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ADP-Aggregation in platelets frozen with glycerol
ANNUAL
MEETING
sions. Two hundred and seven units of frozen platelets were used in 19 thrombocytopenic patients during the past 2 years, of which eight patients had idiopathic thrombocytopenic purpura. No side effects were observed. 74.
Platelet
Cryopreservation
Using
Dimethyl
B. SEIFERT AND D. GREIFF (Department of Pathology, Medical College of Wisconsin, Milwaukee, WI 53233).
SuZjoxide.
Studies by others have used 510% glycerol or dimethyl sulfoxide (DMSO) as the principal protective additives for the cryopreservation of human platelets. We have developed a method for cryopreservation of platelet preparations using 0.1 M DMSO (0.8%) plus 0.3 M sodium glycerophosphate (NaGly-POJ. In our studies, platelets were cooled at lOO”C/min to a terminal temperature of -70°C and thawed at +lO”C. Initial problems of clumping associated with the addition of sodium glycerophosphate were found to be related to the high pH produced. Clumping was eliminated by lowering the pH of the original platelet preparation to 7.2 by adding extra acid-citrate-dextrose (ACD). The added dextrose was also found to be necessary for optimum preservation and minimal clumping. The effectiveness of this new method has been confirmed by phase microscopy, electron microscopy, and by assay of platelet preparations for the enzyme, leucine aminopeptidase. Fully 35% of the thawed platelets remained in their diskoid shape and another 50% showed minor changes, appearing as small spheres. In vivo studies of the effectiveness of these frozen and thawed platelet preparations for correcting elevated bleeding in aspirinized donors will be reported. (Supported, in part, by contract ~$70-2213. National Heart and Lung Institute, NIH.) 75. Reversal Reaction and Viability of Frozen Human Platelets. B. K. Km1 AND M. BALDINI’
(Division of Hematologic Research. The Memorial Hospital, Pawtucket, and Brown University, Providence, RI 02860). We have previously shown that the biphasic platelet reaction to osmotic stress (reversal reaction), measured by changes in light absorbancy of the suspension at 420 nm is a sensitive indicator of platelet integrity [Fed. Proc. 29, 717 (1970)]. This study has been expanded to demonstrate the relationship between the reversal reaction and survival in vivo of fresh and frozen human platelets, Platelets treated with various concentrations (5, 10 or 15%) of dimethylsulfoxide (DMSO). dimethylacetamide or glycerol were cooled at 1°C per min and stored at -79°C for l-2 days. Thaw-
ABSTRACTS
329
ing was done ‘in a 37°C water bath and additives were removed. Freezing without cryoprotective agents caused complete loss of platelet reversal reaction. With cryoprotective agents, various degrees of depression (55100%) were observed. The smallest depression was seen with 5% DMSO. After labeling with =Cr, survival in vivo of fresh platelets or of platelets frozen with 5% DMSO and reversal reaction were measured simultaneously in the same preparations. Correlation between values of survival and of reversal reaction both in fresh and frozen platelets was highly significant (r = 0.81). Viable human platelets could be preserved by freezing with 5% DMSO with a high degree of efficiency. After storage at -79°C for l-2 days recovery values were 44-52% and life span was eorma1 (S-11 days), It was concluded that the reversal reaction is a sensitive test for the prediction of in &JO viability of stored platelets. (Supported by A.E.C. Contract #AT(ll-I)-3234.) 76. ADP-Aggregation in Platelets Frozen with Glycerol. G. DAYIAN: M. COHEN,’ S. N. CHIN,
A. W. ROWE (Laboratory of Cryobiology, The New York Blood Center, 310, East 67th Street, New York, NY 10021).
AND
A freezing procedure reported previously [Cryo8, 393 (1971)] to reduce cryoinjury to intraplatelet lysosomes was further assessed for its efficacy in preserving platelet function as measured by ADP-induced aggregation. Human washed platelets were frozen in 5% glycerol in saline by cooling immediately to -80°C at 30”C/min and then to -196°C at -lOOO”C/min and stored in liquid nitrogen until thawed at -lOOO”C/min. Deglycerolization was accomplished by dialysis against changing glycerol concentrations (in saline) decreasing at a rate of O.l%/min. Aggregation was determined turbidimetrically with platelets suspended in Eagle’s minimal medium containing albumin and fibrinogen. Preincubation with apyrase of platelets made refractory to ADP by the above procedures was essential for demonstrating aggregation. Frozen and thawed platelets always aggregated in the presence of ADP with a rate indicative of expected normal saturation kinetics. When the frozen and thawed platelets were preincubated with higher concentrations of apyrase and more platelet protein was added to the aggregation medium, aggregation could be obtained which was comparable to that of nonfrozen processed platelets. Functional integrity was thereby demonstrated in the recovered (>50%) intact platelets. Damage resulting from the present freezing procedure was due partly to preferential lysis of old platelets, as indicated incomparative studies of cryoinjury between small-light (old) and largebiology
330
ANNUAL
MEETING
heavy (young) enriched platelet suspensions. (Supported by research grants from NIH (HL-09011) and Union Carbide Corporation.) Mechanical Resistance to Volume Reduction in Supercooled Blood Platelets. R. A. KAHN, AND H. T. MERYMAN (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, MD 20014).
77.
We have previously shown that platelets exposed to hypertonic sodium chloride at 37°C decrease predictably in volume until the osmolality of the extracellular solution reaches four times isotonic. At higher osmolalities volume increases and the platelets are irreversibly injured. If platelets are precooled to -5°C and then exposed to hypertonic salt their volume also decreases, but the rate and extent of reduction is not as great as it was with the 37°C platelets and injury does not occur until eight times isotonic. When platelets at -5°C were ,exposed to a hypertonic solution and then returned to a near isotonic environment the volume reduction was found not to be associated with an influx of extracellular solute as we previously postulated. Furthermore, measurements of the intracellular and extracellular osmolality of platelets in a hypertonic environment revealed a considerable discrepancy between the two compartments with the intracellular osmolality as much as 650 mosm less than the extracellular suspending medium. These results are compatible with a mechanical resistance to volume reduction in supercoo)ed platelets. (Supported in part by National Institutes of Health Contract NIH-‘70-2005.) 78. A Preliminary Evaluation of Additives for the Cryopreservation of Mouse Marrow. .K. R. DILLER, AND H. M. PYLEI (Cryogenic Engineering Laboratory, Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 and Blood Research Institute, Boston, MA 02115). A number of additive compounds have been screened for their ability to protect mouse marrow from freezing injury. Parameters investigated include the composition and concentration of the additive and the cooling rate. Two techniques were employed for freezing the cells: marrow suspensions were frozen at accurately controlled cooling rates either in 2-~1 vol between glass coverslips on a cryomicroscope or in S-ml vol in a small, refrigerated dewar. Recovery was assayed for specimens frozen on the cryomicroscope by postfreeze and postthaw cell morphology and for specimens frozen in the dewnr by microscopic counting, supra-
ABSTRACTS
vital staining, electronic analysis of cell size and distribution, and the stem cell colony technique of Till and McCulloch. The cooling rate was measured via a thermocouple placed into the cell suspension. Survival signatures have been generated in this manner for a number of additives. Some general trends can be observed from this data, including the following: nonpenetrating additives function most effectively at higher cooling rates; penetrating additives function most effectively at lower cooling rates; and as the additive concentration is increased, the best recovery occurs at lower cooling rates. (Supported in part by Research Grants from the American Cancer Society and the Damon Runyon Cancer Fund.) 79. Improved Cryopreservation of Bone Mal.row Treated with Gold Salts. M. D. PERSIDSKY (The
Institute
of Medical
Sciences, Pacific Medical
Center, San Francisco, CA).
In our previous study [Persidsky, Cryobiology 8, 482, (1971)] we have shown that inactivation of lysosomal enzymes by in viva treatment with trypan blue significantly increases bone marrow cell recovery after cryopreservation. In the present study, we have investigated in vitro effects of other inhibitors of lysosomal hydrolysis on cryopreservation of rat bone marrow cells. Cells were incubated at 0°C for 45 min with aurothiomalate (Myochrysine) (0.3 mg/ml) in Hanks’ medium containing 20% rat serum. Thereafter, cells were centrifuged, resuspended in Hanks’ medium containing 10% DMSO, equilibrated for 10 min at O”C, frozen according to the Polge procedure. and then thawed rapidly. The criterion of cell viability was incorporation of [1-“CJglycine at 37°C. The controls were cells which were stored without freezing at 0°C in Hanks’ medium containing 20% serum. Cells frozen without DMSO and without gold incorporated less than 0.4% of the radioactivity of the controls. Cells frozen with DMSO and without the gold pretreatment incorporated 35% of the control level. Cells frozen with DMSO after the gold pretreatment incorporated the same amount of label as the controls. Aurothioglucose and suramin have also been tested. The effects of these compounds have been tested, in addition, on cryopreservation of human white blood cells, and these results will be presented. These findings support our hypothesis that lysosomes are primary targets of cell cryoinjury, and that the inactivation of lysosomal enzymes may be a significant factor in the development of methods for long-term banking of cells and organs by cryopreservation and by perfusion. (Supported by U.S.P.H.S. Grant No. 1 R01 (X12754-01.)
MEETING
sions. Two hundred and seven units of frozen platelets were used in 19 thrombocytopenic patients during the past 2 years, of which eight patients had idiopathic thrombocytopenic purpura. No side effects were observed. 74.
Platelet
Cryopreservation
Using
Dimethyl
B. SEIFERT AND D. GREIFF (Department of Pathology, Medical College of Wisconsin, Milwaukee, WI 53233).
SuZjoxide.
Studies by others have used 510% glycerol or dimethyl sulfoxide (DMSO) as the principal protective additives for the cryopreservation of human platelets. We have developed a method for cryopreservation of platelet preparations using 0.1 M DMSO (0.8%) plus 0.3 M sodium glycerophosphate (NaGly-POJ. In our studies, platelets were cooled at lOO”C/min to a terminal temperature of -70°C and thawed at +lO”C. Initial problems of clumping associated with the addition of sodium glycerophosphate were found to be related to the high pH produced. Clumping was eliminated by lowering the pH of the original platelet preparation to 7.2 by adding extra acid-citrate-dextrose (ACD). The added dextrose was also found to be necessary for optimum preservation and minimal clumping. The effectiveness of this new method has been confirmed by phase microscopy, electron microscopy, and by assay of platelet preparations for the enzyme, leucine aminopeptidase. Fully 35% of the thawed platelets remained in their diskoid shape and another 50% showed minor changes, appearing as small spheres. In vivo studies of the effectiveness of these frozen and thawed platelet preparations for correcting elevated bleeding in aspirinized donors will be reported. (Supported, in part, by contract ~$70-2213. National Heart and Lung Institute, NIH.) 75. Reversal Reaction and Viability of Frozen Human Platelets. B. K. Km1 AND M. BALDINI’
(Division of Hematologic Research. The Memorial Hospital, Pawtucket, and Brown University, Providence, RI 02860). We have previously shown that the biphasic platelet reaction to osmotic stress (reversal reaction), measured by changes in light absorbancy of the suspension at 420 nm is a sensitive indicator of platelet integrity [Fed. Proc. 29, 717 (1970)]. This study has been expanded to demonstrate the relationship between the reversal reaction and survival in vivo of fresh and frozen human platelets, Platelets treated with various concentrations (5, 10 or 15%) of dimethylsulfoxide (DMSO). dimethylacetamide or glycerol were cooled at 1°C per min and stored at -79°C for l-2 days. Thaw-
ABSTRACTS
329
ing was done ‘in a 37°C water bath and additives were removed. Freezing without cryoprotective agents caused complete loss of platelet reversal reaction. With cryoprotective agents, various degrees of depression (55100%) were observed. The smallest depression was seen with 5% DMSO. After labeling with =Cr, survival in vivo of fresh platelets or of platelets frozen with 5% DMSO and reversal reaction were measured simultaneously in the same preparations. Correlation between values of survival and of reversal reaction both in fresh and frozen platelets was highly significant (r = 0.81). Viable human platelets could be preserved by freezing with 5% DMSO with a high degree of efficiency. After storage at -79°C for l-2 days recovery values were 44-52% and life span was eorma1 (S-11 days), It was concluded that the reversal reaction is a sensitive test for the prediction of in &JO viability of stored platelets. (Supported by A.E.C. Contract #AT(ll-I)-3234.) 76. ADP-Aggregation in Platelets Frozen with Glycerol. G. DAYIAN: M. COHEN,’ S. N. CHIN,
A. W. ROWE (Laboratory of Cryobiology, The New York Blood Center, 310, East 67th Street, New York, NY 10021).
AND
A freezing procedure reported previously [Cryo8, 393 (1971)] to reduce cryoinjury to intraplatelet lysosomes was further assessed for its efficacy in preserving platelet function as measured by ADP-induced aggregation. Human washed platelets were frozen in 5% glycerol in saline by cooling immediately to -80°C at 30”C/min and then to -196°C at -lOOO”C/min and stored in liquid nitrogen until thawed at -lOOO”C/min. Deglycerolization was accomplished by dialysis against changing glycerol concentrations (in saline) decreasing at a rate of O.l%/min. Aggregation was determined turbidimetrically with platelets suspended in Eagle’s minimal medium containing albumin and fibrinogen. Preincubation with apyrase of platelets made refractory to ADP by the above procedures was essential for demonstrating aggregation. Frozen and thawed platelets always aggregated in the presence of ADP with a rate indicative of expected normal saturation kinetics. When the frozen and thawed platelets were preincubated with higher concentrations of apyrase and more platelet protein was added to the aggregation medium, aggregation could be obtained which was comparable to that of nonfrozen processed platelets. Functional integrity was thereby demonstrated in the recovered (>50%) intact platelets. Damage resulting from the present freezing procedure was due partly to preferential lysis of old platelets, as indicated incomparative studies of cryoinjury between small-light (old) and largebiology
330
ANNUAL
MEETING
heavy (young) enriched platelet suspensions. (Supported by research grants from NIH (HL-09011) and Union Carbide Corporation.) Mechanical Resistance to Volume Reduction in Supercooled Blood Platelets. R. A. KAHN, AND H. T. MERYMAN (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, MD 20014).
77.
We have previously shown that platelets exposed to hypertonic sodium chloride at 37°C decrease predictably in volume until the osmolality of the extracellular solution reaches four times isotonic. At higher osmolalities volume increases and the platelets are irreversibly injured. If platelets are precooled to -5°C and then exposed to hypertonic salt their volume also decreases, but the rate and extent of reduction is not as great as it was with the 37°C platelets and injury does not occur until eight times isotonic. When platelets at -5°C were ,exposed to a hypertonic solution and then returned to a near isotonic environment the volume reduction was found not to be associated with an influx of extracellular solute as we previously postulated. Furthermore, measurements of the intracellular and extracellular osmolality of platelets in a hypertonic environment revealed a considerable discrepancy between the two compartments with the intracellular osmolality as much as 650 mosm less than the extracellular suspending medium. These results are compatible with a mechanical resistance to volume reduction in supercoo)ed platelets. (Supported in part by National Institutes of Health Contract NIH-‘70-2005.) 78. A Preliminary Evaluation of Additives for the Cryopreservation of Mouse Marrow. .K. R. DILLER, AND H. M. PYLEI (Cryogenic Engineering Laboratory, Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 and Blood Research Institute, Boston, MA 02115). A number of additive compounds have been screened for their ability to protect mouse marrow from freezing injury. Parameters investigated include the composition and concentration of the additive and the cooling rate. Two techniques were employed for freezing the cells: marrow suspensions were frozen at accurately controlled cooling rates either in 2-~1 vol between glass coverslips on a cryomicroscope or in S-ml vol in a small, refrigerated dewar. Recovery was assayed for specimens frozen on the cryomicroscope by postfreeze and postthaw cell morphology and for specimens frozen in the dewnr by microscopic counting, supra-
ABSTRACTS
vital staining, electronic analysis of cell size and distribution, and the stem cell colony technique of Till and McCulloch. The cooling rate was measured via a thermocouple placed into the cell suspension. Survival signatures have been generated in this manner for a number of additives. Some general trends can be observed from this data, including the following: nonpenetrating additives function most effectively at higher cooling rates; penetrating additives function most effectively at lower cooling rates; and as the additive concentration is increased, the best recovery occurs at lower cooling rates. (Supported in part by Research Grants from the American Cancer Society and the Damon Runyon Cancer Fund.) 79. Improved Cryopreservation of Bone Mal.row Treated with Gold Salts. M. D. PERSIDSKY (The
Institute
of Medical
Sciences, Pacific Medical
Center, San Francisco, CA).
In our previous study [Persidsky, Cryobiology 8, 482, (1971)] we have shown that inactivation of lysosomal enzymes by in viva treatment with trypan blue significantly increases bone marrow cell recovery after cryopreservation. In the present study, we have investigated in vitro effects of other inhibitors of lysosomal hydrolysis on cryopreservation of rat bone marrow cells. Cells were incubated at 0°C for 45 min with aurothiomalate (Myochrysine) (0.3 mg/ml) in Hanks’ medium containing 20% rat serum. Thereafter, cells were centrifuged, resuspended in Hanks’ medium containing 10% DMSO, equilibrated for 10 min at O”C, frozen according to the Polge procedure. and then thawed rapidly. The criterion of cell viability was incorporation of [1-“CJglycine at 37°C. The controls were cells which were stored without freezing at 0°C in Hanks’ medium containing 20% serum. Cells frozen without DMSO and without gold incorporated less than 0.4% of the radioactivity of the controls. Cells frozen with DMSO and without the gold pretreatment incorporated 35% of the control level. Cells frozen with DMSO after the gold pretreatment incorporated the same amount of label as the controls. Aurothioglucose and suramin have also been tested. The effects of these compounds have been tested, in addition, on cryopreservation of human white blood cells, and these results will be presented. These findings support our hypothesis that lysosomes are primary targets of cell cryoinjury, and that the inactivation of lysosomal enzymes may be a significant factor in the development of methods for long-term banking of cells and organs by cryopreservation and by perfusion. (Supported by U.S.P.H.S. Grant No. 1 R01 (X12754-01.)