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Platelet aggregation during progressive hemorrhagic shock in pigs
Platelet aggregation during progressive hemorrhagic shock in pigs Possible effects on the postperfusion syndrome Dennis M. Meagher, D.V.M., Ph.D.* Donald L. Piermattei, D.V.M., Ph.D.,** and Henry Swan, M.D., D.Sc.,*** Ft. Collins, Colo.
investigating the possible causes of postperfusion deaths in miniature swine, the formation of platelet aggregates in stored pig blood was studied.1 The existence of platelet aggregates in banked blood and in blood collected dur ing exsanguination has long been rec ognized.2"5 Several workers have studied the relationship between platelet aggregation in priming blood and the postperfusion syn drome.612 This study was designed to investigate possible methods that would allow collec tion and storage of aggregate-free pig blood for priming in cardiopulmonary bypass. Blood for priming had been collected from pigs at the time of commercial slaughter.1 In order to investigate the pos sible formation of platelet aggregates during blood collection, serial blood samples were collected during total exsanguination of pigs, and fresh smears were examined miFrom the Surgical Laboratory, College of Veterinary Medicine and Biomédical Sciences, Colorado State University, Ft. Collins, Colo. 80521. Received for publication April 5, 1971. •Supported during this study by a fellowship from the Canadian Medical Research Council. ** Supported during this study by a fellowship from the National Institutes of Health •♦•Professor of Surgery (Research), Department of Clinics and Surgery, Colorado State University, Ft. Collins, Colo. 80521.
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croscopically. Blood collected in varying amounts and stored in a variety of anti coagulants was sampled at 12 hour inter vals for 3 days. Smears from these blood samples were examined for platelet or other cellular aggregation. To obtain a quantita tive measurement of platelet aggregation, the number of platelet aggregates per 100 leukocytes were counted in each blood smear and categorized according to size. During collection, platelet aggregates were present by the time 2,000 ml. of blood had been obtained, but they were not noted immediately after the first 750 ml. had been collected (Table I). The number of ag gregates and their size increased pro gressively until the animal had been totally exsanguinated. In order to study the effects of storage Table I. Mean platelet aggregate counts per 100 white cells in blood smears made during collection of ACD blood from 4 pigs Size of aggregates
Smears taken during collection
0-25
25-100
Over 100
First 5 ml. After 500 ml. After 750 ml. After 2,000 ml. Last 5 ml.
0 0 0 18 24
0 0 0 7 30
0 0 0 0 15
Volume 62 Number 5 November, 1971
Platelet aggregation during hemorrhagic shock
on these platelet aggregates, a series of 750 ml. units was stored in acid citrate dextrose (ACD) either alone or combined with heparin and low molecular weight dextran40 (LMWD 40 ). These studies are displayed in Tables II and III. Platelet aggregates were present in all stored blood within a few hours after col lection; as the storage period was extended, the aggregates became larger and more nu merous. Bacterial cultures from the banked blood failed to demonstrate bacterial con tamination. The first 750 ml. of blood, which was collected from a pig and stored in ACD solution with 75 ml. of LMWD 40 and 7,500 units of heparin added to the bottle, was the most resistant to platelet aggregation (Table II, B). No large platelet aggregates developed in this blood during the first 24 hours of storage. Larger volumes of blood, blood stored in ethylene diaminetetracetic acid (EDTA), or blood collected later dur ing exsanguination all demonstrated marked
823
platelet aggregation. Tables II, A and HI, A and B present the results of platelet ag gregate counts in three consecutive units of 750 ml. of blood collected from 1 pig; the fact that the size and numbers of aggregates increase as bleeding continues is evident. Discussion Platelet aggregation appears to be a sig nificant problem in banked pig blood. Ag gregates appeared in the blood regardless of which anticoagulant was used. The first 750 ml. of blood collected in ACD solution with LMWD 40 and heparin added had the least number and the smallest platelet ag gregates of any of the banked bloods in this study. The presence of platelet aggregates in banked blood almost from the time it is collected demonstrates a potential danger for its use in priming the cardiopulmonary bypass pump. Factors released during collection, espe cially if the donor is bled to the state of shock, are thought to cause an increase in
Table II. Comparative size of platelet aggregates in stored blood First 750 ml. of blood in ACD alone
First 750 ml. of Mood in ACD with heparin and LMWDi0
Size of aggregates Smear taken Beginning of refrigeration After 12 hr. After 24 hr. After 48 hr. After 72 hr.
0-25 7 10 30 44 50
25-100 2 3 9 28 45
Size of aggregates
Over 100 0 0 3 5 15
0-25 5 11 21 37 40
25-100 0 0 0 3 5
Over 100 0 0 0 0 0
Table III. Comparative size of platelet aggregates in stored blood A
B
Second 750 ml. of blood in ACD
Third 750 ml. of blood in ACD
Size of aggregates
Size of Aggregates
Smear taken
0-25
Beginning of refrigeration After 12 hr. After 24 hr. After 48 hr. After 72 hr.
10 80 80 55 45
|
25-100 10 40 50 35 30
| Over 100 0 5 7 12 18
0-25 35 100 70 47 38
I
25-100 10 100 70 62 41
| Over 100 7 15 25 27 36
The Journal of
8 24
Meagher, Piermattei, Swan
Thoracic and Cardiovascular Surgery
platelet adhesiveness resulting in platelet aggregation.5· 13> " In any event, platelet aggregates have been identified in banked blood after very short periods of storage. 3 ' 4 Whether these factors are released from in side the body or from the cellular elements in the blood after collection has not yet been established, but it seems clear that platelet aggregation is initiated after the blood has been collected and that it is progressive with storage time. These phenomena are now shown to be especially prominent in pig blood.
Platelets and Leukocytes and Their Removal by Filtration, N . Engl. J. Med. 265: 728, 1961. 4 Swank, R. L.: Adhesiveness of Platelets and Leukocytes During Acute Exsanguination, Am. I. Physiol. 202: 261, 1962. 5 Swank, R. L., Fellman, J. H., and Hissen, W.: Aggregation of Blood Cells by 5-Hydroxytryptamine (Serotonin) Circ. Res. 13: 392, 1963. 6 Allardyce, D. B., Yoshida, S. H., and Ashmore, P. G.: The importance of Microembolism in the Pathogenesis of Organ Dys function Caused by Prolonged Use of the
Conclusions
7 Ashmore, P. G., Svitek, V., and Ambrose, P.: The Incidence and Effects of Particulate Ag gregation and Microembolism in Pump-Oxy-
1. As a pig is exsanguinated, platelet aggregates appear in the blood in increasing numbers and in increasing size as bleeding continues after the onset of severe shock. 2. As blood stands in storage, platelet aggregates increase in size and number re gardless of the anticoagulant used. 3. In order to diminish the incidence and severity of pulmonary damage following cardiopulmonary bypass, only the first 750 ml. of blood drawn from a single animal should be used. It should be stored in ACD solution, with LMWD (0 and heparin added to the bottle. Preferably, it should be used within 24 hours. REFERENCES 1 Meagher, D . M.: Post-perfusion Pulmonary Hypertension in Miniature Pigs, Ph.D. Thesis, Colorado State University, Ft. Collins, Colo., 1969. 2 Sakai, Y., and Lewis, F . J.: A Study of Platelet Aggregation and Blood Velocity in Vitro, J. THORAC. CARDIOVASC. SURG. 49:
982,
1965. 3 Swank, R. L.: Alteration of Blood on Storage: Measurement of Adhesiveness of "Aging"
Pump
Oxygenator,
SURG. 52: 706,
genation
Systems,
SURG. 55: 691,
J.
THORAC.
CARDIOVASC.
1966.
J.
THORAC.
CARDIOVASC.
1968.
8 Bosher, L. H.: Discussion of Microembolism Following Perfusion, J. T H O R A C CARDIOVASC SURG. 52: 723,
1966.
9 Connolly, J. E.: Respiratory Failure Follow ing Cardiopulmonary Bypass From Cardiac Surgery, Norman, J. C , editor: New York, N. Y., 1967, Meredith Publishing Co. 10 Rossi, P. M., Shae-Chi, Y., Koepke, J., and Spencer, F . C : Pulmonary Injury From Pro longed Oxygénation With Venous Blood, Surg. Forum 15: 277, 1964. 11 Swank, R. L., and Porter, G. A.: Disappear ance of the Microemboli Transfused Into Pa tients During Cardiopulmonary Bypass, Trans fusion 3: 192, 1963. 12 Sykes, M. K., Robinson, B., Melrose, D. G., and Nahas, R.: Pulmonary Changes After Extracorporeal Circulation in Dogs, Br. J. Anesth. 38: 432, 1966. 13 Born, G. V. R.: Aggregation of Blood Plate lets by Adenosine Diphosphate and Its Rever sal, Nature 194: 927, 1962. 14 Shimamoto, T., Ishioka, T., and Fugita, T.: Antithrombotic Effect of Mono-amine Oxidase Inhibitors (Nialamide), Circ. Res. 10: 647, 1962.
investigating the possible causes of postperfusion deaths in miniature swine, the formation of platelet aggregates in stored pig blood was studied.1 The existence of platelet aggregates in banked blood and in blood collected dur ing exsanguination has long been rec ognized.2"5 Several workers have studied the relationship between platelet aggregation in priming blood and the postperfusion syn drome.612 This study was designed to investigate possible methods that would allow collec tion and storage of aggregate-free pig blood for priming in cardiopulmonary bypass. Blood for priming had been collected from pigs at the time of commercial slaughter.1 In order to investigate the pos sible formation of platelet aggregates during blood collection, serial blood samples were collected during total exsanguination of pigs, and fresh smears were examined miFrom the Surgical Laboratory, College of Veterinary Medicine and Biomédical Sciences, Colorado State University, Ft. Collins, Colo. 80521. Received for publication April 5, 1971. •Supported during this study by a fellowship from the Canadian Medical Research Council. ** Supported during this study by a fellowship from the National Institutes of Health •♦•Professor of Surgery (Research), Department of Clinics and Surgery, Colorado State University, Ft. Collins, Colo. 80521.
822
croscopically. Blood collected in varying amounts and stored in a variety of anti coagulants was sampled at 12 hour inter vals for 3 days. Smears from these blood samples were examined for platelet or other cellular aggregation. To obtain a quantita tive measurement of platelet aggregation, the number of platelet aggregates per 100 leukocytes were counted in each blood smear and categorized according to size. During collection, platelet aggregates were present by the time 2,000 ml. of blood had been obtained, but they were not noted immediately after the first 750 ml. had been collected (Table I). The number of ag gregates and their size increased pro gressively until the animal had been totally exsanguinated. In order to study the effects of storage Table I. Mean platelet aggregate counts per 100 white cells in blood smears made during collection of ACD blood from 4 pigs Size of aggregates
Smears taken during collection
0-25
25-100
Over 100
First 5 ml. After 500 ml. After 750 ml. After 2,000 ml. Last 5 ml.
0 0 0 18 24
0 0 0 7 30
0 0 0 0 15
Volume 62 Number 5 November, 1971
Platelet aggregation during hemorrhagic shock
on these platelet aggregates, a series of 750 ml. units was stored in acid citrate dextrose (ACD) either alone or combined with heparin and low molecular weight dextran40 (LMWD 40 ). These studies are displayed in Tables II and III. Platelet aggregates were present in all stored blood within a few hours after col lection; as the storage period was extended, the aggregates became larger and more nu merous. Bacterial cultures from the banked blood failed to demonstrate bacterial con tamination. The first 750 ml. of blood, which was collected from a pig and stored in ACD solution with 75 ml. of LMWD 40 and 7,500 units of heparin added to the bottle, was the most resistant to platelet aggregation (Table II, B). No large platelet aggregates developed in this blood during the first 24 hours of storage. Larger volumes of blood, blood stored in ethylene diaminetetracetic acid (EDTA), or blood collected later dur ing exsanguination all demonstrated marked
823
platelet aggregation. Tables II, A and HI, A and B present the results of platelet ag gregate counts in three consecutive units of 750 ml. of blood collected from 1 pig; the fact that the size and numbers of aggregates increase as bleeding continues is evident. Discussion Platelet aggregation appears to be a sig nificant problem in banked pig blood. Ag gregates appeared in the blood regardless of which anticoagulant was used. The first 750 ml. of blood collected in ACD solution with LMWD 40 and heparin added had the least number and the smallest platelet ag gregates of any of the banked bloods in this study. The presence of platelet aggregates in banked blood almost from the time it is collected demonstrates a potential danger for its use in priming the cardiopulmonary bypass pump. Factors released during collection, espe cially if the donor is bled to the state of shock, are thought to cause an increase in
Table II. Comparative size of platelet aggregates in stored blood First 750 ml. of blood in ACD alone
First 750 ml. of Mood in ACD with heparin and LMWDi0
Size of aggregates Smear taken Beginning of refrigeration After 12 hr. After 24 hr. After 48 hr. After 72 hr.
0-25 7 10 30 44 50
25-100 2 3 9 28 45
Size of aggregates
Over 100 0 0 3 5 15
0-25 5 11 21 37 40
25-100 0 0 0 3 5
Over 100 0 0 0 0 0
Table III. Comparative size of platelet aggregates in stored blood A
B
Second 750 ml. of blood in ACD
Third 750 ml. of blood in ACD
Size of aggregates
Size of Aggregates
Smear taken
0-25
Beginning of refrigeration After 12 hr. After 24 hr. After 48 hr. After 72 hr.
10 80 80 55 45
|
25-100 10 40 50 35 30
| Over 100 0 5 7 12 18
0-25 35 100 70 47 38
I
25-100 10 100 70 62 41
| Over 100 7 15 25 27 36
The Journal of
8 24
Meagher, Piermattei, Swan
Thoracic and Cardiovascular Surgery
platelet adhesiveness resulting in platelet aggregation.5· 13> " In any event, platelet aggregates have been identified in banked blood after very short periods of storage. 3 ' 4 Whether these factors are released from in side the body or from the cellular elements in the blood after collection has not yet been established, but it seems clear that platelet aggregation is initiated after the blood has been collected and that it is progressive with storage time. These phenomena are now shown to be especially prominent in pig blood.
Platelets and Leukocytes and Their Removal by Filtration, N . Engl. J. Med. 265: 728, 1961. 4 Swank, R. L.: Adhesiveness of Platelets and Leukocytes During Acute Exsanguination, Am. I. Physiol. 202: 261, 1962. 5 Swank, R. L., Fellman, J. H., and Hissen, W.: Aggregation of Blood Cells by 5-Hydroxytryptamine (Serotonin) Circ. Res. 13: 392, 1963. 6 Allardyce, D. B., Yoshida, S. H., and Ashmore, P. G.: The importance of Microembolism in the Pathogenesis of Organ Dys function Caused by Prolonged Use of the
Conclusions
7 Ashmore, P. G., Svitek, V., and Ambrose, P.: The Incidence and Effects of Particulate Ag gregation and Microembolism in Pump-Oxy-
1. As a pig is exsanguinated, platelet aggregates appear in the blood in increasing numbers and in increasing size as bleeding continues after the onset of severe shock. 2. As blood stands in storage, platelet aggregates increase in size and number re gardless of the anticoagulant used. 3. In order to diminish the incidence and severity of pulmonary damage following cardiopulmonary bypass, only the first 750 ml. of blood drawn from a single animal should be used. It should be stored in ACD solution, with LMWD (0 and heparin added to the bottle. Preferably, it should be used within 24 hours. REFERENCES 1 Meagher, D . M.: Post-perfusion Pulmonary Hypertension in Miniature Pigs, Ph.D. Thesis, Colorado State University, Ft. Collins, Colo., 1969. 2 Sakai, Y., and Lewis, F . J.: A Study of Platelet Aggregation and Blood Velocity in Vitro, J. THORAC. CARDIOVASC. SURG. 49:
982,
1965. 3 Swank, R. L.: Alteration of Blood on Storage: Measurement of Adhesiveness of "Aging"
Pump
Oxygenator,
SURG. 52: 706,
genation
Systems,
SURG. 55: 691,
J.
THORAC.
CARDIOVASC.
1966.
J.
THORAC.
CARDIOVASC.
1968.
8 Bosher, L. H.: Discussion of Microembolism Following Perfusion, J. T H O R A C CARDIOVASC SURG. 52: 723,
1966.
9 Connolly, J. E.: Respiratory Failure Follow ing Cardiopulmonary Bypass From Cardiac Surgery, Norman, J. C , editor: New York, N. Y., 1967, Meredith Publishing Co. 10 Rossi, P. M., Shae-Chi, Y., Koepke, J., and Spencer, F . C : Pulmonary Injury From Pro longed Oxygénation With Venous Blood, Surg. Forum 15: 277, 1964. 11 Swank, R. L., and Porter, G. A.: Disappear ance of the Microemboli Transfused Into Pa tients During Cardiopulmonary Bypass, Trans fusion 3: 192, 1963. 12 Sykes, M. K., Robinson, B., Melrose, D. G., and Nahas, R.: Pulmonary Changes After Extracorporeal Circulation in Dogs, Br. J. Anesth. 38: 432, 1966. 13 Born, G. V. R.: Aggregation of Blood Plate lets by Adenosine Diphosphate and Its Rever sal, Nature 194: 927, 1962. 14 Shimamoto, T., Ishioka, T., and Fugita, T.: Antithrombotic Effect of Mono-amine Oxidase Inhibitors (Nialamide), Circ. Res. 10: 647, 1962.