- Email: [email protected]
The effect of glycerol on the uptake of serotonin by platelets
ABSTRACTS,
16th ANNUAL
Aliyuot Freezing for the Neonate. M. L. FRANKS AND J. A. GRIEP (Indiana University School of Medicine, 1100 East Michigan Street, Indianapolis, Indiana 46223).
57. Small
The Frozen Blood Laboratory adapted a technique for freezing and deglycerolizing small aliquots of blood in third packs for replacement therapy for the neonate. The plan made exclusive use of group 0, Rh-negative cells because of its versatility. The third packs were subdivided into syringes and dispensed uncrossmatched after appropriate testing for irregular antibodies had been completed on mother and neonate. Prior to starting the program, 15 units were tested for quality of the deglycerolized product. Average yield of cells per third pack was between 50 and 65 ml. The mean average recovery was 87%. The mean free hemoglobin of the final supernatant solution was 15 mg/dl, with a mean osmolality of 200 mosmolikg. The potassium concentration in the supernatant was 0.56 meq/liter and had a pH of 6.6 at room temperature. After 1 year of clinical use, post-transfusion data were collected on approximately 200 neonates. Replacement volume ranged between 10 and 12% of the neonates’ total blood volume. Postinfusion hemoglobin increment averaged 2 g, neg blood pH was -0.01 pH unit, and no significant change occurred in PO2 or PCO,. In \*i\w. survival was studied by means of the Ashby Hemagglutination Test. The results indicated that the transfused cells survived approximately the same as the neonate’s own cells after 24 hr postinfusion. As a sterility check, 20 syringes were cultured and appeared to be negative after 24 hr at 4°C. Antibody studies performed on 60 neonates detected cold agglutinins from a few of the samples. There have been no reports of postinfusion complications due to the transfusions. The major advantages to the blood bank were better availability of fresh blood for the newborn, and small aliquots of blood could be thawed as needed which resulted in more efficient blood utilization. 58. Studies
ofthe
Srtspe~~sio~. NIKOLOV,
Eqrtilibrcrtion L. Tsv.
TSONEV, TSVETKOV,
o.f MeJO N.
in (I Platelet
ALEKSIEV, AND
AL.
CH. MILEV
(Central Problem Laboratory for Freeze-Drying and Cryobiology, Sofia, Bulgaria). In the following investigation an attempt was made for a correlated determination of the penetration time of a preserving solution (5% Me,SO + 2.5% glucose in plasma) into a platelet suspension, intended for a low-temperature preservation. Two methods were used: an osmometric and a spectrometric one. The results obtained by the two methods agreed quite satisfactorily. This allowed us to fix the equilibration time between the 14th and the 22nd minute. After completing the equilibration period deviations from the established final values were observed (increasing and decreasing of osmotic concentration and of light absorb-
MEETING
601
tion). Platelet suspensions were program-frozen after equilibration. The results obtained illustrate a successful low-temperature preservation. Vitality and functional qualities of platelets were determined by the traditional methods: platelet number, reversible reaction to hypotonic stress, and clot retraction. 59. Cryoprotectant ensation.
Combinations
fk-
Platelet
Prrs-
J. L. BURTON AND H. T. MERYMAN (Cryobiology Laboratory, American Red Cross, 9312 Old Georgetown Road, Bethesda, Maryland 20014).
Human platelets frozen at the optimum rate of I-3”Cimin in 5% (0.7 M) dimethylsulfoxide (Me,SO) will remain stable at -80°C for several months. Platelets frozen in a similar concentration of glycerol at an optimum rate of approximately 30”Cimin are unstable at -80°C and must be stored using liquid nitrogen. In order to improve the stability at -80°C using glycerol, the effect of higher concentrations was investigated. The feasibility of using higher concentrations was found to be severely limited by the slow rate of glycerol permeation into the cells. The time required for glycerolizing and deglycerolizing became excessive and the many dilutions required for the stepwise changes in concentration resulted in unmanageable volumes requiring sedimentation steps that in turn resulted in cell loss through clumping. The difference in optimum cooling rates for Me,SO and glycerol implies that the former solute reduces cell hydraulic permeability, which might also explain the increased stability of MeSO-frozen platelets at -80°C. The possibility was therefore explored that low concentrations of Me,SO added to glycerol might enhance cell storage stability. Using morphological scoring as an assay, it was found that the addition of only 150 mM dimethylsulfoxide (Me,SO) to 1.5 M glycerol increased the post-thaw morphological score from 22 to 65% of control after storage at -80°C for 3 days. The addition of 50 mM urea to this solution further increased the post-thaw score to 70% after 3 days at -80°C. After 7 and 15 days platelets frozen with glycerol, Me,SO, and urea remained unchanged while those frozen with glycerol and Me,SO only scored 46 and 0%. respectively. 60. The Effect of G/yc,erol on the Uptake o,f Serotonin by Platelets. W. J. ARMITAGE (MRC Medical
Cryobiology Group, University Department of Surgery, Cambridge, United Kingdom). The transport of serotonin by platelets is a membrane-based, energy-dependent process that is amenable to kinetic analysis. In this study, the rate of uptake of serotonin was measured and the effect of glycerol evaluated. Platelet-rich plasma was separated from fresh human blood and the platelets were incubated with either saline or glycerol (1 moliliter) at
602
ABSTRACTS,
16th
37°C. Tritiated serotonin was added (0.5 to 2.0 PmoV liter substrate concentration) and the uptake stopped after 10 set with ice-cold EDTA-saline to ensure that only the initial rate of uptake was measured. The activity in the platelets was determined by liquid scintillation counting and the rate of uptake expressed as picomoles per 10” cells per 10 sec. The Michaelis constant, K,, , and the theoretical maximum rate of uptake, V,,,, were determined from a double reciprocal plot of the rate of uptake against substrate concentration. After 2 min of incubation with saline at 37”C, the K,,, was 1.2 ? 0.2 pmoiliter and the V,,, 32.1 2 3.0 pmol/lO* cells/l0 sec. These values were unaffected by 30 min of incubation. When platelets were incubated for 2 min with glycerol, V,,, was reduced to 17.5 2 2.6 pmol/lO* cells/l0 set but K,,, was unchanged at 1.2 + 0.2 WmoVliter. This implied that the uptake of serotonin was inhibited noncompetitively. The inhibition lessened with time so that after 30 min of incubation with glycerol, V,,, had reached 29.2 2 1.4 pmol/lO* cells/l0 set, implying that the effect was osmotic and that it was nullified by the movement of glycerol into the platelets. WORKSHOP
3.
GAMETE
AND
EMBRYO
PRESERVATION
61. Comparati~~e
Aspects
of Freezing
Spermatozoa.
B. G. CRABO (Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108). In successful freezing of spermatozoa, cryoprotective systems, freeze rate, and method of thawing play an important role and have to be adjusted for each species. Differences in response are likely to depend on variations in sperm membrane composition. The importance and behavior of seminal proteins in freezing and for fertilization will be described, using swine as an example. Fertility trials are the only reliable assay of the outcome of freezing. This is complicated by specifics in the reproductive anatomy and physiology of particular species. Laboratory assays must evaluate many different functions of the fertilizing mechanisms in the spermatozoa to be of value for assessing the fertilizing capacity in some cases. Attempts to evaluate membrane function in the laboratory are discussed. 62. Status
of Rrseurch in the Cryopresenwtion Spermutozou. J. K. SHERMAN
of
(Department of Anatomy, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72201). H1rt77un
A historical synopsis of research in the cryobiology of human spermatozoa is presented, to illustrate their contribution to basic and applied aspects of the discipline, especially in cryobanking. The type, extent, and results of recent and current investigations in this labo-
ANNUAL
MEETING
ratory and in others are discussed and evaluated. Contemporary methods of cryopreservation used in cryobanks here in the United States and abroad are described and compared. Finally, the future of cryobiology of human spermatozoa is discussed in terms of basic and applied research avenues and efforts. 63. The Importance tion in Farm
of Embryo or Oocyte PreservaAnimal Production and Research.
K. J. BETTERIDGE (Animal Pathology Directorate, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute, P.O. Box 11300, Station H, Nepean, Ontario K2H 8P9, Canada). The current status of general embryo transfer procedures is briefly reviewed in order to give perspective to the need for preserving embryos and/or oocytes. The development of short- and long-term preservation methods to date is summarized with indications of their efficacy but without methodological details. Present and potential applications of cryopreservation of embryos and oocytes to farm animal production (e.g., in “banking” embryos; moving livestock internationally; holding embryos pending the results of sexing or other analyses) and research (e.g., comparison of foundation and improved stock in genetic work) are introduced. It is anticipated that subsequent reports will describe details of freezing and thawing methods and amplify selected examples of how preserved embryos are being used in transfer programmes. 64. Freezing, Bovine
an Alternative to Discarding Embryos. ROBERT D. BAKER
Embryos, Inc., Middleville,
Excess
(American Michigan 49333).
Effective methods have been developed to superovulate cattle and to recover embryos under commercial conditions. However, one out of every seven recoveries results in an excess of embryos, that is, more embryos than should or could be transferred into synchronized recipients. Procedures used to freeze the excess embryos for long-term storage or export are under investigation. Acceptable conception rates using frozen-thawed embryos have not yet been achieved. Rates over 40% will likely be required for extensive commercial use of frozen-thawed bovine embryos. 65. The Application cial Bovine
of Cryopreservation Embryo Transfer.
to Commer-
HARLEY J. SCHNEIDER, JR. (Rio Vista International, Inc., San Antonio, Texas).
The use of embryo cryopreservation on a commercial basis is discussed. Selected topics for discussion are: selection of a freezing program, i.e., long vs short
16th ANNUAL
Aliyuot Freezing for the Neonate. M. L. FRANKS AND J. A. GRIEP (Indiana University School of Medicine, 1100 East Michigan Street, Indianapolis, Indiana 46223).
57. Small
The Frozen Blood Laboratory adapted a technique for freezing and deglycerolizing small aliquots of blood in third packs for replacement therapy for the neonate. The plan made exclusive use of group 0, Rh-negative cells because of its versatility. The third packs were subdivided into syringes and dispensed uncrossmatched after appropriate testing for irregular antibodies had been completed on mother and neonate. Prior to starting the program, 15 units were tested for quality of the deglycerolized product. Average yield of cells per third pack was between 50 and 65 ml. The mean average recovery was 87%. The mean free hemoglobin of the final supernatant solution was 15 mg/dl, with a mean osmolality of 200 mosmolikg. The potassium concentration in the supernatant was 0.56 meq/liter and had a pH of 6.6 at room temperature. After 1 year of clinical use, post-transfusion data were collected on approximately 200 neonates. Replacement volume ranged between 10 and 12% of the neonates’ total blood volume. Postinfusion hemoglobin increment averaged 2 g, neg blood pH was -0.01 pH unit, and no significant change occurred in PO2 or PCO,. In \*i\w. survival was studied by means of the Ashby Hemagglutination Test. The results indicated that the transfused cells survived approximately the same as the neonate’s own cells after 24 hr postinfusion. As a sterility check, 20 syringes were cultured and appeared to be negative after 24 hr at 4°C. Antibody studies performed on 60 neonates detected cold agglutinins from a few of the samples. There have been no reports of postinfusion complications due to the transfusions. The major advantages to the blood bank were better availability of fresh blood for the newborn, and small aliquots of blood could be thawed as needed which resulted in more efficient blood utilization. 58. Studies
ofthe
Srtspe~~sio~. NIKOLOV,
Eqrtilibrcrtion L. Tsv.
TSONEV, TSVETKOV,
o.f MeJO N.
in (I Platelet
ALEKSIEV, AND
AL.
CH. MILEV
(Central Problem Laboratory for Freeze-Drying and Cryobiology, Sofia, Bulgaria). In the following investigation an attempt was made for a correlated determination of the penetration time of a preserving solution (5% Me,SO + 2.5% glucose in plasma) into a platelet suspension, intended for a low-temperature preservation. Two methods were used: an osmometric and a spectrometric one. The results obtained by the two methods agreed quite satisfactorily. This allowed us to fix the equilibration time between the 14th and the 22nd minute. After completing the equilibration period deviations from the established final values were observed (increasing and decreasing of osmotic concentration and of light absorb-
MEETING
601
tion). Platelet suspensions were program-frozen after equilibration. The results obtained illustrate a successful low-temperature preservation. Vitality and functional qualities of platelets were determined by the traditional methods: platelet number, reversible reaction to hypotonic stress, and clot retraction. 59. Cryoprotectant ensation.
Combinations
fk-
Platelet
Prrs-
J. L. BURTON AND H. T. MERYMAN (Cryobiology Laboratory, American Red Cross, 9312 Old Georgetown Road, Bethesda, Maryland 20014).
Human platelets frozen at the optimum rate of I-3”Cimin in 5% (0.7 M) dimethylsulfoxide (Me,SO) will remain stable at -80°C for several months. Platelets frozen in a similar concentration of glycerol at an optimum rate of approximately 30”Cimin are unstable at -80°C and must be stored using liquid nitrogen. In order to improve the stability at -80°C using glycerol, the effect of higher concentrations was investigated. The feasibility of using higher concentrations was found to be severely limited by the slow rate of glycerol permeation into the cells. The time required for glycerolizing and deglycerolizing became excessive and the many dilutions required for the stepwise changes in concentration resulted in unmanageable volumes requiring sedimentation steps that in turn resulted in cell loss through clumping. The difference in optimum cooling rates for Me,SO and glycerol implies that the former solute reduces cell hydraulic permeability, which might also explain the increased stability of MeSO-frozen platelets at -80°C. The possibility was therefore explored that low concentrations of Me,SO added to glycerol might enhance cell storage stability. Using morphological scoring as an assay, it was found that the addition of only 150 mM dimethylsulfoxide (Me,SO) to 1.5 M glycerol increased the post-thaw morphological score from 22 to 65% of control after storage at -80°C for 3 days. The addition of 50 mM urea to this solution further increased the post-thaw score to 70% after 3 days at -80°C. After 7 and 15 days platelets frozen with glycerol, Me,SO, and urea remained unchanged while those frozen with glycerol and Me,SO only scored 46 and 0%. respectively. 60. The Effect of G/yc,erol on the Uptake o,f Serotonin by Platelets. W. J. ARMITAGE (MRC Medical
Cryobiology Group, University Department of Surgery, Cambridge, United Kingdom). The transport of serotonin by platelets is a membrane-based, energy-dependent process that is amenable to kinetic analysis. In this study, the rate of uptake of serotonin was measured and the effect of glycerol evaluated. Platelet-rich plasma was separated from fresh human blood and the platelets were incubated with either saline or glycerol (1 moliliter) at
602
ABSTRACTS,
16th
37°C. Tritiated serotonin was added (0.5 to 2.0 PmoV liter substrate concentration) and the uptake stopped after 10 set with ice-cold EDTA-saline to ensure that only the initial rate of uptake was measured. The activity in the platelets was determined by liquid scintillation counting and the rate of uptake expressed as picomoles per 10” cells per 10 sec. The Michaelis constant, K,, , and the theoretical maximum rate of uptake, V,,,, were determined from a double reciprocal plot of the rate of uptake against substrate concentration. After 2 min of incubation with saline at 37”C, the K,,, was 1.2 ? 0.2 pmoiliter and the V,,, 32.1 2 3.0 pmol/lO* cells/l0 sec. These values were unaffected by 30 min of incubation. When platelets were incubated for 2 min with glycerol, V,,, was reduced to 17.5 2 2.6 pmol/lO* cells/l0 set but K,,, was unchanged at 1.2 + 0.2 WmoVliter. This implied that the uptake of serotonin was inhibited noncompetitively. The inhibition lessened with time so that after 30 min of incubation with glycerol, V,,, had reached 29.2 2 1.4 pmol/lO* cells/l0 set, implying that the effect was osmotic and that it was nullified by the movement of glycerol into the platelets. WORKSHOP
3.
GAMETE
AND
EMBRYO
PRESERVATION
61. Comparati~~e
Aspects
of Freezing
Spermatozoa.
B. G. CRABO (Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108). In successful freezing of spermatozoa, cryoprotective systems, freeze rate, and method of thawing play an important role and have to be adjusted for each species. Differences in response are likely to depend on variations in sperm membrane composition. The importance and behavior of seminal proteins in freezing and for fertilization will be described, using swine as an example. Fertility trials are the only reliable assay of the outcome of freezing. This is complicated by specifics in the reproductive anatomy and physiology of particular species. Laboratory assays must evaluate many different functions of the fertilizing mechanisms in the spermatozoa to be of value for assessing the fertilizing capacity in some cases. Attempts to evaluate membrane function in the laboratory are discussed. 62. Status
of Rrseurch in the Cryopresenwtion Spermutozou. J. K. SHERMAN
of
(Department of Anatomy, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72201). H1rt77un
A historical synopsis of research in the cryobiology of human spermatozoa is presented, to illustrate their contribution to basic and applied aspects of the discipline, especially in cryobanking. The type, extent, and results of recent and current investigations in this labo-
ANNUAL
MEETING
ratory and in others are discussed and evaluated. Contemporary methods of cryopreservation used in cryobanks here in the United States and abroad are described and compared. Finally, the future of cryobiology of human spermatozoa is discussed in terms of basic and applied research avenues and efforts. 63. The Importance tion in Farm
of Embryo or Oocyte PreservaAnimal Production and Research.
K. J. BETTERIDGE (Animal Pathology Directorate, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute, P.O. Box 11300, Station H, Nepean, Ontario K2H 8P9, Canada). The current status of general embryo transfer procedures is briefly reviewed in order to give perspective to the need for preserving embryos and/or oocytes. The development of short- and long-term preservation methods to date is summarized with indications of their efficacy but without methodological details. Present and potential applications of cryopreservation of embryos and oocytes to farm animal production (e.g., in “banking” embryos; moving livestock internationally; holding embryos pending the results of sexing or other analyses) and research (e.g., comparison of foundation and improved stock in genetic work) are introduced. It is anticipated that subsequent reports will describe details of freezing and thawing methods and amplify selected examples of how preserved embryos are being used in transfer programmes. 64. Freezing, Bovine
an Alternative to Discarding Embryos. ROBERT D. BAKER
Embryos, Inc., Middleville,
Excess
(American Michigan 49333).
Effective methods have been developed to superovulate cattle and to recover embryos under commercial conditions. However, one out of every seven recoveries results in an excess of embryos, that is, more embryos than should or could be transferred into synchronized recipients. Procedures used to freeze the excess embryos for long-term storage or export are under investigation. Acceptable conception rates using frozen-thawed embryos have not yet been achieved. Rates over 40% will likely be required for extensive commercial use of frozen-thawed bovine embryos. 65. The Application cial Bovine
of Cryopreservation Embryo Transfer.
to Commer-
HARLEY J. SCHNEIDER, JR. (Rio Vista International, Inc., San Antonio, Texas).
The use of embryo cryopreservation on a commercial basis is discussed. Selected topics for discussion are: selection of a freezing program, i.e., long vs short