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A proposed mechanism for PVP cryoprotection
ABSTRACTS,
13TH
hemolysis that would be expected at different steps in a given dilution procedure. Red cells were then subjected to these dilution procedures, and the amount of hemolysis produced by each step of the dilution was determined experimentally. The agreement between the predicted amount of hemolysis and that actually observed is good. Research sponsored by the Energy Research and Development Administration under contract with the Union Carbide Corporation. The United States Governqent’s right to retain a nonex&sive, royalty-free license in and to copyright covering this abstract is acknowledged. 29. The Effects of the Anticoagulants ACD and CPD on the Glycerol Permeability of Human Erythrocytes. T. PAPANEK,* M. ROBBIN,* C. E. HUGGIKS, AND E. G. CRAVALHO. (Cryogenic Engineering Laboratory, hlassachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Surgery, Harvard Medical School, Low Temperature Surgical Unit and Blood Bank Transfusion Service, Massachusetts General Hospital, Boston, Massachusetts 02114). The pre-freeze addition and post-thaw removal of glycerol are vital steps in the clinical storage of frozen human blood. Experience at the Massachusetts General Hospital Blood Bank has suggested that the glycerol permeability of human erythrocytes may be differentially altered by the anticoagulants ACD (acid citrate-dextrose) and CPD ( citrate phosphate-dextrose). The glycerol permeability of human red cells suspended in ( 1) normal saline, (2) normal saline with ACD, and (3) normal saline with CPD has been measured using a stop-flow apparatus developed in this laboratory. This apparatus permits introduction of a step change in extracellular solute concentration and subsequent monitoring of the resulting changes in cell volume by measurements of the intensity of forward scattered light from the cell suspension. Qualitative comparisons of permeability are obtained by visual examination of the measured cell volume vs time curves. Quantitative values of the membrane parameters were obtained via a computer fit of the measured cell volumes to those obtained from theoretical models of coupled water and solute fluxes. Data obtained at low (0.5 M) and high (3.0 M) glycerol concentrations indicate that the glycerol and water permeabilities of human erythrocytes are not significantly affected by the anticoagulant medium. Future work in this area will focus on the hemolysis kinetics of red cells during clinical glycerolization and deglycerolization protocols.
ANNUAL
MEETING
653
Supported iti part by the National Heart and Lung Institute, Grant No. HL-14322, and the Undergraduate Research Opportunities Program, M.I.T. 30. A Proposed Mechanism for PVP Cryoprotection. R. J. WILLIAMS. (American National Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Maryland 20014). It is well known that polyvinylpyrollidone (PVP) and certain other polymeric solutes induce partial rather than complete hemolysis of hypotonically stressed red cells, implying that the polymers accelerate the resealing of the cell membrane before all hemoglobin has escaped. Evidence has been acquired that, rather than stimulating a resealing of the plasma membrane, PVP substitutes for lost membrane materials at a site of defect. Packed red cell hemolysates mix readily with aqueous solutions of dimethyl sulfoxide ( DMSO ), glycerol, or sucrose, but in PVP solutions above 1.25% stable droplets are formed. The contact angle of these droplets on a polyethylene substrate are greater than 90”, demonstrating that the surface energy of the PVP solution is lower than that of the hemolysate. The cosine of the hemolysate contact angle is inversely related to the log PVP concentration, as predicted by the Gibbs surface energy equation. It is suggested that the low surface energy of PVP solutions causes it to seal defects in red cell membranes, preventing the loss of hemoglobin. This sealing could disappear when cells protected washed and would thus by PVP are subsequently account for the continuing hemolysis seen on repeated washing of red cells frozen in PVP solutions. 31. Ultrastructural
Studies
on Red
Cells
Cryo-
preserved with Hydroxyethyl Starch. E. D. ALLEN, L. WEATHERBEE,* AND P. A. PERMOAD.” (Veterans Administration Hospital, 2215 Fuller Road, Ann Arbor, Michigan 48105, and University of Michigan, Medical Center, Ann Arbor, Michigan 48104).
Studies have been directed toward ultrastructural examination of morphological changes of the red blood cell frozen and thawed in the presence of 14% hydroxyethyl starch (HES ). Earlier studies indicated that such cells underwent additional damage (as measured by increased levels of supernatant hemoglobin) when the thawed mixture was diluted with saline or low ionic strength buffers. To determine whether this increased hemoglobin is caused by “intact” cells leaking hemoglobin or by complete destruction of some cells,
13TH
hemolysis that would be expected at different steps in a given dilution procedure. Red cells were then subjected to these dilution procedures, and the amount of hemolysis produced by each step of the dilution was determined experimentally. The agreement between the predicted amount of hemolysis and that actually observed is good. Research sponsored by the Energy Research and Development Administration under contract with the Union Carbide Corporation. The United States Governqent’s right to retain a nonex&sive, royalty-free license in and to copyright covering this abstract is acknowledged. 29. The Effects of the Anticoagulants ACD and CPD on the Glycerol Permeability of Human Erythrocytes. T. PAPANEK,* M. ROBBIN,* C. E. HUGGIKS, AND E. G. CRAVALHO. (Cryogenic Engineering Laboratory, hlassachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Surgery, Harvard Medical School, Low Temperature Surgical Unit and Blood Bank Transfusion Service, Massachusetts General Hospital, Boston, Massachusetts 02114). The pre-freeze addition and post-thaw removal of glycerol are vital steps in the clinical storage of frozen human blood. Experience at the Massachusetts General Hospital Blood Bank has suggested that the glycerol permeability of human erythrocytes may be differentially altered by the anticoagulants ACD (acid citrate-dextrose) and CPD ( citrate phosphate-dextrose). The glycerol permeability of human red cells suspended in ( 1) normal saline, (2) normal saline with ACD, and (3) normal saline with CPD has been measured using a stop-flow apparatus developed in this laboratory. This apparatus permits introduction of a step change in extracellular solute concentration and subsequent monitoring of the resulting changes in cell volume by measurements of the intensity of forward scattered light from the cell suspension. Qualitative comparisons of permeability are obtained by visual examination of the measured cell volume vs time curves. Quantitative values of the membrane parameters were obtained via a computer fit of the measured cell volumes to those obtained from theoretical models of coupled water and solute fluxes. Data obtained at low (0.5 M) and high (3.0 M) glycerol concentrations indicate that the glycerol and water permeabilities of human erythrocytes are not significantly affected by the anticoagulant medium. Future work in this area will focus on the hemolysis kinetics of red cells during clinical glycerolization and deglycerolization protocols.
ANNUAL
MEETING
653
Supported iti part by the National Heart and Lung Institute, Grant No. HL-14322, and the Undergraduate Research Opportunities Program, M.I.T. 30. A Proposed Mechanism for PVP Cryoprotection. R. J. WILLIAMS. (American National Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Maryland 20014). It is well known that polyvinylpyrollidone (PVP) and certain other polymeric solutes induce partial rather than complete hemolysis of hypotonically stressed red cells, implying that the polymers accelerate the resealing of the cell membrane before all hemoglobin has escaped. Evidence has been acquired that, rather than stimulating a resealing of the plasma membrane, PVP substitutes for lost membrane materials at a site of defect. Packed red cell hemolysates mix readily with aqueous solutions of dimethyl sulfoxide ( DMSO ), glycerol, or sucrose, but in PVP solutions above 1.25% stable droplets are formed. The contact angle of these droplets on a polyethylene substrate are greater than 90”, demonstrating that the surface energy of the PVP solution is lower than that of the hemolysate. The cosine of the hemolysate contact angle is inversely related to the log PVP concentration, as predicted by the Gibbs surface energy equation. It is suggested that the low surface energy of PVP solutions causes it to seal defects in red cell membranes, preventing the loss of hemoglobin. This sealing could disappear when cells protected washed and would thus by PVP are subsequently account for the continuing hemolysis seen on repeated washing of red cells frozen in PVP solutions. 31. Ultrastructural
Studies
on Red
Cells
Cryo-
preserved with Hydroxyethyl Starch. E. D. ALLEN, L. WEATHERBEE,* AND P. A. PERMOAD.” (Veterans Administration Hospital, 2215 Fuller Road, Ann Arbor, Michigan 48105, and University of Michigan, Medical Center, Ann Arbor, Michigan 48104).
Studies have been directed toward ultrastructural examination of morphological changes of the red blood cell frozen and thawed in the presence of 14% hydroxyethyl starch (HES ). Earlier studies indicated that such cells underwent additional damage (as measured by increased levels of supernatant hemoglobin) when the thawed mixture was diluted with saline or low ionic strength buffers. To determine whether this increased hemoglobin is caused by “intact” cells leaking hemoglobin or by complete destruction of some cells,