The Effect of Agitation on In Vitro Metabolism of Erythrocytes Stored in CPD-Adenine T. A. BENSINGER, J. METRO,AND E. BEUTLER From the Department ojHematology. City ojHope Medical Center. Duarte. Cali/ornia Agitation of blood stored in plastic containers has been reported to lead to improved posttransfusion survival and it has been found that, in some media, agitation has improv'ed erythrocyte 2.3diphosphoglycerate (2, I D P G ) levels. Using CPD I1 media (CPD with 277.5 m M glucose and 2.04 m M adenine), we were not able to identify any improvement in levelsof adenosine triphosphate, 2J-DPG or glucose in whole blood under various agitation conditions when compared with the nonagitated control. The 2,3-DPG level was moderately improved through 23 days in the 90 per cent hematocrit packed erythrocytes but the results were not considered to be significantly beneficial to warrant agitation. Thus, the application of agitation to the CPD I1 blood storage system was of no great benefit in improving metabolic intermediatelevels.
AGITATIONof blood during storage in plastic containers has been reported to lead More to improved posttransfusion ~iability.~ recently, the preservation of erythrocyte 2.3diphosphoglycerate (2,3-DPG) has been shown to be improved in certain media as a result of a g i t a t i ~ n In . ~ a previous paper, we reported that erythrocytes packed to 90 per cent and agitated weekly could maintain ATP at greater than 2.0 pmole/gram Hb for up to 42 days of storage if 2.04 mM adenine and 277.5 mM glucose were present in the storage medium.' It seemed advisable to undertake a study in which various conditions of agitation were applied to blood stored both as whole blood and packed units in CPD adenine.
CPD modified by increasing the glucose concentration to 277.5 mM and adding adenine at a concentration of 2.04 mM (CPD II).' Blood was collected in the ratio of 50 ml of blood for each 7 ml of the storage medium and was intermittently mixed with the medium by gentle manual inversion of the bags during the four- to eight-minute collection period. All blood was allowed to stand for one-half hour at room temperature (22 to 25 C) and then was placed in a blood storage refrigerator at 4 C. Within three hours after collection the units chosen for packing were centrifuged at 4500 x g for five minutes. The supernatant plasma was pressed off to obtain hematocrits of approximately 90 per cent. The full units of blood, either as whole blood or 90 per cent packed hematocrit erythrocytes, were divided into the following categories for agitation: 1) continuous agitation in a mixer rotating at 4.5 revolutions per minute, 2) agitation by vigorous manual inversion for one minute either three or seven times per week, and 3) no agitation except at the time of sample collection. Measurements I n Vitro Hematocrits were measured by a standard microtechnique. Erythrocyte ATP and glucose concentrations were determined by the hexokinase method.l2,3-DPG levels were measured by a modification of the technique of Krimsky.l
Results and Discussion Levels of glucose adequate for metabolism in the red blood cells were present for the full storage time of 42 days in all units, both whole blood and packed cells, under the various conditions of agitation. No major differences in ATP levels were apparent in either the whole blood or packed erythrocytes subjected to the various agitation schedules although ATP levels were higher in whole blood than in the packed erythrocytes (Figs. 1 and 2). Erythrocyte 2,3-DPG levels were somewhat higher in the packed units subjected to
Materials and Methods All blood donors were adults, hematologically normal and free from any illness for at least three months prior to donation. Blood was collected aseptically into Fenwal polyvinyl chloride bags containing conventional Received for publication August 26, 1974; accepted October 7. 1974 This work was supported, in part, by Army Contract No.D A D A 17-70-C-0078 and by NIH grant HL 07449.
140 Transfusion Mar.-Apr. 1975
Volume I 5 Number 2
I
600
Tim;
141
agitated per week
GI ucose mgmldeciliter 500
400
300
200
100
I
1
8
7
14
21
1
28
35
I
42
Days
FIG. I . Level of ATP and glucose present in whole blood collected in CPD 11. The units were subjected to agitation continuously, three or seven times per week or only at the time of sample collection. The means at various intervals of collection are plotted but the standard deviations have been omitted for clarity.
15
Times agitated per week
2.3 DPG pM1g Hb
7x
-x-x-x-x
Continuous
-0-0-
10
1
5
0
0 Time (Days) FIG. 2. Level of 2,3-DPG in whole blood collected in CPD I I and subjected to agitation continuously, three or seven times per week or only at time of sample collection. The means and standard deviations are plotted.
142
Times agitated per week
6001
JX
Continuous
-x-x-x-x -0-0-
Glucose mgmldeci I iter
ATP pM/g H b
7
I
I
I
I
14
21 Days
28
35
42
FIG.3. Level of ATP and glucose present in 90 per cent Hct packed erythrocytes collected in C P D 11. The units were subjected to agitation continuously, three or seven times per week or only at the time of sample collection. The means are plotted but the standard deviations have been omitted for clarity.
15
JX
2.3 DPG pM/g
-x-X-X-x
Hb 10
5
0 J
14
21 Time (Days)
28
35
42
FIG.4. Level of 2.3-DPG in 90 per cent Hct packed erythrocytes collected in C P D 11. The units were subjected to agitation continuously, three or seven times per week or only at the time of sample collection. The means and standard deviations are plotted.
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143
AGITATION OF ERYTHROCYTES
continuous agitation through 28 days of storage when compared with agitation of either seven or three times per week or in blood not agitated (Figs. 3 and 4). However, the 2,3-DPG levels were not significantly different after 28 days of storage. This difference in 2,3-DPG levels was not demonstrated in the whole blood units subjected to various schedules of agitation. In a comparable preservation system, ACD adenine, agitation also was found to exert very little beneficial effect on ATP levels.' However, there was some improvement in 2,3DPG levels. The application of agitation to whole blood stored in CPD I1 has no beneficial effect in maintenance of either ATP or 2,3DPG. When packed erythrocytes were studied, minimal improvement in 2,3-DPG levels was noted in the units that were continuously agitated. The beneficial effect of agitation on 2,3-DPG levels seems, therefore, largely to be limited to experimental preservative media designed to improve 2,3-DPG preservation.
References Bensinger, T. A., J. Metro, and E. Beutler: In vitro metabolism of packed erythrocytes stored in C P D adenine. Transfusion 15:135, 1975. 2. Beutler, E.: Red Cell Metabolism: A Manual of Biochemical Methods, 2nd ed. New York, Grune and Stratton, 1975. 3. Dern, R. J., J. J. Wiorowski. and T. Matsuda: Studies on the preservation o f human blood. V. The effect of mixing anticoagulated blood during storage on the poststorage erythrocyte survival. J. Lab. Clin. Med. 75:37. 1970. 4. Wood, L. A., and E. Beutler: The effect of periodic mixing on the preservation of 2.3diphosphoglycerate (2.3-DPG) levels in stored blood. Blood 42:17, 1973. I.
T. A. Bensinger, M.D., MAJ MC, Letterman Army Institute of Research, Presidio of San Francisco, Calif. 94129. J. Metro, B.S., Research Technician, City of Hope Medical Center, Duarte. Calif. 91010. E. Beutler, M.D., Chairman, Division of Medicine, and Director, Department of Hematology, City of Hope Medical Center; Clinical Professor of Medicine, University of Southern California.
AGITATIONof blood during storage in plastic containers has been reported to lead More to improved posttransfusion ~iability.~ recently, the preservation of erythrocyte 2.3diphosphoglycerate (2,3-DPG) has been shown to be improved in certain media as a result of a g i t a t i ~ n In . ~ a previous paper, we reported that erythrocytes packed to 90 per cent and agitated weekly could maintain ATP at greater than 2.0 pmole/gram Hb for up to 42 days of storage if 2.04 mM adenine and 277.5 mM glucose were present in the storage medium.' It seemed advisable to undertake a study in which various conditions of agitation were applied to blood stored both as whole blood and packed units in CPD adenine.
CPD modified by increasing the glucose concentration to 277.5 mM and adding adenine at a concentration of 2.04 mM (CPD II).' Blood was collected in the ratio of 50 ml of blood for each 7 ml of the storage medium and was intermittently mixed with the medium by gentle manual inversion of the bags during the four- to eight-minute collection period. All blood was allowed to stand for one-half hour at room temperature (22 to 25 C) and then was placed in a blood storage refrigerator at 4 C. Within three hours after collection the units chosen for packing were centrifuged at 4500 x g for five minutes. The supernatant plasma was pressed off to obtain hematocrits of approximately 90 per cent. The full units of blood, either as whole blood or 90 per cent packed hematocrit erythrocytes, were divided into the following categories for agitation: 1) continuous agitation in a mixer rotating at 4.5 revolutions per minute, 2) agitation by vigorous manual inversion for one minute either three or seven times per week, and 3) no agitation except at the time of sample collection. Measurements I n Vitro Hematocrits were measured by a standard microtechnique. Erythrocyte ATP and glucose concentrations were determined by the hexokinase method.l2,3-DPG levels were measured by a modification of the technique of Krimsky.l
Results and Discussion Levels of glucose adequate for metabolism in the red blood cells were present for the full storage time of 42 days in all units, both whole blood and packed cells, under the various conditions of agitation. No major differences in ATP levels were apparent in either the whole blood or packed erythrocytes subjected to the various agitation schedules although ATP levels were higher in whole blood than in the packed erythrocytes (Figs. 1 and 2). Erythrocyte 2,3-DPG levels were somewhat higher in the packed units subjected to
Materials and Methods All blood donors were adults, hematologically normal and free from any illness for at least three months prior to donation. Blood was collected aseptically into Fenwal polyvinyl chloride bags containing conventional Received for publication August 26, 1974; accepted October 7. 1974 This work was supported, in part, by Army Contract No.D A D A 17-70-C-0078 and by NIH grant HL 07449.
140 Transfusion Mar.-Apr. 1975
Volume I 5 Number 2
I
600
Tim;
141
agitated per week
GI ucose mgmldeciliter 500
400
300
200
100
I
1
8
7
14
21
1
28
35
I
42
Days
FIG. I . Level of ATP and glucose present in whole blood collected in CPD 11. The units were subjected to agitation continuously, three or seven times per week or only at the time of sample collection. The means at various intervals of collection are plotted but the standard deviations have been omitted for clarity.
15
Times agitated per week
2.3 DPG pM1g Hb
7x
-x-x-x-x
Continuous
-0-0-
10
1
5
0
0 Time (Days) FIG. 2. Level of 2,3-DPG in whole blood collected in CPD I I and subjected to agitation continuously, three or seven times per week or only at time of sample collection. The means and standard deviations are plotted.
142
Times agitated per week
6001
JX
Continuous
-x-x-x-x -0-0-
Glucose mgmldeci I iter
ATP pM/g H b
7
I
I
I
I
14
21 Days
28
35
42
FIG.3. Level of ATP and glucose present in 90 per cent Hct packed erythrocytes collected in C P D 11. The units were subjected to agitation continuously, three or seven times per week or only at the time of sample collection. The means are plotted but the standard deviations have been omitted for clarity.
15
JX
2.3 DPG pM/g
-x-X-X-x
Hb 10
5
0 J
14
21 Time (Days)
28
35
42
FIG.4. Level of 2.3-DPG in 90 per cent Hct packed erythrocytes collected in C P D 11. The units were subjected to agitation continuously, three or seven times per week or only at the time of sample collection. The means and standard deviations are plotted.
Volume IS Number 2
143
AGITATION OF ERYTHROCYTES
continuous agitation through 28 days of storage when compared with agitation of either seven or three times per week or in blood not agitated (Figs. 3 and 4). However, the 2,3-DPG levels were not significantly different after 28 days of storage. This difference in 2,3-DPG levels was not demonstrated in the whole blood units subjected to various schedules of agitation. In a comparable preservation system, ACD adenine, agitation also was found to exert very little beneficial effect on ATP levels.' However, there was some improvement in 2,3DPG levels. The application of agitation to whole blood stored in CPD I1 has no beneficial effect in maintenance of either ATP or 2,3DPG. When packed erythrocytes were studied, minimal improvement in 2,3-DPG levels was noted in the units that were continuously agitated. The beneficial effect of agitation on 2,3-DPG levels seems, therefore, largely to be limited to experimental preservative media designed to improve 2,3-DPG preservation.
References Bensinger, T. A., J. Metro, and E. Beutler: In vitro metabolism of packed erythrocytes stored in C P D adenine. Transfusion 15:135, 1975. 2. Beutler, E.: Red Cell Metabolism: A Manual of Biochemical Methods, 2nd ed. New York, Grune and Stratton, 1975. 3. Dern, R. J., J. J. Wiorowski. and T. Matsuda: Studies on the preservation o f human blood. V. The effect of mixing anticoagulated blood during storage on the poststorage erythrocyte survival. J. Lab. Clin. Med. 75:37. 1970. 4. Wood, L. A., and E. Beutler: The effect of periodic mixing on the preservation of 2.3diphosphoglycerate (2.3-DPG) levels in stored blood. Blood 42:17, 1973. I.
T. A. Bensinger, M.D., MAJ MC, Letterman Army Institute of Research, Presidio of San Francisco, Calif. 94129. J. Metro, B.S., Research Technician, City of Hope Medical Center, Duarte. Calif. 91010. E. Beutler, M.D., Chairman, Division of Medicine, and Director, Department of Hematology, City of Hope Medical Center; Clinical Professor of Medicine, University of Southern California.
