- Email: [email protected]
Rapid freezing containers and simplified slow freeze technique
tumor in the femoral region was then destroyed by freezing in order to observe the response of the lymphnodes. Group 3: In vitro experiments were designed in order to observe the reaction of the lymphocytes to the tumor cells. Results: In Group 1, inflammatory cell infiltration and degenerated tumor cells were found in the trunor which was not frozen. In Group 2, similar reactions were observed in the metastatic lymphnodes. In Group 3, it \vas observed that the lymphocytes of the rats which underwent cryosurgery killed many tumor cells. From these experimental findings, it was revealed that the destruction of tremor by cryosurgery induced the specific immrmologic rt‘action on the cellular immm~e system of the host, which inhibited the m&static site. 18.
Ar;oi&Lllce
Of
Bleeding
in
Hqmtic
Cryo.w~ely
hy an Improccment
in the Technique. PURSESDU DUTTA .~SD ASOHEW A. GACE (Vetcrans Administration IIospital, Ruffalo, Sen York 14215). Large volumes of canine liv.er can 1~~ frozen and destroyed by spraying liquid nitrogen ( -196°C) on the surface or by contact with a cryoprobt: cooled by the same. The principal problem following such techniques is incessant post-thaw bleeding from deep cracks in the liver produced by intense freezing. In experiments with 20 dogs, this complication was overcome by a ne\v “pour technique” whercaby the liver srrrface was protected by covering it with two to three layers of Dacron velour cloth impregnated with a water-soluble jelly and pouring liquid nitrogeu through a polystyrene foam barrel (5-cm louver opening) placed on the gel cloth, thus providing a better seal and preventing spillage. This permitted a higher and safer surface temperature (-120°C) on the liver, while allowing a slow but crtcnsive freezing of a large volume of tissue ( 400 ml) in 30 min. Tissue temperatures were monitored during cryosurgery. At l- and 2-cl11 depths the temperature frequently reached -100 and -40°C respectively, in 15 min. An hour after thawing, there were statistically significant decreases in platelet count and an increase in activated clotting time, prothrombin time, and partial thromboplnstin time, while the hemoglobin, hematocrit, and fibrinogen levels remained nracticallv unaltered. These minima1 defects in coagulation appeared after rcest&lishment of circulation through the thawed tissue without giving rise to any bleeding tendencies locally or distally. All dogs survived with uu<
19. Experimental
Cryogenic Injury of tire Palate. (Department of Surgery, Veterans Pldministrntion Hospital, Buffalo, New York 14215). ASDREW
GAGE
Early rcpoi-ts on cryosurgery, written soon after modern cryosurgical equipment became available, stated that -20°C vans lethal for tissue. This tenperature proved inadequate for the treatment of cancer and prompted experimentation to clarify fruther factors in cryosurgical technique for tumor destruction. Experiments were performed on the canine palate by three time schedules, that is, a single freeze of 3-min duration, a double free-/c of 3-min diiration each, and a single freezing of 6 min. At each time schedule, observations were made at four different cryoprobe temperatures, --l(iO, -120, -80, and -40°C. Surface, deep tiswere recorded. The sue, aud nasal temperatures experiments showed that a single 3-min freezing at tissue temperatures colder than -100°C can 1~ produced with relatively little damage to tisSIICS. \yhen the freezing period was prolonged to 6 mm, tissue temperatrues of -100°C were associated vsith extensive damage. At either the 3- or 6-min exposure, tissric temperatures of -50 to -60°C produced onlv, sunerficial ulcerative injuries of the palate. Iii sharp contrast, with a double freezing cycle, temperatures of -50°C or colder were associated with extensive tissue dcincluding sequestration of bone. Eastruction, traordinary sensitivity of mclanocytes to cold injury \\-a~ observed. At the vvarmer temperatures, that is, -3 to -14°C tissue temperature, loss of pigmentation \vas produced without epithelial loss. These cxperimeuts show that, in clinical cryosurgery, it probably is necessary to achieve tumor tissue temperature of -60°C or colder and that the freezing must be repetitive for certainty of cell death. The seusitivity of the nrclanocytes to cold may bc exploited for sclectivc cell dcstruction in clinical disease. 20. Rapid Freezing Contuiner.s and Sim&icd Slora Freeze Technique. SAJIO SUMIDA (National Fukuokn Central IIospitnl, Jonni 2-2, Pukuoka, Japan ). Packed red cells cryoprotected by the use of an equal volume of a 3070 glycerol solution containing 27; mannitol, 2% sorbitol, and 0.64$ KaCl \vere rapidly frozen in liquid nitrogen at -196°C. Habio bags showed the lowest recovery rate and the highest hemolysis. UCAR-Hemoflcs Ijags liad the middle values of these parameters ljetweeu tlrc stainless steel and Hahia. With tlrc* iiiipro\elrrcrit of frrezirig procedures rrsiug -Is?; glycerol solrrtion for Ilabia bags, tlrc recovery
GSS
ABSTRACTS,
14TI-I
increased and hemolysis decreased. ATP and DPG values were satisfactory. About 16% or more of the UCAR bags ruptured in the water bath at +4O”C. Stainless steel and IIabia containers did not burst. This is a very important property. The high glycerol slow freeze procedure reported by Tullis and Meryman (Transfusion 17, 438, 1977) was modified and repeated. An equal volume of a 60% glycerol soIntion was added to the red cells in a bag having a maximurn capacity of 1000 ml with violent shaking. The 60% glyccrol solution contained 2% sodium lactate, 0.002>& KCI, and 0.2% sodium phosphate. The glycerolized cells were centrifuged at 41OOg for 5 min. The supernatant excess glycerol solution was discarded and the bag containing packed glycerolized cells was then frozen in a -80°C mechanical freezer. The bags were thawed in 40°C wrater for 5-6 min. Deglycerolization was performed by use of batch \vashing. This simplified method is now used at Fnknoka Red Cross Blood Center. The washing solutions are: 1, 7% NaCl (100 ml) + 0.9% NaCl (500 ml); 2 and 3, 0.9% NaCl (700-800 ml). The recovery after thcsc three washes was 87.9% or more.
21. Thermographic Studies of Phantom and Canine Kidneys Thauxxl by Microu;atie Ra&don. S. h4. KIL~WSKI,* E. F. GRAHAM, AND R. C. LILLEHEI (University of hlinnesota, St. Paul, Minnesota
55108).
Phantom kidneys composed of gelatin, KCl, and polyethylene glycol dissolved in distilled water and allowed to set in a kidney-shaped mold were frozen, packed in Styrofoam containers, and placed in a microwave chamber for rewarming. The chamber was irradiated by pulsed 2450-MHz md&ion. Radiation was of long pulse (on time, 0.24 src; off time, 0.22 see) repeated four times. The kidney halves were separated immediately and thermograms were taken of the surface heat distribution. An ACA Thermovision was cmployed. Kidneys from five dogs were selected on the basis of size compatibility with the phantom models and perfused with cryoprecipitated plasma with 12.5% of one of the following cryoprotectams: DhlSO, glycerol, or ethylene glycol. Five kidneys were treated as the phantom models and fi\,e were packed in the gelatin material. Again thermograms were taken of the surface heat distribution. Kidney-shaped phantoms experienced intense hot spots diie to the focusing effect of the parabolic interior surface of the kidney. The effect of changing conductivity of the models 011 llic iutensily of the hot spots prodiiccd wits also tl~,tlloltstl.~,(c‘d. Cairirrc kidlrcys packed iii styrof0ii11l ah c\tr<~ricrrcc~d intcirhc hot spots. llowever,
all gelatin packed kidneys except the one with ethylene glycol in the plasma showed unifornr thawing with all areas above freezing hut helo\\ lrody temperature. SYMPOSIIJM
II. VIABILITY
ASSAYS
A. Cellrtlur Conctitnents in Rlootl. STELLA K~mrrr
B. C.
I>.
E:.
(Clinical R(,search Ccntre, IIarrow, England). ‘I’issrce Ctdtlrrc. J. E. SIIANMN (American Type Culture Collection, Washington, D.C.). S~Jc’mcltozOa. E. F. GliAHAhl (University of ,\linnesota, Minneapolis, Minnesota). Oca. 1). WHITTIXCHAM (Medical Research Council, Mammalian Development Unit, London, England ) Plants. J. P. PALTA, J. LEVITT, AXD E. J. SrAIIELAIASN ( University of Slinnesota, Minntapolis, llinnesota). SESSION
C. PLAIXTS
22. Resistance of the Fungtcs Rhizoctonia Solmi to Low Temperatww. S. Bauson * AND J. NATH (Division of Plant Sciences, West ~iirginia Virginia
University, 26506).
hlorgantown,
West
The resistance of living organisms to adverse mviromnental influences is largely determined by their rates of metabolism. Generally the lower the metabolism the greater the resistance and the longer the viability. A direct functional relationship between metabolic rates and longevity remains to be shown. In the experiments reported here, using Rhizoctonia sold, the quiescent state (at extremely low to zero metabolism) brought about by desiccation of active hyphal tips, was tested and compared at room temperature and at -196°C for vialibility and lags. Air-dried quiescent hyphnl tips held at room temperature rcniain viable and grow out when returned to suitable media, as if their growth had not been interrupted, until up to 3 months of drying. After that a lag period develops, usually of 1 to 2 days, Irefore initiation of visible growM~. With increasing drying time there is a tendency for increased lag time and loss of viability. The changes may be conceived as zero lag+ l-day lag-+ 2-day lag+ 3 day lug+ loss of viability. The lag phase development can be accelerated by heating the dried quiescent forms. Dried quiescent tips with or without heat treatment wcrc very resistant to change at -196°C. In duplicate cq~eriirrrnts with lrcat-treated cliiicscetrt tips of 5 ain 7 woc~ks ~IIration iu liqlrid nitr-ogru, 74’/0 viability ‘~riirl 92%~ lags \\ rrc attniricd conrparcd with 61 ‘/o viability
Ar;oi&Lllce
Of
Bleeding
in
Hqmtic
Cryo.w~ely
hy an Improccment
in the Technique. PURSESDU DUTTA .~SD ASOHEW A. GACE (Vetcrans Administration IIospital, Ruffalo, Sen York 14215). Large volumes of canine liv.er can 1~~ frozen and destroyed by spraying liquid nitrogen ( -196°C) on the surface or by contact with a cryoprobt: cooled by the same. The principal problem following such techniques is incessant post-thaw bleeding from deep cracks in the liver produced by intense freezing. In experiments with 20 dogs, this complication was overcome by a ne\v “pour technique” whercaby the liver srrrface was protected by covering it with two to three layers of Dacron velour cloth impregnated with a water-soluble jelly and pouring liquid nitrogeu through a polystyrene foam barrel (5-cm louver opening) placed on the gel cloth, thus providing a better seal and preventing spillage. This permitted a higher and safer surface temperature (-120°C) on the liver, while allowing a slow but crtcnsive freezing of a large volume of tissue ( 400 ml) in 30 min. Tissue temperatures were monitored during cryosurgery. At l- and 2-cl11 depths the temperature frequently reached -100 and -40°C respectively, in 15 min. An hour after thawing, there were statistically significant decreases in platelet count and an increase in activated clotting time, prothrombin time, and partial thromboplnstin time, while the hemoglobin, hematocrit, and fibrinogen levels remained nracticallv unaltered. These minima1 defects in coagulation appeared after rcest&lishment of circulation through the thawed tissue without giving rise to any bleeding tendencies locally or distally. All dogs survived with uu<
19. Experimental
Cryogenic Injury of tire Palate. (Department of Surgery, Veterans Pldministrntion Hospital, Buffalo, New York 14215). ASDREW
GAGE
Early rcpoi-ts on cryosurgery, written soon after modern cryosurgical equipment became available, stated that -20°C vans lethal for tissue. This tenperature proved inadequate for the treatment of cancer and prompted experimentation to clarify fruther factors in cryosurgical technique for tumor destruction. Experiments were performed on the canine palate by three time schedules, that is, a single freeze of 3-min duration, a double free-/c of 3-min diiration each, and a single freezing of 6 min. At each time schedule, observations were made at four different cryoprobe temperatures, --l(iO, -120, -80, and -40°C. Surface, deep tiswere recorded. The sue, aud nasal temperatures experiments showed that a single 3-min freezing at tissue temperatures colder than -100°C can 1~ produced with relatively little damage to tisSIICS. \yhen the freezing period was prolonged to 6 mm, tissue temperatrues of -100°C were associated vsith extensive damage. At either the 3- or 6-min exposure, tissric temperatures of -50 to -60°C produced onlv, sunerficial ulcerative injuries of the palate. Iii sharp contrast, with a double freezing cycle, temperatures of -50°C or colder were associated with extensive tissue dcincluding sequestration of bone. Eastruction, traordinary sensitivity of mclanocytes to cold injury \\-a~ observed. At the vvarmer temperatures, that is, -3 to -14°C tissue temperature, loss of pigmentation \vas produced without epithelial loss. These cxperimeuts show that, in clinical cryosurgery, it probably is necessary to achieve tumor tissue temperature of -60°C or colder and that the freezing must be repetitive for certainty of cell death. The seusitivity of the nrclanocytes to cold may bc exploited for sclectivc cell dcstruction in clinical disease. 20. Rapid Freezing Contuiner.s and Sim&icd Slora Freeze Technique. SAJIO SUMIDA (National Fukuokn Central IIospitnl, Jonni 2-2, Pukuoka, Japan ). Packed red cells cryoprotected by the use of an equal volume of a 3070 glycerol solution containing 27; mannitol, 2% sorbitol, and 0.64$ KaCl \vere rapidly frozen in liquid nitrogen at -196°C. Habio bags showed the lowest recovery rate and the highest hemolysis. UCAR-Hemoflcs Ijags liad the middle values of these parameters ljetweeu tlrc stainless steel and Hahia. With tlrc* iiiipro\elrrcrit of frrezirig procedures rrsiug -Is?; glycerol solrrtion for Ilabia bags, tlrc recovery
GSS
ABSTRACTS,
14TI-I
increased and hemolysis decreased. ATP and DPG values were satisfactory. About 16% or more of the UCAR bags ruptured in the water bath at +4O”C. Stainless steel and IIabia containers did not burst. This is a very important property. The high glycerol slow freeze procedure reported by Tullis and Meryman (Transfusion 17, 438, 1977) was modified and repeated. An equal volume of a 60% glycerol soIntion was added to the red cells in a bag having a maximurn capacity of 1000 ml with violent shaking. The 60% glyccrol solution contained 2% sodium lactate, 0.002>& KCI, and 0.2% sodium phosphate. The glycerolized cells were centrifuged at 41OOg for 5 min. The supernatant excess glycerol solution was discarded and the bag containing packed glycerolized cells was then frozen in a -80°C mechanical freezer. The bags were thawed in 40°C wrater for 5-6 min. Deglycerolization was performed by use of batch \vashing. This simplified method is now used at Fnknoka Red Cross Blood Center. The washing solutions are: 1, 7% NaCl (100 ml) + 0.9% NaCl (500 ml); 2 and 3, 0.9% NaCl (700-800 ml). The recovery after thcsc three washes was 87.9% or more.
21. Thermographic Studies of Phantom and Canine Kidneys Thauxxl by Microu;atie Ra&don. S. h4. KIL~WSKI,* E. F. GRAHAM, AND R. C. LILLEHEI (University of hlinnesota, St. Paul, Minnesota
55108).
Phantom kidneys composed of gelatin, KCl, and polyethylene glycol dissolved in distilled water and allowed to set in a kidney-shaped mold were frozen, packed in Styrofoam containers, and placed in a microwave chamber for rewarming. The chamber was irradiated by pulsed 2450-MHz md&ion. Radiation was of long pulse (on time, 0.24 src; off time, 0.22 see) repeated four times. The kidney halves were separated immediately and thermograms were taken of the surface heat distribution. An ACA Thermovision was cmployed. Kidneys from five dogs were selected on the basis of size compatibility with the phantom models and perfused with cryoprecipitated plasma with 12.5% of one of the following cryoprotectams: DhlSO, glycerol, or ethylene glycol. Five kidneys were treated as the phantom models and fi\,e were packed in the gelatin material. Again thermograms were taken of the surface heat distribution. Kidney-shaped phantoms experienced intense hot spots diie to the focusing effect of the parabolic interior surface of the kidney. The effect of changing conductivity of the models 011 llic iutensily of the hot spots prodiiccd wits also tl~,tlloltstl.~,(c‘d. Cairirrc kidlrcys packed iii styrof0ii11l ah c\tr<~ricrrcc~d intcirhc hot spots. llowever,
all gelatin packed kidneys except the one with ethylene glycol in the plasma showed unifornr thawing with all areas above freezing hut helo\\ lrody temperature. SYMPOSIIJM
II. VIABILITY
ASSAYS
A. Cellrtlur Conctitnents in Rlootl. STELLA K~mrrr
B. C.
I>.
E:.
(Clinical R(,search Ccntre, IIarrow, England). ‘I’issrce Ctdtlrrc. J. E. SIIANMN (American Type Culture Collection, Washington, D.C.). S~Jc’mcltozOa. E. F. GliAHAhl (University of ,\linnesota, Minneapolis, Minnesota). Oca. 1). WHITTIXCHAM (Medical Research Council, Mammalian Development Unit, London, England ) Plants. J. P. PALTA, J. LEVITT, AXD E. J. SrAIIELAIASN ( University of Slinnesota, Minntapolis, llinnesota). SESSION
C. PLAIXTS
22. Resistance of the Fungtcs Rhizoctonia Solmi to Low Temperatww. S. Bauson * AND J. NATH (Division of Plant Sciences, West ~iirginia Virginia
University, 26506).
hlorgantown,
West
The resistance of living organisms to adverse mviromnental influences is largely determined by their rates of metabolism. Generally the lower the metabolism the greater the resistance and the longer the viability. A direct functional relationship between metabolic rates and longevity remains to be shown. In the experiments reported here, using Rhizoctonia sold, the quiescent state (at extremely low to zero metabolism) brought about by desiccation of active hyphal tips, was tested and compared at room temperature and at -196°C for vialibility and lags. Air-dried quiescent hyphnl tips held at room temperature rcniain viable and grow out when returned to suitable media, as if their growth had not been interrupted, until up to 3 months of drying. After that a lag period develops, usually of 1 to 2 days, Irefore initiation of visible growM~. With increasing drying time there is a tendency for increased lag time and loss of viability. The changes may be conceived as zero lag+ l-day lag-+ 2-day lag+ 3 day lug+ loss of viability. The lag phase development can be accelerated by heating the dried quiescent forms. Dried quiescent tips with or without heat treatment wcrc very resistant to change at -196°C. In duplicate cq~eriirrrnts with lrcat-treated cliiicscetrt tips of 5 ain 7 woc~ks ~IIration iu liqlrid nitr-ogru, 74’/0 viability ‘~riirl 92%~ lags \\ rrc attniricd conrparcd with 61 ‘/o viability