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Improvements in frozen blood stored in liquid nitrogen
ABSTRACTS-ELEVENTH d to 0nc: ~~tltld glycerol over a pcriotl of 7 nlin and to the other adcled it in 1 min, II significant difference was found for the gs Hh in thcs sl~prrnatant; 0.130 i 0.045 and 0.065 t 0.019, respectively. For pooled units of packed cells the relation between degree of hemolysis and addition time was confirmed. The addition of glycerol in less than I min is now part of our routine procedure, resulting in a level of hemolysis hclow 0.1 gT& Hb in the supernatant.
3. Reciew
of Hydroxyethyl Sturch us cm Ertmcellular Cryoprotectice Agent of Red Blood Cell.~. L. WEAWEHHEE,’ E. ALLEX,” H. H. SPEXCEH, S. M. LIN~ENAUER,* ANI) I?. A. PE~w~AU.* (Veterans Administrntion Hospital, 2215 Fuller Rd., Ann Arbor, Michigan and University of Michigan Mcdical Center, Ann Arbor, Michigan 48105).
Cryoprotective properties of hydroxyethyl starch (HES) were first examined when it became available as a plasma expander since clextran and PVP were similar in structure and had shown promise as extracellular protective agents. Early reports suggested that coagulopathies might result from the use of HES but transfusion studies in this laboratory using primates indicated that coagulation problems did not occur with low molecular weight hydronyethyl starch. Hydroxyethyl starch in a final concentration of 14% offered the best red cell protection and a molecular weight fraction averaging 60,000 demonstrated improved cryoprotective properties (high molecular weight HES was found to precipitate some plasma proteins). Rapid freezing and thawing are required with HES preserved red cells and these are conditions satisfied with agitation during freezing in liquid nitrogen and during thawing in a 48°C water bath. Hemoflex plastic bags have replaced the metal containers which were originally used. Most freezing experiments have been carried out with 30-50 ml freezing mixtures. Freezitlg of 24 large units (385 ml total volume) gave satisfactory results assessed by in oitro postthaw studies. Evaluation in humans has heen limited to date. Recent studies have centered on red cell washing either before or after freezing with HES. Prefreeze washing of red cells has been found to improve the postthaw saline stability and is probably related to removing plasma. HES preserved red cells compare favourably to other existing methods with respect to in &ro tests and red cell survival. The development of methocls of processing and the assessment of large unit techniques is underway. (Supported in part by
ANNUAL
4.
MEETING
537
prcservution of Humun Red Cells in Liquid Nitrogen with Hydroxyethyl Starch. F. J. LIUNETT~ AND S. M. HUNT.’ (Center for Blood Research, 800 Huntington Avcnuc, Boston, !vlassachusctts 02115).
Erythrocytes were frozen in Hemoflex plastic bags in liquid nitrogen with 14% (W/V) hydroxyethylstarch (HES). Both a small volume ( 25.0 ml hematocrit 37) and a full unit system (405 1111, hematocrit, 4244 ) were employed. SllldI vohnne freezings were investigated to optimize container confignration, conditions, composition of media, and postthawed stability of cells. Based on this experience a method for full unit freezing of packed erythrocytes was devised. The results of the ten most recent experiments with 25.0 ml freezing gave postthawed cell recoveries of 99.2 2 0.3%. Stability for 1 hr in 0.15 hl NaCl at 22.O”C. ~a.$ 85.9 * 4.3%. The cells gained I7 mequiv. of Na’ and lost 21 mequiv. of K’. There was a 23% decrease in ATP while 2.3 DPG was unchanged. In the large volume method all the packed red cells in a unit after removal of platelet rich plasma and bnffy coat were frozen for 80-90 set in 14% HES in liquid nitrogen. A single bag (Nemoflex 7450-3 ) was sandwiched between large perforated aluminum plates. Thawing was done at 48°C. for 50-55 sec. The results of eight recent freezing and short term storage in liquid nitrogen vapor gave cell recoveries of 97.2 % l.l%, and stabilities in saline of 75.7 f. 1.8%. The ATP was decreased 33% and 2, 3 DPG unchanged. The cells gained 33 mequiv. of Na’ and lost 31 mequiv. of K’. Cell size distributions obtained electronically were consistent with the cell chemistry. As yet no animal transfusions have been made. (Snpportcd hy ONR contract NOOO1473c-0100.)
5. Improcements in Frozen Blood Stored in Liquid Kitrogen. D. S. PEPPER,* K. ,4hlER,” A. G. E. ALLAN* AND J. MCTAVISH.” (s. E. Scotland Regional Blood Transfusion Service, Royal Infirmary, Edinburgh EH3 9HB, Scotland). Introduced by J. Farrant.
The optimum conditions for freezing, recovery and subsequent storage of blood have been investigated. The 650 ml Printal aerosol can in current European use has been replaced by a 550 ml metal box can with smaller dimensions giving
5x3
ABSTRACTS-ELEVENTH
a tighter neck seal, reduced air space, and reduced incidence of explosion. A 250 liter refrigerator will take 204 cans in two layers. Up to 280 ml of cells are mixed with 200 ml of 45% glycerol at pH 6.7 and washed with 19% sorbitol and saline. Manual washing in a 5-port bag and automatic washing (IBM 2991) gave recoveries of 93 and 95%, respectively. The supernatant Hb was found to be 370 mg% in saline resuspended cells, (50% HCT), 270 :n 5% albumin, 98 in ACD-saline, and 60 in ACDalbumin. Using the latter medium, cells remained transfuscable for at least 9 days postthaw. This blood had a pH of 6.2, an aluminum content of d 0.3 ppm, glycerol and sorbitol contents of 0.76 and 0.58 g per unit. A unit costs about f8. to freeze, store, and recover (excluding labour), and takes about 20 nun to wash subsequent to thawing. 6. Transfusion of Frozen Red Ccl1 and Serum Hepatitis. S. SUMIDA AND M. SUMIDA.” (Division of Cardiovascular Surgery and Frozen Blood Section, Division of Dermatology, National Fukuoka Central Hospital, Jonai 2-2, Fukuoka, 810 Japan).
Since 1965, we have transfused the red blood cells preserved by freezing after Huggins’ and Rowe’s procedure. Five thousand or more units of those erythrocytes processed by freeze-thawdeglycerolization wash were administered into 670 patients to treat their anemia from various causes. About 2% of the recipients revealed posttransfusion hepatitis, of whom Australia antigen test was positive in recent three cases. The use of banked ACD blood revealed 7% or more incidence of posttransfusion hepatitis. We processed the banked ACD blood having the positive Austraha antigen by the same procedure previously mentioned. However, it was extremely difficult to wash out Australia antigen. Judging from the above mentioned results, we can’t eliminate serum hepatitis perfectly even if we use the frozen red cells. 7. Frozen Blood-Ten Years of Clinical Experience. C. E. HUGGINS. (Massachusetts GenBoston, Massachusetts eral Hospital, 02114).
Since 1964 almost 50,000 units of packed human blood cells have been treated with 8.6 M glycerol solution, frozen and stored at --85”C, deglycerolized by agglomeration, resuspended in saline and transfused at Massachusetts General Hospital.
ANNUAL
Mi?ETlNG
Between 1964 and 1969, frozen-deglycerolized blood cells were generally transfused on specific clinical indications. In the 1970’s frozen blood h,dS been used in ever increasing quantities to regulate inventory and assure adequate amounts of blood cells for transfusion at all times. P.lrallel increase in the usage of freshly frozen plasma, cryoprecipitate, and platelet concentrate permits optimum use of all components. Outdating loss of blood has fallen from 9% in 1964 to 0.47; for the past six months. Freezing techniques actively applied in a hospital setting can be used to solve most of the clinical problems of blood transfusion therapy. SESSION
8. The
B. EXPERIMENTAL FREEZING
RED
CELL
Effect of PlaA7na on Red Blood Cells Frozen with Hydroxyethyl Starch (HES). L. WEATHERBEE," E. ALLEN,* H. H. SPENCEH, S. M. LINENAUER,~ AND I?. A. PERAIOAD." (Veterans Administration Hospital, 2215 Fuller Rd., Ann Arbor, Michigan and University of Michigan Medical Center, Ann Arbor, Michigan 48105).
In a continuing series of experiments we have attempted to improve the quality of red cells frozen with the cryoprotective agent, hydroxyethyl starch. The methods previously used consisted of centrifuging whole blood and removing the supernatant plasma and buffy coat. The packed red cells were then mixed with 40% HES solution in a ratio of 65 to 35. This yielded a 14% starch concentration and a hematocrit near 40% but the hematocrit would vary depending on the amount of plasma present in the packed cells. Since plasma is present in the packed cells, its effect on the red cells during the freeze-thaw cycle was of major interest. This effect was examined using a freezing mixture of 30 ml with a hematocrit of 40% and a starch concentration of 14%. Washing the red cells before freezing to remove the plasma increased the saline stability after thawing from 75.3 2 3.9% to 82.9 2 1.5% while the red cells recoveries (98%) and supernatant hemoglobin (300 mg”/o ) remained nearly unchanged. Although all of these in oitro tests measure the quality of the thawed red cells, the saline stability is an accurate in titro indicator of how the cells will survive in tiivo. If the cells from which the surrounding plasma has been removed by washing are resuspended in the plasma, the saline stability drops to values nearly identical to the unwashed cells (75.0 -t 2.4). The presence of plasma reduces the saline stability of red cells frozen throughout a hematocrit
3. Reciew
of Hydroxyethyl Sturch us cm Ertmcellular Cryoprotectice Agent of Red Blood Cell.~. L. WEAWEHHEE,’ E. ALLEX,” H. H. SPEXCEH, S. M. LIN~ENAUER,* ANI) I?. A. PE~w~AU.* (Veterans Administrntion Hospital, 2215 Fuller Rd., Ann Arbor, Michigan and University of Michigan Mcdical Center, Ann Arbor, Michigan 48105).
Cryoprotective properties of hydroxyethyl starch (HES) were first examined when it became available as a plasma expander since clextran and PVP were similar in structure and had shown promise as extracellular protective agents. Early reports suggested that coagulopathies might result from the use of HES but transfusion studies in this laboratory using primates indicated that coagulation problems did not occur with low molecular weight hydronyethyl starch. Hydroxyethyl starch in a final concentration of 14% offered the best red cell protection and a molecular weight fraction averaging 60,000 demonstrated improved cryoprotective properties (high molecular weight HES was found to precipitate some plasma proteins). Rapid freezing and thawing are required with HES preserved red cells and these are conditions satisfied with agitation during freezing in liquid nitrogen and during thawing in a 48°C water bath. Hemoflex plastic bags have replaced the metal containers which were originally used. Most freezing experiments have been carried out with 30-50 ml freezing mixtures. Freezitlg of 24 large units (385 ml total volume) gave satisfactory results assessed by in oitro postthaw studies. Evaluation in humans has heen limited to date. Recent studies have centered on red cell washing either before or after freezing with HES. Prefreeze washing of red cells has been found to improve the postthaw saline stability and is probably related to removing plasma. HES preserved red cells compare favourably to other existing methods with respect to in &ro tests and red cell survival. The development of methocls of processing and the assessment of large unit techniques is underway. (Supported in part by
ANNUAL
4.
MEETING
537
prcservution of Humun Red Cells in Liquid Nitrogen with Hydroxyethyl Starch. F. J. LIUNETT~ AND S. M. HUNT.’ (Center for Blood Research, 800 Huntington Avcnuc, Boston, !vlassachusctts 02115).
Erythrocytes were frozen in Hemoflex plastic bags in liquid nitrogen with 14% (W/V) hydroxyethylstarch (HES). Both a small volume ( 25.0 ml hematocrit 37) and a full unit system (405 1111, hematocrit, 4244 ) were employed. SllldI vohnne freezings were investigated to optimize container confignration, conditions, composition of media, and postthawed stability of cells. Based on this experience a method for full unit freezing of packed erythrocytes was devised. The results of the ten most recent experiments with 25.0 ml freezing gave postthawed cell recoveries of 99.2 2 0.3%. Stability for 1 hr in 0.15 hl NaCl at 22.O”C. ~a.$ 85.9 * 4.3%. The cells gained I7 mequiv. of Na’ and lost 21 mequiv. of K’. There was a 23% decrease in ATP while 2.3 DPG was unchanged. In the large volume method all the packed red cells in a unit after removal of platelet rich plasma and bnffy coat were frozen for 80-90 set in 14% HES in liquid nitrogen. A single bag (Nemoflex 7450-3 ) was sandwiched between large perforated aluminum plates. Thawing was done at 48°C. for 50-55 sec. The results of eight recent freezing and short term storage in liquid nitrogen vapor gave cell recoveries of 97.2 % l.l%, and stabilities in saline of 75.7 f. 1.8%. The ATP was decreased 33% and 2, 3 DPG unchanged. The cells gained 33 mequiv. of Na’ and lost 31 mequiv. of K’. Cell size distributions obtained electronically were consistent with the cell chemistry. As yet no animal transfusions have been made. (Snpportcd hy ONR contract NOOO1473c-0100.)
5. Improcements in Frozen Blood Stored in Liquid Kitrogen. D. S. PEPPER,* K. ,4hlER,” A. G. E. ALLAN* AND J. MCTAVISH.” (s. E. Scotland Regional Blood Transfusion Service, Royal Infirmary, Edinburgh EH3 9HB, Scotland). Introduced by J. Farrant.
The optimum conditions for freezing, recovery and subsequent storage of blood have been investigated. The 650 ml Printal aerosol can in current European use has been replaced by a 550 ml metal box can with smaller dimensions giving
5x3
ABSTRACTS-ELEVENTH
a tighter neck seal, reduced air space, and reduced incidence of explosion. A 250 liter refrigerator will take 204 cans in two layers. Up to 280 ml of cells are mixed with 200 ml of 45% glycerol at pH 6.7 and washed with 19% sorbitol and saline. Manual washing in a 5-port bag and automatic washing (IBM 2991) gave recoveries of 93 and 95%, respectively. The supernatant Hb was found to be 370 mg% in saline resuspended cells, (50% HCT), 270 :n 5% albumin, 98 in ACD-saline, and 60 in ACDalbumin. Using the latter medium, cells remained transfuscable for at least 9 days postthaw. This blood had a pH of 6.2, an aluminum content of d 0.3 ppm, glycerol and sorbitol contents of 0.76 and 0.58 g per unit. A unit costs about f8. to freeze, store, and recover (excluding labour), and takes about 20 nun to wash subsequent to thawing. 6. Transfusion of Frozen Red Ccl1 and Serum Hepatitis. S. SUMIDA AND M. SUMIDA.” (Division of Cardiovascular Surgery and Frozen Blood Section, Division of Dermatology, National Fukuoka Central Hospital, Jonai 2-2, Fukuoka, 810 Japan).
Since 1965, we have transfused the red blood cells preserved by freezing after Huggins’ and Rowe’s procedure. Five thousand or more units of those erythrocytes processed by freeze-thawdeglycerolization wash were administered into 670 patients to treat their anemia from various causes. About 2% of the recipients revealed posttransfusion hepatitis, of whom Australia antigen test was positive in recent three cases. The use of banked ACD blood revealed 7% or more incidence of posttransfusion hepatitis. We processed the banked ACD blood having the positive Austraha antigen by the same procedure previously mentioned. However, it was extremely difficult to wash out Australia antigen. Judging from the above mentioned results, we can’t eliminate serum hepatitis perfectly even if we use the frozen red cells. 7. Frozen Blood-Ten Years of Clinical Experience. C. E. HUGGINS. (Massachusetts GenBoston, Massachusetts eral Hospital, 02114).
Since 1964 almost 50,000 units of packed human blood cells have been treated with 8.6 M glycerol solution, frozen and stored at --85”C, deglycerolized by agglomeration, resuspended in saline and transfused at Massachusetts General Hospital.
ANNUAL
Mi?ETlNG
Between 1964 and 1969, frozen-deglycerolized blood cells were generally transfused on specific clinical indications. In the 1970’s frozen blood h,dS been used in ever increasing quantities to regulate inventory and assure adequate amounts of blood cells for transfusion at all times. P.lrallel increase in the usage of freshly frozen plasma, cryoprecipitate, and platelet concentrate permits optimum use of all components. Outdating loss of blood has fallen from 9% in 1964 to 0.47; for the past six months. Freezing techniques actively applied in a hospital setting can be used to solve most of the clinical problems of blood transfusion therapy. SESSION
8. The
B. EXPERIMENTAL FREEZING
RED
CELL
Effect of PlaA7na on Red Blood Cells Frozen with Hydroxyethyl Starch (HES). L. WEATHERBEE," E. ALLEN,* H. H. SPENCEH, S. M. LINENAUER,~ AND I?. A. PERAIOAD." (Veterans Administration Hospital, 2215 Fuller Rd., Ann Arbor, Michigan and University of Michigan Medical Center, Ann Arbor, Michigan 48105).
In a continuing series of experiments we have attempted to improve the quality of red cells frozen with the cryoprotective agent, hydroxyethyl starch. The methods previously used consisted of centrifuging whole blood and removing the supernatant plasma and buffy coat. The packed red cells were then mixed with 40% HES solution in a ratio of 65 to 35. This yielded a 14% starch concentration and a hematocrit near 40% but the hematocrit would vary depending on the amount of plasma present in the packed cells. Since plasma is present in the packed cells, its effect on the red cells during the freeze-thaw cycle was of major interest. This effect was examined using a freezing mixture of 30 ml with a hematocrit of 40% and a starch concentration of 14%. Washing the red cells before freezing to remove the plasma increased the saline stability after thawing from 75.3 2 3.9% to 82.9 2 1.5% while the red cells recoveries (98%) and supernatant hemoglobin (300 mg”/o ) remained nearly unchanged. Although all of these in oitro tests measure the quality of the thawed red cells, the saline stability is an accurate in titro indicator of how the cells will survive in tiivo. If the cells from which the surrounding plasma has been removed by washing are resuspended in the plasma, the saline stability drops to values nearly identical to the unwashed cells (75.0 -t 2.4). The presence of plasma reduces the saline stability of red cells frozen throughout a hematocrit