- Email: [email protected]
41. The effect of supercooling on frozen erythrocytes
ABSTRACTS-TENTH ANNUAL MEETING cenfly, large differences in the freezing and melting points have also been detected in the blood serums of several northern fishes such as the winter flounder, Pseudopleuronectes americanus, the short horn sculpin, Myoxocephalus scorpius, the tomcod, Microgadus tomcod, and the smelt, Osmerus mordax. The differences range from 0.4 to 0.8 ° C, and as with the antarctic fishes result from the presence of glycoproteins. These glycoproteins, however, differ greatly from the antarctic glycoproteins in amino acid and carbohydrate composition. Also, in contrast to the antarctic fishes which possess antifreeze compounds throughout the year, the northern fishes synthesize their's only during the winter. A mechanism is suggested whereby different molecules from distantly related fishes give rise to the same antifreeze effect. SECTION ON FREEZING OF BLOOD 40. Effects of Rapid Freezing of Erythrocytes. ToYdo NEI (Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan ). The effects of high cooling rates (10~-10 ~° C/ rain) upon human red blood cells suspended in various cryoprotectants such as glycerol, ammonium acetate, sucrose, sodium chloride, and PVP were examined from both morphological and physiological points of view. Experiments were designed to observe the freezing patterns of frozen cells with the freeze-etching technique and to estimate the extent of hemolysis after rapid thawing. In particular, the relationship between intracellular ice crystal formation and hemolysis was investigated. Results obtained from the present experiment were rather complex and not easily interpreted. The size of intracellular ice crystals was dependent upon cooling rates and showed some correlation with hemolysis. The shape of the ice crystals observed varied with different additives. Only high concentrations of permeable additives such as glycerol and ammonium acetate prevented intracellular ice formation and hemolysis at extremely rapid freezing rates.
619
A depression of the transition cooling rate for intracellular ice formation has been demonstrated experimentally by increasing the degree of supercooling prior to nucleation. Fresh human whole blood was collected in ACD and frozen on the stage of a cryomicroscope. The cell suspension was initially cooled in the liquid state to a preselected degree of supercooling and then held at that temperature by the thermal control system, Extracellular ice was seeded by touching the specimen with a liquid nitrogen cooled copper wire. The temperature controller was programmed to further cool the specimen at constant cooling rate immediately following nucleation (i.e., before any significant volume change could occur at constant temperature); The cells were continuously monitored visually through the microscope to detect whether or not intracellular ice formed. Preliminary results show that one order of magnitude increase in supercooling (from --1 ° C to --10 ° C) decreases the transition cooling rate for intracellular freezing in human erythrocytes by approximately two orders of magnitude (from several thousand --° C/rain to - 1 0 ° C/min). These resuits are supported by an analytic model for freezing and supercooling of cells developed recently. (Supported in part by USPHS Grant 1 PO1 HL14322-01 from NHLI.) 42. Volumetric Changes in Human Erythrocytes
During Freezing at Constant Cooling Rates. W. W. WATSOn, K. R. DmLEn, E. G. CaAVALHO, AND C. E. HUC~INS (Cryogenic Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, Mass. 02139; and Department of Surgery, Harvard Medical School, Surgical Low Temperature Unit and Blood Bank Transfusion Service, Massachusetts General Hospital 02114).
Human erythrocytes collected in ACD anticoagulant were frozen at constant cooling rates on a cryomicroscope, and the major diameters of cells were recorded at known temperatures by sequential photomicrography. These diameters were measured and used to calculate cell volumes. Correlation of cell major diameter and cell volume was 41. The Effect of Supercooling on Frozen Erythro- achieved by a separate set of experiments that emcytes. K. R. DILLER,E. G. CP~VALrI0, AND ployed a photographic method similar to that deC. E. HuCCINS (Cryogenic Engineering veloped by Canham and Burton (Circ. Res. 22, Laboratory, Massachusetts Institute of 405-422, 1968). Erythrocytes were suspended in Technology, Cambridge, Mass. 02139; and saline solutions of increasing tonieity and were the Department of Surgery, Harvard Medi- photographed in cross section with the aid of Nocal School, Surgical Low Temperature Unit marskii interference microscopy. Cell volumes and Blood Bank Transfusion Service, Mas- were calculated by revolving the cross section about the central axis. The resulting volumetric sachusetts General Hospital, Boston, Massadata were correlated with the measured major cell chusetts 02114).
619
A depression of the transition cooling rate for intracellular ice formation has been demonstrated experimentally by increasing the degree of supercooling prior to nucleation. Fresh human whole blood was collected in ACD and frozen on the stage of a cryomicroscope. The cell suspension was initially cooled in the liquid state to a preselected degree of supercooling and then held at that temperature by the thermal control system, Extracellular ice was seeded by touching the specimen with a liquid nitrogen cooled copper wire. The temperature controller was programmed to further cool the specimen at constant cooling rate immediately following nucleation (i.e., before any significant volume change could occur at constant temperature); The cells were continuously monitored visually through the microscope to detect whether or not intracellular ice formed. Preliminary results show that one order of magnitude increase in supercooling (from --1 ° C to --10 ° C) decreases the transition cooling rate for intracellular freezing in human erythrocytes by approximately two orders of magnitude (from several thousand --° C/rain to - 1 0 ° C/min). These resuits are supported by an analytic model for freezing and supercooling of cells developed recently. (Supported in part by USPHS Grant 1 PO1 HL14322-01 from NHLI.) 42. Volumetric Changes in Human Erythrocytes
During Freezing at Constant Cooling Rates. W. W. WATSOn, K. R. DmLEn, E. G. CaAVALHO, AND C. E. HUC~INS (Cryogenic Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, Mass. 02139; and Department of Surgery, Harvard Medical School, Surgical Low Temperature Unit and Blood Bank Transfusion Service, Massachusetts General Hospital 02114).
Human erythrocytes collected in ACD anticoagulant were frozen at constant cooling rates on a cryomicroscope, and the major diameters of cells were recorded at known temperatures by sequential photomicrography. These diameters were measured and used to calculate cell volumes. Correlation of cell major diameter and cell volume was 41. The Effect of Supercooling on Frozen Erythro- achieved by a separate set of experiments that emcytes. K. R. DILLER,E. G. CP~VALrI0, AND ployed a photographic method similar to that deC. E. HuCCINS (Cryogenic Engineering veloped by Canham and Burton (Circ. Res. 22, Laboratory, Massachusetts Institute of 405-422, 1968). Erythrocytes were suspended in Technology, Cambridge, Mass. 02139; and saline solutions of increasing tonieity and were the Department of Surgery, Harvard Medi- photographed in cross section with the aid of Nocal School, Surgical Low Temperature Unit marskii interference microscopy. Cell volumes and Blood Bank Transfusion Service, Mas- were calculated by revolving the cross section about the central axis. The resulting volumetric sachusetts General Hospital, Boston, Massadata were correlated with the measured major cell chusetts 02114).