Critical Importance of Citrate-Blood Ratio in Platelet Aggregation Studies CHUNG-HSIN TS'AO, P H . D . , ROSE L O ,
M T (ASCP),
AND JANE RAYMOND,
M T (ASCP)
From the Coagulation Laboratory, Department of Pathology, Northwestern University, McGaw Medical Center, Chicago, Illinois
ABSTRACT
studies have become increasingly important in assisting the diagnosis of bleeding disorders and in assessment of the effects of medication on platelet reactivity. In clinical laboratories, these studies are carried out on plateletrich plasma, usually prepared from citrated whole blood. T h e ratio of blood to citrate is usually 9 to 1 (vol./vol.). T h e concentratio of citrate in the blood chelates most ionized calcium so that the clotting processes are interrupted. T h e remaining unbound calcium ions are adequate for platelets to perform their in-vitro functions. On a unit basis, blood collected by routine PLATELET AGGREGATION
Received May 29, 1975; received revised manuscript July 29, 1975; accepted for publication July 29, 1975. Supported in part by a Special Coagulation Research Fund, 4271-115-05 of the Northwestern University Pathology Group. Address reprint requests to Dr. Ts'ao: Department of Pathology, Northwestern Memorial Hospital Wesley Pavilion, 250 East Superior Street, Chicago, Illinois 60611.
blood bank procedure contains more citrate than that obtained by the laboratory procedure. In our studies of the in-vitro functions of platelets stored in an acidcitrate-dextrose environment, we found that relatively slight variations in ACD concentration significantly affect platelet aggregation by physiologic agents such as adenosine diphosphate and epinephrine. We have now performed experiments on platelets freshly prepared from blood of normal subjects anticoagulated with different amounts of citrate. T h e purpose of this paper is to call attention to the importance of standardizing the anticoagulant concentration in a given laboratory in which platelet aggregation studies are performed, and among laboratories when platelet aggregation results are compared. Materials and Methods Platelets obtained from young, healthy volunteers who had not taken aspirin-con-
518
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Ts'ao, Chung-hsin, Lo, Rose, and Raymond, Jane: Critical importance of citrate-blood ratio in platelet aggregation studies. Am J Clin Pathol 65: 518— 522, 1976. T h e effects of citrate concentration on adenosinediphosphate-, epinephrine-, collagen-, and ristocetin-induced human platelet aggregation were investigated. Relatively small increments in citrate concentration markedly inhibited platelet aggregation by all three physiologic agents. T h e inhibitory effect was greatest on epinephrine-induced aggregation, and least on collagen-induced aggregation. Ristocetin-induced aggregation was not affected by excess citrate anticoagulation. These findings indicate the importance of controlling the citrate:blood ratio in clinical platelet aggregation studies and in the assessment of antiplatelet drugs. (Keywords: ACD; Sodium citrate; Platelet aggregation; ADP; Collagen; Epinephrine; Ristocetin.)
April 1976
CITRATE CONCENTRATION AND PLATELET CLUMPING
Results Platelet-aggregating effects of adenosinediphosphate (ADP), epinephrine, and collagen were profoundly affected by the amounts of citrate used to anticoagulate
the blood. Regardless of whether ACD or trisodium citrate was the anticoagulant, the greater the amount of anticoagulant, the slower the aggregation, the lesser the extent of aggregation, and the longer the collagen aggregation time. However, ristocetin-induced aggregation was not appreciably affected by increased citrate concentration. The aggregabilities of platelets of individual subjects varied with respect to given concentrations of aggregating agents. Similarly, the extents of the effects of increased ACD or trisodium citrate on platelet aggregation also differed among platelets from different donors. For some, the differences in ADP-, epinephrine-, and collagen-induced platelet aggregations were small (25%). In several instances, platelets prepared from blood containing 0.133 ml. per ACD ml. blood failed to clump after exposure to epinephrine. Epinephrine-induced aggregation was absent in all samples anticoagulated with 0.177 ml. ACD per ml. blood, while ADP- and collageninduced aggregations of the same samples remained discernible. In one instance, the extent of aggregation by ristocetin was the least in the sample containing the lowest concentration of ACD. In most cases, ristocetin-induced aggregations of the low citrate-containing specimens were comparable to those of specimens containing greater amounts of citrate. These individual differences were apparently not related to hematocrit or platelet count. The hematocrits of our donors' blood specimens ranged from 40 to 48%. Platelet counts of all subjects were within normal limits. For any given blood sample anticoagulated with various amounts of citrate, platelet concentrations in PRP specimens were comparable. Figure 1 shows platelet aggregation tracings of PRP specimens and-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
taining drugs in the preceding two weeks were studied. Blood was drawn from a vein in the antecubital region with a plastic syringe; aiiquots of the blood were mixed with various amounts of ACD (NIH formula A) or 3.8% trisodium citrate in polystyrene tubes. T h e amounts of anticoagulant in ml. per ml. blood were 0.097, 0.111, 0.133, 0.155, and 0.177. Plateletrich plasma (PRP) was prepared by centrifugation of blood at 110 X g for 15 minutes at room temperature, platelet-poor plasma (PPP) by centrifuging the remaining blood at 1,100 X g for another 15 minutes. Platelet count in each PRP sample was determined by phase-contrast microscopy. Hematocrit of the blood was measured by the microhematocrit technic. Both PRP and PPP were kept covered (at 25 C.) as much as possible during an experiment to minimize escape of C 0 2 . Platelet aggregation studies were performed on a Chrono-log platelet aggregometer equipped with a recorder. 21 Optimal concentrations of adenosinediphosphate (ADP, Sigma Chemicals), epinephrine (Upjohn Co.), purified human skin collagen, 4 or ristocetin (kindly supplied by Mr. G. Barlow of Abbott Lab.) were chosen for their ability to elicit marked aggregation of individual PRP containing the lowest concentration of anticoagulant (i.e., 0.097 ml. citrate per ml. blood). T h e same aggregating agents were used to aggregate PRP containing greater amounts of citrate. The rate of aggregation (assessed by extent of aggregation 30 seconds after addition of an aggregating agent), maximum aggregation at any time during the 5-minute recording period and at the end of 5 minutes, and collagen aggregation time were analyzed.
519
520
TS'AO, LO AND RAYMOND ADP
A.J.C.P. —Vol. 65
Ml
coagulated with different amounts of ACD. Despite the variation of effects of increased citrate on platelet aggregation of individual PRP specimens, analysis of the data obtained from the subjects included in the study disclosed a definitive pattern. When the average maximum aggregations from four to seven individual experiments were plotted against the amounts of ACD in the blood (Fig. 2), it was evident that higher concentrations of citrate exerted no effect on ristocetin-induced aggregation, the least effect on collagen-induced aggregation, and the greatest effect on epinephrineinduced aggregation. Discussion T h e aggregability of human platelets is affected by factors such as platelet count, the time interval between venipuncture and testing, temperature, pH of the environment, and type and concentration of anticoagulant. O'Brien 13 has shown that
PRP stored at 37 C. failed to respond to epinephrine after 90 minutes, to ADP after 180-240 minutes, and to thrombin after 200 minutes. Platelets stored at room temperature lose their responsiveness to aggregating agents at a slower rate. 6,12 Recently, Warlow and associates22 and Rossi and Louis 18 have found that epinephrine-induced platelet aggregation actually increases progressively as citrated PRP is left at room temperature for a period as long as 2 hours. The temperature-dependent nature of ADP-induced platelet aggregation has been demonstrated by Odegard and co-workers 15 and by Praga and Pogliani.16 Platelet aggregation reactions require the presence of free calcium ions. T h e inhibitory effect of sodium citrate, which binds ionized calcium, on human platelet aggregation was demonstrated by O'Brien and colleagues, 14 and more recently, by Han and Ardlie. 5 T h e latter investigators suggest that 0.1 M, rather than 0 . 1 2 9 M (3.8%) of sodium citrate
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
FIG. 1. Light transmission tracings depicting platelet aggregation induced by ADP, epinephrine (EPI), collagen (COL) and ristocetin (RIST). a-e denote concentrations of ACD used to anticoagulate the blood: a 0.097,6 0.111, c 0.133, d 0.155, and e 0.177 ml. ACD per ml. blood.
April 1976
CITRATE CONCENTRATION AND PLATELET CLUMPING
let aggregation reactions, the precise ratio between citrate and blood becomes crucial. T h e effect of excess citrate was greatest on epinephrine-induced aggregation, and least on collagen-induced aggregation, with the three physiologic aggregating agents we used. High concentrations of citrate affected not only the secondary phases of ADP- and epinephrine-induced aggregations, but also the primary phases. It appears, however, that secondary-phase aggregation, which is induced by materials released from platelets themselves, is more susceptible to increased citrate concentration. Apparently, epinephrine-induced platelet release is more readily suppressed by sodium citrate than collagen-induced release. Ristocetin-induced aggregation was not influenced by increased amounts of citrate. It has been shown that calcium ions are not essential for this reaction. Indeed, platelets in EDTA PRP, not responsive to physiologic agents without the addition of exogenous calcium, are quite aggregable by ristocetin. 7 ' 8,21 Use of the platelet aggregation test by
100i—
RIST
Fie. 2. Maximum platelet aggregation, i.e., the highest light transmittance during the 5-minute recording period, plotted against concentrations of ACD used to anticoagulate the blood. Each point represents the average from 4 to 7 experiments. It is clear that the greatest effect of increased ACD is on epinephrine-induced aggregation. Ristocetin-induced aggregation is not affected by increments of ACD.
0.10
0.1S
A C D ml/ml blOOd
0.20
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
should be used to anticoagulate blood for platelet aggregation studies. Increased concentrations of sodium citrate would have two effects: (1) Large quantities of Ca + + would be chelated. As a result, the amount of Ca + + left in PRP would be insufficient for platelets to function properly. (2) T h e pH of the PRP would be reduced. Aggregation of platelets by most agents will not take place in EDTA PRP, in which practically all free calcium ions are bound to EDTA. T h e reaction is also abolished below pH 6.4 and above pH 10.0 in citrated PRP 3.H.20 ~phe aggregability of platelets is markedly influenced by the pH environment without alteration of the Ca + + concentration in PRP.9.io,i7 In the present study, we have demonstrated substantial inhibition of platelet aggregation by relatively small increments of citrate concentration. Overfilling or underfilling a tube with fixed amounts of anticoagulants is, unfortunately, not uncommon. For coagulation tests this may not be critical, because calcium ions are supplied by an exogenous source. For plate-
521
522
TS'AO, LO AND RAYMOND
References 1. Danta G: Secondary phase platelet aggregation induced by adenosine diphosphate in patients with cerebral occlusive disease and in control subjects. T h r o m b Diath Haemorrh 23:159169, 1970 2. Davis JW: Defective platelet disaggregation associated with occlusive arterial diseases. Angiology 24:391-397, 1973 3. Flatow FA Jr, Freireich EJ: T h e increased effectiveness of platelet concentrates in acidified plasma. Blood 27:449-459, 1966 4. Green D, Dunne B, Schmid FR, et al: A study of the variable response of human platelets to collagen: Relation to aspirin-induced inhibition of aggregation. Am J Clin Pathol 60:920-926, 1973 5. Han P, Ardlie NG: T h e influence of pH, temperature, and calcium on platelet aggregation. Maintenance of environmental pH and platelet function for in vitro studies in plasma stored at 37 C. Br J Haematol 26:373-389, 1974 6. Harrison MJG, Emmons PR, Mitchell JRA: T h e variability of human platelet aggregation. J Atheroscler Res 7:197-205, 1967 7. Howard MA, Firkin BG: Ristocetin — A new tool
65
in the investigation of platelet aggregation. Thromb Diath Haemorrh 25:362-369, 1971 8. Jenkins CSP, Meyer D, Dreyfus MD, et al: Willebrand factor and ristocetin. I. Mechanism of ristocetin-induced platelet aggregation. Br J Haematol 28:561-578, 1974 9. Kerby GP, Taylor SM: The influence of buffers on human platelet aggregation and energy metabolism. Proc Soc Exp Biol Med 136:452456, 1971 10. Lamber E L J r , W a n i n e r RA III, Batchelor ED: Effect of metabolic acidosis and alkalosis on h u m a n platelet aggregation induced by epinephrine and ADP. Proc Soc Exp Biol Med 145:743-746, 1974 11. McLean JR, Veloso H: Change of shape without aggregation caused by ADP in rabbit platelets allow pH. LifeSci 6:1983-1986, 1967 12. Murphy S, Gardner FH: Platelet storage at 22 C: Metabolic, morphologic and functional studies. J Clin Invest 50:370-377, 1971 13. O'Brien JR: A comparison of platelet aggregation produced by seven compounds and a comparison of their inhibitors. J Clin Pathol 17: 2 7 5 - 2 8 1 , 1964 14. O'Brien JR, Shoobridge SM, Finch WJ: Comparison of the effect of heparin and citrate on platelet aggregation. J Clin Pathol 2 2 : 2 8 31, 1969 • 15. Odegard AE, Skalhegg BA, Hellem AJ: Investigation on adenosine diphosphate (ADP) induced platelet adhesiveness in vitro. Part III. T h e inactivation of ADP in plasma. Thromb Diath Haemorrh 11:317-326, 1964 16. Praga CA, Pogliani EM: Effect of temperature on ADP-induced platelet aggregation. Its significance in studying ami-aggregating drugs. T h r o m b Diath Haemorrh 29:183-189, 1973 17. Rogers AB: T h e effect of pH on human platelet aggregation induced by epinephrine and ADP. Proc Soc Exp Biol Med 139:11001103, 1972 18. Rossi EC, Louis G: A time-dependent increase in the responsiveness of platelet-rich plasma to epinephrine. J Lab Clin Med 85:300-306, 1975 19. Salky N, Dugdale M: Platelet abnormalities in ischemic heart disease. Am J Cardiol 32:612617, 1973 20. Skoza L, Zucker MB, Jerushalmy Z, et al: Kinetic studies of platelet aggregation induced by adenosine diphosphate and its inhibition by chelating agents, quanidino compounds and adenosine. T h r o m b Diath Haemorrh 18:713725, 1967 21. Ts'ao C: Platelet aggregation by ristocetin in EDTA plasma. Extensive aggregation with high concentrations of EDTA. Haemostasis 1:315-318, 1973 22. Warlow C, Corna A, Ogston D, et al: T h e relationship between platelet aggregation and time interval after venipuncture. T h r o m b Diath Haemorrh 31:133-141, 1974 23. Zahavi J, Dreyfuss F: An abnormal pattern of adenosine-diphosphate induced platelet aggregation in acute myocardial infarction. Thromb Diath Haemorrh 21:76-88, 1970
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
clinical laboratories has been increasing. Platelet aggregation is a valuable tool in assisting in the diagnosis of several disorders, notably hemorrhagic diathesis. Increased platelet aggregation and altered platelet disaggregation have been reported to occur in blood of patients who have cardiovascular and cerebral vascular diseases.1,2
M T (ASCP),
AND JANE RAYMOND,
M T (ASCP)
From the Coagulation Laboratory, Department of Pathology, Northwestern University, McGaw Medical Center, Chicago, Illinois
ABSTRACT
studies have become increasingly important in assisting the diagnosis of bleeding disorders and in assessment of the effects of medication on platelet reactivity. In clinical laboratories, these studies are carried out on plateletrich plasma, usually prepared from citrated whole blood. T h e ratio of blood to citrate is usually 9 to 1 (vol./vol.). T h e concentratio of citrate in the blood chelates most ionized calcium so that the clotting processes are interrupted. T h e remaining unbound calcium ions are adequate for platelets to perform their in-vitro functions. On a unit basis, blood collected by routine PLATELET AGGREGATION
Received May 29, 1975; received revised manuscript July 29, 1975; accepted for publication July 29, 1975. Supported in part by a Special Coagulation Research Fund, 4271-115-05 of the Northwestern University Pathology Group. Address reprint requests to Dr. Ts'ao: Department of Pathology, Northwestern Memorial Hospital Wesley Pavilion, 250 East Superior Street, Chicago, Illinois 60611.
blood bank procedure contains more citrate than that obtained by the laboratory procedure. In our studies of the in-vitro functions of platelets stored in an acidcitrate-dextrose environment, we found that relatively slight variations in ACD concentration significantly affect platelet aggregation by physiologic agents such as adenosine diphosphate and epinephrine. We have now performed experiments on platelets freshly prepared from blood of normal subjects anticoagulated with different amounts of citrate. T h e purpose of this paper is to call attention to the importance of standardizing the anticoagulant concentration in a given laboratory in which platelet aggregation studies are performed, and among laboratories when platelet aggregation results are compared. Materials and Methods Platelets obtained from young, healthy volunteers who had not taken aspirin-con-
518
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Ts'ao, Chung-hsin, Lo, Rose, and Raymond, Jane: Critical importance of citrate-blood ratio in platelet aggregation studies. Am J Clin Pathol 65: 518— 522, 1976. T h e effects of citrate concentration on adenosinediphosphate-, epinephrine-, collagen-, and ristocetin-induced human platelet aggregation were investigated. Relatively small increments in citrate concentration markedly inhibited platelet aggregation by all three physiologic agents. T h e inhibitory effect was greatest on epinephrine-induced aggregation, and least on collagen-induced aggregation. Ristocetin-induced aggregation was not affected by excess citrate anticoagulation. These findings indicate the importance of controlling the citrate:blood ratio in clinical platelet aggregation studies and in the assessment of antiplatelet drugs. (Keywords: ACD; Sodium citrate; Platelet aggregation; ADP; Collagen; Epinephrine; Ristocetin.)
April 1976
CITRATE CONCENTRATION AND PLATELET CLUMPING
Results Platelet-aggregating effects of adenosinediphosphate (ADP), epinephrine, and collagen were profoundly affected by the amounts of citrate used to anticoagulate
the blood. Regardless of whether ACD or trisodium citrate was the anticoagulant, the greater the amount of anticoagulant, the slower the aggregation, the lesser the extent of aggregation, and the longer the collagen aggregation time. However, ristocetin-induced aggregation was not appreciably affected by increased citrate concentration. The aggregabilities of platelets of individual subjects varied with respect to given concentrations of aggregating agents. Similarly, the extents of the effects of increased ACD or trisodium citrate on platelet aggregation also differed among platelets from different donors. For some, the differences in ADP-, epinephrine-, and collagen-induced platelet aggregations were small (25%). In several instances, platelets prepared from blood containing 0.133 ml. per ACD ml. blood failed to clump after exposure to epinephrine. Epinephrine-induced aggregation was absent in all samples anticoagulated with 0.177 ml. ACD per ml. blood, while ADP- and collageninduced aggregations of the same samples remained discernible. In one instance, the extent of aggregation by ristocetin was the least in the sample containing the lowest concentration of ACD. In most cases, ristocetin-induced aggregations of the low citrate-containing specimens were comparable to those of specimens containing greater amounts of citrate. These individual differences were apparently not related to hematocrit or platelet count. The hematocrits of our donors' blood specimens ranged from 40 to 48%. Platelet counts of all subjects were within normal limits. For any given blood sample anticoagulated with various amounts of citrate, platelet concentrations in PRP specimens were comparable. Figure 1 shows platelet aggregation tracings of PRP specimens and-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
taining drugs in the preceding two weeks were studied. Blood was drawn from a vein in the antecubital region with a plastic syringe; aiiquots of the blood were mixed with various amounts of ACD (NIH formula A) or 3.8% trisodium citrate in polystyrene tubes. T h e amounts of anticoagulant in ml. per ml. blood were 0.097, 0.111, 0.133, 0.155, and 0.177. Plateletrich plasma (PRP) was prepared by centrifugation of blood at 110 X g for 15 minutes at room temperature, platelet-poor plasma (PPP) by centrifuging the remaining blood at 1,100 X g for another 15 minutes. Platelet count in each PRP sample was determined by phase-contrast microscopy. Hematocrit of the blood was measured by the microhematocrit technic. Both PRP and PPP were kept covered (at 25 C.) as much as possible during an experiment to minimize escape of C 0 2 . Platelet aggregation studies were performed on a Chrono-log platelet aggregometer equipped with a recorder. 21 Optimal concentrations of adenosinediphosphate (ADP, Sigma Chemicals), epinephrine (Upjohn Co.), purified human skin collagen, 4 or ristocetin (kindly supplied by Mr. G. Barlow of Abbott Lab.) were chosen for their ability to elicit marked aggregation of individual PRP containing the lowest concentration of anticoagulant (i.e., 0.097 ml. citrate per ml. blood). T h e same aggregating agents were used to aggregate PRP containing greater amounts of citrate. The rate of aggregation (assessed by extent of aggregation 30 seconds after addition of an aggregating agent), maximum aggregation at any time during the 5-minute recording period and at the end of 5 minutes, and collagen aggregation time were analyzed.
519
520
TS'AO, LO AND RAYMOND ADP
A.J.C.P. —Vol. 65
Ml
coagulated with different amounts of ACD. Despite the variation of effects of increased citrate on platelet aggregation of individual PRP specimens, analysis of the data obtained from the subjects included in the study disclosed a definitive pattern. When the average maximum aggregations from four to seven individual experiments were plotted against the amounts of ACD in the blood (Fig. 2), it was evident that higher concentrations of citrate exerted no effect on ristocetin-induced aggregation, the least effect on collagen-induced aggregation, and the greatest effect on epinephrineinduced aggregation. Discussion T h e aggregability of human platelets is affected by factors such as platelet count, the time interval between venipuncture and testing, temperature, pH of the environment, and type and concentration of anticoagulant. O'Brien 13 has shown that
PRP stored at 37 C. failed to respond to epinephrine after 90 minutes, to ADP after 180-240 minutes, and to thrombin after 200 minutes. Platelets stored at room temperature lose their responsiveness to aggregating agents at a slower rate. 6,12 Recently, Warlow and associates22 and Rossi and Louis 18 have found that epinephrine-induced platelet aggregation actually increases progressively as citrated PRP is left at room temperature for a period as long as 2 hours. The temperature-dependent nature of ADP-induced platelet aggregation has been demonstrated by Odegard and co-workers 15 and by Praga and Pogliani.16 Platelet aggregation reactions require the presence of free calcium ions. T h e inhibitory effect of sodium citrate, which binds ionized calcium, on human platelet aggregation was demonstrated by O'Brien and colleagues, 14 and more recently, by Han and Ardlie. 5 T h e latter investigators suggest that 0.1 M, rather than 0 . 1 2 9 M (3.8%) of sodium citrate
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
FIG. 1. Light transmission tracings depicting platelet aggregation induced by ADP, epinephrine (EPI), collagen (COL) and ristocetin (RIST). a-e denote concentrations of ACD used to anticoagulate the blood: a 0.097,6 0.111, c 0.133, d 0.155, and e 0.177 ml. ACD per ml. blood.
April 1976
CITRATE CONCENTRATION AND PLATELET CLUMPING
let aggregation reactions, the precise ratio between citrate and blood becomes crucial. T h e effect of excess citrate was greatest on epinephrine-induced aggregation, and least on collagen-induced aggregation, with the three physiologic aggregating agents we used. High concentrations of citrate affected not only the secondary phases of ADP- and epinephrine-induced aggregations, but also the primary phases. It appears, however, that secondary-phase aggregation, which is induced by materials released from platelets themselves, is more susceptible to increased citrate concentration. Apparently, epinephrine-induced platelet release is more readily suppressed by sodium citrate than collagen-induced release. Ristocetin-induced aggregation was not influenced by increased amounts of citrate. It has been shown that calcium ions are not essential for this reaction. Indeed, platelets in EDTA PRP, not responsive to physiologic agents without the addition of exogenous calcium, are quite aggregable by ristocetin. 7 ' 8,21 Use of the platelet aggregation test by
100i—
RIST
Fie. 2. Maximum platelet aggregation, i.e., the highest light transmittance during the 5-minute recording period, plotted against concentrations of ACD used to anticoagulate the blood. Each point represents the average from 4 to 7 experiments. It is clear that the greatest effect of increased ACD is on epinephrine-induced aggregation. Ristocetin-induced aggregation is not affected by increments of ACD.
0.10
0.1S
A C D ml/ml blOOd
0.20
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
should be used to anticoagulate blood for platelet aggregation studies. Increased concentrations of sodium citrate would have two effects: (1) Large quantities of Ca + + would be chelated. As a result, the amount of Ca + + left in PRP would be insufficient for platelets to function properly. (2) T h e pH of the PRP would be reduced. Aggregation of platelets by most agents will not take place in EDTA PRP, in which practically all free calcium ions are bound to EDTA. T h e reaction is also abolished below pH 6.4 and above pH 10.0 in citrated PRP 3.H.20 ~phe aggregability of platelets is markedly influenced by the pH environment without alteration of the Ca + + concentration in PRP.9.io,i7 In the present study, we have demonstrated substantial inhibition of platelet aggregation by relatively small increments of citrate concentration. Overfilling or underfilling a tube with fixed amounts of anticoagulants is, unfortunately, not uncommon. For coagulation tests this may not be critical, because calcium ions are supplied by an exogenous source. For plate-
521
522
TS'AO, LO AND RAYMOND
References 1. Danta G: Secondary phase platelet aggregation induced by adenosine diphosphate in patients with cerebral occlusive disease and in control subjects. T h r o m b Diath Haemorrh 23:159169, 1970 2. Davis JW: Defective platelet disaggregation associated with occlusive arterial diseases. Angiology 24:391-397, 1973 3. Flatow FA Jr, Freireich EJ: T h e increased effectiveness of platelet concentrates in acidified plasma. Blood 27:449-459, 1966 4. Green D, Dunne B, Schmid FR, et al: A study of the variable response of human platelets to collagen: Relation to aspirin-induced inhibition of aggregation. Am J Clin Pathol 60:920-926, 1973 5. Han P, Ardlie NG: T h e influence of pH, temperature, and calcium on platelet aggregation. Maintenance of environmental pH and platelet function for in vitro studies in plasma stored at 37 C. Br J Haematol 26:373-389, 1974 6. Harrison MJG, Emmons PR, Mitchell JRA: T h e variability of human platelet aggregation. J Atheroscler Res 7:197-205, 1967 7. Howard MA, Firkin BG: Ristocetin — A new tool
65
in the investigation of platelet aggregation. Thromb Diath Haemorrh 25:362-369, 1971 8. Jenkins CSP, Meyer D, Dreyfus MD, et al: Willebrand factor and ristocetin. I. Mechanism of ristocetin-induced platelet aggregation. Br J Haematol 28:561-578, 1974 9. Kerby GP, Taylor SM: The influence of buffers on human platelet aggregation and energy metabolism. Proc Soc Exp Biol Med 136:452456, 1971 10. Lamber E L J r , W a n i n e r RA III, Batchelor ED: Effect of metabolic acidosis and alkalosis on h u m a n platelet aggregation induced by epinephrine and ADP. Proc Soc Exp Biol Med 145:743-746, 1974 11. McLean JR, Veloso H: Change of shape without aggregation caused by ADP in rabbit platelets allow pH. LifeSci 6:1983-1986, 1967 12. Murphy S, Gardner FH: Platelet storage at 22 C: Metabolic, morphologic and functional studies. J Clin Invest 50:370-377, 1971 13. O'Brien JR: A comparison of platelet aggregation produced by seven compounds and a comparison of their inhibitors. J Clin Pathol 17: 2 7 5 - 2 8 1 , 1964 14. O'Brien JR, Shoobridge SM, Finch WJ: Comparison of the effect of heparin and citrate on platelet aggregation. J Clin Pathol 2 2 : 2 8 31, 1969 • 15. Odegard AE, Skalhegg BA, Hellem AJ: Investigation on adenosine diphosphate (ADP) induced platelet adhesiveness in vitro. Part III. T h e inactivation of ADP in plasma. Thromb Diath Haemorrh 11:317-326, 1964 16. Praga CA, Pogliani EM: Effect of temperature on ADP-induced platelet aggregation. Its significance in studying ami-aggregating drugs. T h r o m b Diath Haemorrh 29:183-189, 1973 17. Rogers AB: T h e effect of pH on human platelet aggregation induced by epinephrine and ADP. Proc Soc Exp Biol Med 139:11001103, 1972 18. Rossi EC, Louis G: A time-dependent increase in the responsiveness of platelet-rich plasma to epinephrine. J Lab Clin Med 85:300-306, 1975 19. Salky N, Dugdale M: Platelet abnormalities in ischemic heart disease. Am J Cardiol 32:612617, 1973 20. Skoza L, Zucker MB, Jerushalmy Z, et al: Kinetic studies of platelet aggregation induced by adenosine diphosphate and its inhibition by chelating agents, quanidino compounds and adenosine. T h r o m b Diath Haemorrh 18:713725, 1967 21. Ts'ao C: Platelet aggregation by ristocetin in EDTA plasma. Extensive aggregation with high concentrations of EDTA. Haemostasis 1:315-318, 1973 22. Warlow C, Corna A, Ogston D, et al: T h e relationship between platelet aggregation and time interval after venipuncture. T h r o m b Diath Haemorrh 31:133-141, 1974 23. Zahavi J, Dreyfuss F: An abnormal pattern of adenosine-diphosphate induced platelet aggregation in acute myocardial infarction. Thromb Diath Haemorrh 21:76-88, 1970
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
clinical laboratories has been increasing. Platelet aggregation is a valuable tool in assisting in the diagnosis of several disorders, notably hemorrhagic diathesis. Increased platelet aggregation and altered platelet disaggregation have been reported to occur in blood of patients who have cardiovascular and cerebral vascular diseases.1,2
