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Preservation of human platelets for transfusion at +22 and −196 °C and the use of prostaglandin E1
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
or without PGE, stored at +22”C for 24 hr. PGEl improved the life span of frozen PC. The addition of 100 ng of PGE1/ml of whole blood produced an improvement in the life span of either liquid-stored PC or previously frozen PC. Sumida, S. Preservation of human platelets for transfusion at +22” and -196”C, and prostaglandin E1. Low Temp. Med. 2, 131-135 (1976). Dayian, G., and Rowe, A. W. Cryopreservation of human platelets for transfusion. Cryobiology 13, l-8 ( 1976). Valeri, C. R., et al. Prostaglandin in the preparation of blood components. Science 175, 539542 ( 1972). SESSION
S7. pH
F. SPERMATOZOA, AND EMBRYOS
OVA,
Change of Bovine Semen Bugler with Temperature. R. S. JEYENDRAN AND E. F. GRAHAM (University of Minnesota, St. Paul, Minnesota 55108).
The pH change due to temperature was measured for a combination of Good’s buffers, Tes and Tris (TEST), with and without egg yolk containing 2 or 6% of 19 different cryoprotective compounds. The pH at 5°C was significantly higher than at room temperature. The addition of egg yolk and/or cryoprotective compound did not alter the pH significantly during cooling, even though a slight drop in pH was noted with the addition of 20% egg yolk, indicating that the change in pH is primarily due to the buffer. The pH change of 10 different buffering systems with temperature from 22 to 5°C was examined. Of these, three were conventional buffers, which included phosphate yolk, citrate yolk, and skim milk, and seven were Goods buffers with egg yolk, which included Tes, Tris, Bes, Mops, Pipes, Mes, and TEST. The results showed that the pH of the three conventional buffers did not change with decreasing temperature, but Good’s buffers showed an increase in pH with decreasing temperature from 22 to 5°C even though they had an excellent buffering capacity. (pK, of Good’s buffers changed very slightly with temperature. ) The pH of TEST yolk buffer (pH 7.2 at room temperature) was measured con’tinuously from 37°C to below freezing (-19°C). The pH increased linearly with decreasing temperature to pH 8.0 C 0.2 from 37°C to about -14°C at which point it dropped abruptly to pH 6.5 -t 0.2. The change in pH observed with decreasing temperature did not alter the viability of bovine spermatozoa during freezing and rewarming.
ANNUAL
IrlEETING
701
58.1 Effect of Freezing on Bovine Semen Dduter Components. CHRISTOPHER P. D'ALLEINXE* ASD C. P. MERILAN (University of hfissonri, Columbia, Missouri 65201). 59. Relation between Different Assays for Sperm Quality and Fertility of Frozen Boar Semen. &I. K. PAVELKO,* S. EIXARSSON," ASD B. G. CRABO (Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108). Fertility of boar semen appears to be poorly related to currently used laboratory techniques. This study attempted to correlate the fertility of frozen boar semen with an in vitro test measuring the uptake of ‘=I-labelled boar seminal proteins added to the thawing fluid. Semen from eight boars was frozen according to the Beltsville concentrated technique and thawed in OLEP (Larsson and Einarsson, Acta Vet. Scud. 17, 43, 1976), BTS (Purse1 and Johnson, J. Anim. Sci. 40, 99, 1975), and TESNaK. The first ‘two fluids have resulted in good fertility and TESNaK in zero fertility (Crabo, Larson, and Graham, Cryobiology 9, 331, 1972). Sperm thawed in OLEP featured the highest uptake of radiolabelled proteins (874%) of the plasma activity and those in TESNaK the lowest (402%). There was no difference in the percentage of normal acrosome (21-25s) or osmotically reactive cells (21-25s) between the fluids but the sperm motility was lower in TESNaK (8 vs 26% ). Furthermore, the fertility of the eight boars was assessed after insemination of 16-37 sows per boar with semen frozen as described above and thawed in BTS. The fertility varied from 23 to 71% between boars. The correlation between fertility of the individual boars and sperm motility was 0.19, between fertility and percentage normal acrosome ridges 0.29, and between fertility and the “protein uptake” in BTS 0.43. However, a correlation coefficient of 0.77 was obtained between fertility and the protein uptake in TESNaK. Thus, it appears that the less beneficial thawing fluid aided in disclosing differences between boars. It is possible that the composition of the thawing fluids affects the binding mechanisms of surface coating macromolecules in the sperm membrane. Alterations of the sperm surface may result in rapid elimination of the spermatozoa from the female tract. Differences between boars in this respect may account for loss of fertility by certain boars after freezing of the semen. 60. Acrosornal Proteolytic Actioity of FrozenThawed Boar Spermutozoa. K. I. LARS~~N * (Span. B. G. Crabo) (Department of Ob-
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
or without PGE, stored at +22”C for 24 hr. PGEl improved the life span of frozen PC. The addition of 100 ng of PGE1/ml of whole blood produced an improvement in the life span of either liquid-stored PC or previously frozen PC. Sumida, S. Preservation of human platelets for transfusion at +22” and -196”C, and prostaglandin E1. Low Temp. Med. 2, 131-135 (1976). Dayian, G., and Rowe, A. W. Cryopreservation of human platelets for transfusion. Cryobiology 13, l-8 ( 1976). Valeri, C. R., et al. Prostaglandin in the preparation of blood components. Science 175, 539542 ( 1972). SESSION
S7. pH
F. SPERMATOZOA, AND EMBRYOS
OVA,
Change of Bovine Semen Bugler with Temperature. R. S. JEYENDRAN AND E. F. GRAHAM (University of Minnesota, St. Paul, Minnesota 55108).
The pH change due to temperature was measured for a combination of Good’s buffers, Tes and Tris (TEST), with and without egg yolk containing 2 or 6% of 19 different cryoprotective compounds. The pH at 5°C was significantly higher than at room temperature. The addition of egg yolk and/or cryoprotective compound did not alter the pH significantly during cooling, even though a slight drop in pH was noted with the addition of 20% egg yolk, indicating that the change in pH is primarily due to the buffer. The pH change of 10 different buffering systems with temperature from 22 to 5°C was examined. Of these, three were conventional buffers, which included phosphate yolk, citrate yolk, and skim milk, and seven were Goods buffers with egg yolk, which included Tes, Tris, Bes, Mops, Pipes, Mes, and TEST. The results showed that the pH of the three conventional buffers did not change with decreasing temperature, but Good’s buffers showed an increase in pH with decreasing temperature from 22 to 5°C even though they had an excellent buffering capacity. (pK, of Good’s buffers changed very slightly with temperature. ) The pH of TEST yolk buffer (pH 7.2 at room temperature) was measured con’tinuously from 37°C to below freezing (-19°C). The pH increased linearly with decreasing temperature to pH 8.0 C 0.2 from 37°C to about -14°C at which point it dropped abruptly to pH 6.5 -t 0.2. The change in pH observed with decreasing temperature did not alter the viability of bovine spermatozoa during freezing and rewarming.
ANNUAL
IrlEETING
701
58.1 Effect of Freezing on Bovine Semen Dduter Components. CHRISTOPHER P. D'ALLEINXE* ASD C. P. MERILAN (University of hfissonri, Columbia, Missouri 65201). 59. Relation between Different Assays for Sperm Quality and Fertility of Frozen Boar Semen. &I. K. PAVELKO,* S. EIXARSSON," ASD B. G. CRABO (Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108). Fertility of boar semen appears to be poorly related to currently used laboratory techniques. This study attempted to correlate the fertility of frozen boar semen with an in vitro test measuring the uptake of ‘=I-labelled boar seminal proteins added to the thawing fluid. Semen from eight boars was frozen according to the Beltsville concentrated technique and thawed in OLEP (Larsson and Einarsson, Acta Vet. Scud. 17, 43, 1976), BTS (Purse1 and Johnson, J. Anim. Sci. 40, 99, 1975), and TESNaK. The first ‘two fluids have resulted in good fertility and TESNaK in zero fertility (Crabo, Larson, and Graham, Cryobiology 9, 331, 1972). Sperm thawed in OLEP featured the highest uptake of radiolabelled proteins (874%) of the plasma activity and those in TESNaK the lowest (402%). There was no difference in the percentage of normal acrosome (21-25s) or osmotically reactive cells (21-25s) between the fluids but the sperm motility was lower in TESNaK (8 vs 26% ). Furthermore, the fertility of the eight boars was assessed after insemination of 16-37 sows per boar with semen frozen as described above and thawed in BTS. The fertility varied from 23 to 71% between boars. The correlation between fertility of the individual boars and sperm motility was 0.19, between fertility and percentage normal acrosome ridges 0.29, and between fertility and the “protein uptake” in BTS 0.43. However, a correlation coefficient of 0.77 was obtained between fertility and the protein uptake in TESNaK. Thus, it appears that the less beneficial thawing fluid aided in disclosing differences between boars. It is possible that the composition of the thawing fluids affects the binding mechanisms of surface coating macromolecules in the sperm membrane. Alterations of the sperm surface may result in rapid elimination of the spermatozoa from the female tract. Differences between boars in this respect may account for loss of fertility by certain boars after freezing of the semen. 60. Acrosornal Proteolytic Actioity of FrozenThawed Boar Spermutozoa. K. I. LARS~~N * (Span. B. G. Crabo) (Department of Ob-