect to morphological changes, the effect of cooling rate, and of critical temperature. The results were as follows: (a) Human red cells treated with chlorpromazine hemolyzed drastically in isotonic solution when they were cooled from 37 to 0°C. (b) Triton X-100 could cause thermal shock hemolysis, but SDS or Tween 80 could not. (c) Phospholipase C (Clostridium perfringens) could cause thermal shock hemolysis but phopholipase AZ could not. (d) Vinblastine could cause thermal shock hemolysis, but cholchicine could not. (e) Chemicals which made cells susceptible to isotonic thermal shock changed the cell shape from biconcave to cup shape or spherocyte. (f) Light-scattering studies showed that hemolysis began when cells were cooled down to a temperature between 15 and 10°C. (g) Light microscope observation showed that stomatocytic or spherocytic cells treated with chlorpromazine lost hemoglobin without any change of shape when they were cooled below 10°C. In contrast, hypertonically stressed biconcave cells became spherocytes when undergoing thermal shock. (h) During hypertonic thermal shock using either NaCl or sucrose, rapid cooling caused more hemolysis than slow cooling. The influence of cooling rate was independent of the duration of suspension in the hypertonic solution prior to cooling. In isotonic thermal shock, the effect of cooling rate was less. No clear pattern emerges from these observations and the mechanism of sensitization to thermal shock remains obscure. 49. Unusual Freeze Fracture Pattern of Unprotected Red Cells. E. D. ALLEN, L. WEATHERBEE,AND
P. A. PERMOAD (Veterans Administration Medical Center, 2215 Fuller Road, Ann Arbor, Michigan). We have examined the ultrastructure of red cells using the freeze fracture technique. To gain a better understanding of the cell damage which might occur during freezing, we examined cells frozen in the absence of cryoprotective agents. Cells were suspended in saline, placed in Hemoflex bags (30-ml final volume), and frozen by agitation in liquid nitrogen. Identical units when thawed indicate that greater than 95% of the cells are damaged (saline stability test following thaw). Replicas prepared from frozen units indicate an unusual appearance of the half-membrane faces. The PF half-membrane face (face closest to protoplast) appears to possess large deposits of material scattered over its surface. In contrast, the EF face (halfmembrane face closest to extracellular region) has gaps or holes scattered over its surface. Etching the
MEETING
fractured surfaces indicates that the deposits are not caused by irregular cell shapes or by ice crystals penetrating the cells. Etching appears to expose the external surface of the cell (ES surface) and to indicate that the deposits are below the external surface of the cell but above the PF face. If so, these deposits may represent the partial passage of cellular contents (hemoglobin) through the cell membrane or the redistribution of membrane components. The smooth appearance of the deposits suggest that they are not hemoglobin. 50. The Effect of Citrate on Red Cell Volume and on Freeze-Thaw Hemolysis. H. T. MERYMAN
AND M. ST. J. DOUGLAS(Cryobiology Laboratory, ARC Blood Services Labs, 9312 Old Georgetown Road, Bethesda, Maryland) Red cells frozen and thawed for clinical use undergo progressive hemolysis following deglycerolization with the supernatant hemoglobin concentration roughly doubling each 24 hr. This progressive lysis can be totally prevented by the addition of sodium citrate at a concenration of 20 mh4 or greater. Red cells suspended in buffered isotonic NaCl either unprotected or protected with glycerol and frozen to a temperature producing about 50% hemolysis, will suffer 15 to 30% less hemolysis when isotonic buffered sodium citrate is substituted for the sodium chloride. Studies of the volume distribution of red cells frozen and thawed in sodium chloride show a substantial population of swollen cells following washing to remove ghosts. After freezing in sodium citrate, no swollen cells are evident. The shape of the volume distribution curve is virtually identical to that of control cells in NaCl but is shifted slightly in the direction of smaller volume. Fresh red cells suspended in isotonic citrate solution are progressively reduced in volume, approaching equilibrium in a matter of hours. On resuspension in isotonic NaC 1, the mean cell volume returns to normal in about 24 hr. These observations suggest that, following freezing and thawing in NaCl, there are four categories of cells: those that are unaffected; those that have experienced some influx of extracellular solute and are swollen but remain intact and at less than lytic volume; those that have suffered too much solute influx and lyse hypotonically on thawing; and perhaps a group with irreversible membrane damage. On this basis, it can be hypothesized that citrate in some way alters the permeability of the cell membrane, increasing its defense against solute influx. As a result, the influx of extracellular solute is postponed to higher osmolalities and freezing injury becomes an all-ornone phenomenon with cells either unaffected or lysed. The ability of citrate to prevent continued swelling and lysis of deglycerolized red cells indicates that it can also reverse the increased membrane permeability that permits this colloid osmotic lysis. The shrinking of cells suspended in isotonic citrate also