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Cryoprotection of human erythrocytes with potassium chloride
ABSTRACTS,
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and will leak hemoglobin to the external environrnent. Depending on the suspension solution used, this leakage will occur immediately or be delayed for several hours. Supported by the Medical Research Service of the Veterans Administration.
81. Cryoprotection of Human Err&rocytes with Potassium Chloride. ROBERT J. WILLIAMS STEPHEN K. SHAW * (American Red Cross Blood Research Laboratory, Bethesda, Maryland 20014).
ASD
It has been reported that human red cells begin to manifest injury when the concentration of nonpenetrating extracellular solute exceeds 4.5 times isotonic. Both the extracellular salt concentration above 0.8 1~1 and the reduction of cell volume below 60% of normal volume which occur at this level of osmotic stress have been invoked as the source of injury. Because of the high covariance between extracellular solute concentration and cell volume, it has been difficult to distinguish between these proposed mechanisms of injury. We incubated red cells in an 800 mOsm KC1 solution containing valinomycin, which facilitates passage of potassium across the membrane. Under these conditions, the shrunken cells absorb extracellular solution and swell to normal volume. Thus, treated cells became isotonic at 800 mOsm. When treated cells were subsequently exposed to 4000 mOsm solutions, they remained larger than 60% of normal volume, and little lysis was observed. Untreated control cells decreased below 6070 volume and considerable lysis occurred. Treatment deferred thermal shock hemolysis and freezing hemolysis to higher solute concentrations. Despite the difference in the concentration at which lysis began in control or in treated cells, in each case lysis began when the extracellular solution had reached 4.5 to 5 times the isotonic concentration. These experiments support the contention that cell injury is related to reduction in cell size but not to the absolute concentration of any solute. Supported in part by NIH Grant GSI 17959.
82. Effect of Nucleation Temperature on Hemolysis in Blood Frozen at Various Temperatures. G. RAPATZ, K. J. khrhrmsrm,* AND E. F. GRAHAhl (Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108). Small quantities of ACD blood contained in fine capillary tubes consistently supercooled to about -15°C before nucleation occurs. Diller et al. (Cryobiology 9, 316, 1972) reported that
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intracellular freezing was observed in all arythrocytes when thin films of blood were cooled at rates of more than 17”C/min. However, if supercooling of the extracellular medium was zero, intracellular freezing first appeared at a cooling rate of 840”C/min (Fifth International Cryogenic Engineering Conference, Kyoto, Japan, 1974). In this study we have compared the extent of hemolysis in blood frozen by immersion into baths at various low temperatures (-20 to -120°C) in samples which were nucleated at -2°C with those that were allowed to supercool until spontaneous nucleation occurred. Samples nucleated at -2°C and then cooled at rates obtained by immersion into baths at temperatures ranging from -20 to -50°C had higher hemolysis values than those which were permitted to nucleate spontaneously in this temperature range. Conversely, in the temperature range of -70 to --120°C the extent of injury to specimens nucleated at -2°C was remarkably lower than in those which were permitted to nucleate spontaneously in this range. These results suggest that extensive supercooling of small volumes of blood combined with rapid cooling to low temperatures promotes damaging intracellular ice formation. Extracellular nucleation at temperatures near the freezing point prevents the very rapid removal of the heat of fusion as occurs in the nucleation of a supercooled medium and thereby reduces the likelihood of intracellular freezing during rapid cooling. This work was supported by NIH Grant No. CM 20531. 83. Effect of Extra- and Intracellular IVater Distribution on Optimal Hydroxyethyl Starch
Concentrations for Different Cooling
Red Blood Cells at and Thawing Rates.
hl~x W. SCHEI~E * AXD HANS E. Nn:x* (Spon. S. Seidl) ( Helmholtz-Inst. fiir Biomed. Technik, Aachen, West Germany). Fresh ACD-blood was vvashed twice in phosphate-buffered saline ( PBS ) . Packed erythrocytes were mixed with hydroxyethyl starch (HES)PBS combinations by short shaking and successive gentle rolling for 5 min. Twenty microliters of this mixture were pipetted into glass capillaries. Together with a reference sample for temperature measurement, four samples were frozen at a time in liquid nitrogen or frigen with controlled cooling rates ranging from 80 to 17,00O”K/ min, thawed at 50 to 24,00O”K/min, and submerged into 5 ml of PBS. Supernatant hemoglobin was then measured photometrically after 8 min. In order to vary the water content of the starting medium, HES concentrations were selected be-
14TH
and will leak hemoglobin to the external environrnent. Depending on the suspension solution used, this leakage will occur immediately or be delayed for several hours. Supported by the Medical Research Service of the Veterans Administration.
81. Cryoprotection of Human Err&rocytes with Potassium Chloride. ROBERT J. WILLIAMS STEPHEN K. SHAW * (American Red Cross Blood Research Laboratory, Bethesda, Maryland 20014).
ASD
It has been reported that human red cells begin to manifest injury when the concentration of nonpenetrating extracellular solute exceeds 4.5 times isotonic. Both the extracellular salt concentration above 0.8 1~1 and the reduction of cell volume below 60% of normal volume which occur at this level of osmotic stress have been invoked as the source of injury. Because of the high covariance between extracellular solute concentration and cell volume, it has been difficult to distinguish between these proposed mechanisms of injury. We incubated red cells in an 800 mOsm KC1 solution containing valinomycin, which facilitates passage of potassium across the membrane. Under these conditions, the shrunken cells absorb extracellular solution and swell to normal volume. Thus, treated cells became isotonic at 800 mOsm. When treated cells were subsequently exposed to 4000 mOsm solutions, they remained larger than 60% of normal volume, and little lysis was observed. Untreated control cells decreased below 6070 volume and considerable lysis occurred. Treatment deferred thermal shock hemolysis and freezing hemolysis to higher solute concentrations. Despite the difference in the concentration at which lysis began in control or in treated cells, in each case lysis began when the extracellular solution had reached 4.5 to 5 times the isotonic concentration. These experiments support the contention that cell injury is related to reduction in cell size but not to the absolute concentration of any solute. Supported in part by NIH Grant GSI 17959.
82. Effect of Nucleation Temperature on Hemolysis in Blood Frozen at Various Temperatures. G. RAPATZ, K. J. khrhrmsrm,* AND E. F. GRAHAhl (Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108). Small quantities of ACD blood contained in fine capillary tubes consistently supercooled to about -15°C before nucleation occurs. Diller et al. (Cryobiology 9, 316, 1972) reported that
ANNUAL
\lEETING
TO!,
intracellular freezing was observed in all arythrocytes when thin films of blood were cooled at rates of more than 17”C/min. However, if supercooling of the extracellular medium was zero, intracellular freezing first appeared at a cooling rate of 840”C/min (Fifth International Cryogenic Engineering Conference, Kyoto, Japan, 1974). In this study we have compared the extent of hemolysis in blood frozen by immersion into baths at various low temperatures (-20 to -120°C) in samples which were nucleated at -2°C with those that were allowed to supercool until spontaneous nucleation occurred. Samples nucleated at -2°C and then cooled at rates obtained by immersion into baths at temperatures ranging from -20 to -50°C had higher hemolysis values than those which were permitted to nucleate spontaneously in this temperature range. Conversely, in the temperature range of -70 to --120°C the extent of injury to specimens nucleated at -2°C was remarkably lower than in those which were permitted to nucleate spontaneously in this range. These results suggest that extensive supercooling of small volumes of blood combined with rapid cooling to low temperatures promotes damaging intracellular ice formation. Extracellular nucleation at temperatures near the freezing point prevents the very rapid removal of the heat of fusion as occurs in the nucleation of a supercooled medium and thereby reduces the likelihood of intracellular freezing during rapid cooling. This work was supported by NIH Grant No. CM 20531. 83. Effect of Extra- and Intracellular IVater Distribution on Optimal Hydroxyethyl Starch
Concentrations for Different Cooling
Red Blood Cells at and Thawing Rates.
hl~x W. SCHEI~E * AXD HANS E. Nn:x* (Spon. S. Seidl) ( Helmholtz-Inst. fiir Biomed. Technik, Aachen, West Germany). Fresh ACD-blood was vvashed twice in phosphate-buffered saline ( PBS ) . Packed erythrocytes were mixed with hydroxyethyl starch (HES)PBS combinations by short shaking and successive gentle rolling for 5 min. Twenty microliters of this mixture were pipetted into glass capillaries. Together with a reference sample for temperature measurement, four samples were frozen at a time in liquid nitrogen or frigen with controlled cooling rates ranging from 80 to 17,00O”K/ min, thawed at 50 to 24,00O”K/min, and submerged into 5 ml of PBS. Supernatant hemoglobin was then measured photometrically after 8 min. In order to vary the water content of the starting medium, HES concentrations were selected be-