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Influence of DMSO on the response of platelets to osmotic stress
700
ABSTRACTS,
14TH
medium the level of this glycoprotein returned to normal and the correction was proportional to the amount of DMSO added. To determine if the effect of DMSO was directed specifically to the membrane as opposed to an intracellular action, we first isolated membranes and then froze them to -10°C in the presence or absence of DMSO. Identical results were achieved in this experiment as with intact platelets. That is, freezing injury was limited to one glycoprotein and the lesion was prevented (or reversed) by DMSO. These results indicate that the nature of freezing injury in platelets is, at least in part, a biochemical alteration of a platelet membrane glycoprotein. DMSO has the ability to reverse or prevent this lesion. 54. Influence of DMSO on the Response of Platelets to Osmotic Stress. RICHARD A. KAHN (Missouri-Illinois Regional Red Cross, St. Louis, Missouri 63108). This study was initiated to gain further nnderstanding of the mechanism of cryoprotection by DMSO. Blood platelets were frozen in the presence of DMSO (2-15s) to various subzero temperatures. Upon thawing, recovery in vitro was assayed by serotonin uptake capacity. It was found that the protective effect of low concentrations of DMSO was due in part to noncolligative mechanism(s), since post-thaw recovery was significantly greater than expected if the presence of DMSO only served to reduce the salt concentration produced at subzero temperatures. Higher starting concentrations of DMSO (lo15%) resulted in recoveries lower than one would expect if DMSO were purely colligative, and it could be demonstrated that recovery was inversely proportional to the concentration of DMSO produced at the final subzero temperature. The noncolligative mechanism(s) could not be ascribed to a lowering of the critical minimum volume of these cells or to an increased resis#tance to shrinkage upon exposure to hypertonic stress without freezing. In addition, platelets exposed to hypertonic stress without freezing succumb at the same osmolality in ‘the presence of DMSO as in its absence. Thus, at low temperatures and with low concentrations of DMSO, platelets can withstand higher concentrations of salt than predicted. 55. Separation of Platelets by Centrifugal Elutriation. MAXIM D. PERSIDSKY AND WILLIAM M. BUCHHOLZ * (Department of Cryobiology, Institutes of Medical Sciences, San Francisco, California 94115). Current methods for separation of platelets involve prolonged centrifugation at high g force,
ANNUAL
MEETING
pelleting, and contact with foreign surfaces, such as in chromatography columns, and with each other. These factors are known to be damaging to platelets. In the present study, centrifugal elutriation was adapted to the separation of platelets utilizing the Beckman JE-6 elutriator rotor in which its small separation chamber (4.5 ml) was substituted by a specially constructed larger chamber (11 ml). In the process of centrifugal elutriation sedimentation of platelets by the centrifugal force is counteracted by the opposing force of liquid flow, so that platelets remain in suspension. This results in minimal contact with foreign surfaces and no pelleting. Separation of platelets with the original chamber requires a higher speed of the rotor (5000 rpm) and a longer time (10 to 15 min), while the new chamber requires 2500 rpm and takes 5 to 7 min to complete separation. Platelets isolated with the larger chamber have normal morphology and apparently normal function. Yields close to 100% have been obtained. Isolation by the smaller chamber is damaging to platelets. Procedural details and the results of functional evaluation of platelets were presented. This work was supported by NIH Grant No. ROl HL20788-01. 56. Preservation of Human Platelets for Tramfusion at $22 and -196°C and the Use of Prostaglandin E,. SAJIO SUMIDA ( National Fukuoka Central Hospital, Jonai 2-2, Fukuoka, 810, Japan ). Blood (200 ml) was drawn from healthy volunteers into blood bags containing CPD solution. Prostaglandin El (100 rig/ml) was added to the blood bags and mixed with CPD solution before blood collection. Other units of blood without added PGEl were treated in the same manner. The platelet concentrate (PC ) prepared by centrifngation was resuspended in a minimum quantity of its residual plasma at room temperature (+22”C) and an equal volume of the cryoprotective solution ( 10% glycerol, 1% mannitol, and 0.75% NaCl in water) was added. The life span of stored platelets was studied using a Yr labeling method. PC was stored in the blood bags with or without PGEl at +22”C for 24 hr. PC prepared with a 5% glycerol solution in a UCAR or a Habia bag were stored in the frozen state for 24 hr. Liquid-stored PC and at -196°C frozen-thawed PC were transfused back into the same healthy donors. The life span of fresh PC prepared with PGEl was similar to that of fresh PC without PGEI. PC treated with PGEl and stored at +22”C for 24 hr had better survival in viva than did PC without PGEI. The life span of frozen PC had better survival than did PC with
ABSTRACTS,
14TH
medium the level of this glycoprotein returned to normal and the correction was proportional to the amount of DMSO added. To determine if the effect of DMSO was directed specifically to the membrane as opposed to an intracellular action, we first isolated membranes and then froze them to -10°C in the presence or absence of DMSO. Identical results were achieved in this experiment as with intact platelets. That is, freezing injury was limited to one glycoprotein and the lesion was prevented (or reversed) by DMSO. These results indicate that the nature of freezing injury in platelets is, at least in part, a biochemical alteration of a platelet membrane glycoprotein. DMSO has the ability to reverse or prevent this lesion. 54. Influence of DMSO on the Response of Platelets to Osmotic Stress. RICHARD A. KAHN (Missouri-Illinois Regional Red Cross, St. Louis, Missouri 63108). This study was initiated to gain further nnderstanding of the mechanism of cryoprotection by DMSO. Blood platelets were frozen in the presence of DMSO (2-15s) to various subzero temperatures. Upon thawing, recovery in vitro was assayed by serotonin uptake capacity. It was found that the protective effect of low concentrations of DMSO was due in part to noncolligative mechanism(s), since post-thaw recovery was significantly greater than expected if the presence of DMSO only served to reduce the salt concentration produced at subzero temperatures. Higher starting concentrations of DMSO (lo15%) resulted in recoveries lower than one would expect if DMSO were purely colligative, and it could be demonstrated that recovery was inversely proportional to the concentration of DMSO produced at the final subzero temperature. The noncolligative mechanism(s) could not be ascribed to a lowering of the critical minimum volume of these cells or to an increased resis#tance to shrinkage upon exposure to hypertonic stress without freezing. In addition, platelets exposed to hypertonic stress without freezing succumb at the same osmolality in ‘the presence of DMSO as in its absence. Thus, at low temperatures and with low concentrations of DMSO, platelets can withstand higher concentrations of salt than predicted. 55. Separation of Platelets by Centrifugal Elutriation. MAXIM D. PERSIDSKY AND WILLIAM M. BUCHHOLZ * (Department of Cryobiology, Institutes of Medical Sciences, San Francisco, California 94115). Current methods for separation of platelets involve prolonged centrifugation at high g force,
ANNUAL
MEETING
pelleting, and contact with foreign surfaces, such as in chromatography columns, and with each other. These factors are known to be damaging to platelets. In the present study, centrifugal elutriation was adapted to the separation of platelets utilizing the Beckman JE-6 elutriator rotor in which its small separation chamber (4.5 ml) was substituted by a specially constructed larger chamber (11 ml). In the process of centrifugal elutriation sedimentation of platelets by the centrifugal force is counteracted by the opposing force of liquid flow, so that platelets remain in suspension. This results in minimal contact with foreign surfaces and no pelleting. Separation of platelets with the original chamber requires a higher speed of the rotor (5000 rpm) and a longer time (10 to 15 min), while the new chamber requires 2500 rpm and takes 5 to 7 min to complete separation. Platelets isolated with the larger chamber have normal morphology and apparently normal function. Yields close to 100% have been obtained. Isolation by the smaller chamber is damaging to platelets. Procedural details and the results of functional evaluation of platelets were presented. This work was supported by NIH Grant No. ROl HL20788-01. 56. Preservation of Human Platelets for Tramfusion at $22 and -196°C and the Use of Prostaglandin E,. SAJIO SUMIDA ( National Fukuoka Central Hospital, Jonai 2-2, Fukuoka, 810, Japan ). Blood (200 ml) was drawn from healthy volunteers into blood bags containing CPD solution. Prostaglandin El (100 rig/ml) was added to the blood bags and mixed with CPD solution before blood collection. Other units of blood without added PGEl were treated in the same manner. The platelet concentrate (PC ) prepared by centrifngation was resuspended in a minimum quantity of its residual plasma at room temperature (+22”C) and an equal volume of the cryoprotective solution ( 10% glycerol, 1% mannitol, and 0.75% NaCl in water) was added. The life span of stored platelets was studied using a Yr labeling method. PC was stored in the blood bags with or without PGEl at +22”C for 24 hr. PC prepared with a 5% glycerol solution in a UCAR or a Habia bag were stored in the frozen state for 24 hr. Liquid-stored PC and at -196°C frozen-thawed PC were transfused back into the same healthy donors. The life span of fresh PC prepared with PGEl was similar to that of fresh PC without PGEI. PC treated with PGEl and stored at +22”C for 24 hr had better survival in viva than did PC without PGEI. The life span of frozen PC had better survival than did PC with