Pre- and Post-Bypass Platelet Function Testing With Multiple Electrode Aggregometry and TEG Platelet Mapping in Cardiac Surgery Seema Agarwal, BM, BS, MA, FRCA, Robert Ian Johnson, PGDip, and Bilal Haneef Kirmani, BSc Hons, MB ChB, MRCS Objectives: To investigate the differences between preand post-cardiopulmonary bypass platelet function tests and to examine whether post-bypass tests could predict postoperative bleeding. Design: A prospective cohort study. Setting: A tertiary cardiothoracic center. Participants: Fifty-four patients presenting for cardiac surgery. Interventions: Pre- and post-bypass platelet function tests with TEG Platelet Mapping (PM) and Multiple Electrode Aggregometry (Multiplate - MEA) were performed. Measurements and Main Results: The results of the paired samples were compared. Outcomes including blood loss in the first 4 and 12 hours postoperatively and requirement for resternotomy also were recorded and regression analyses performed to establish predictive factors. Tests using adenosine diphosphate (ADP) and arachidonic acid as stimulators

for platelet aggregation showed significant differences preand post-bypass, with both MEA and PM showing greater inhibition in platelet function after bypass (p o 0.01). However, there was no significant change in MEA TRAP values with cardiopulmonary bypass (p ¼ 0.79) There was no relationship between post-bypass platelet function and the reduction in measured platelet function and blood loss in the postoperative period. Conclusions: Post-bypass platelet function testing showed reductions in ADP and arachidonic acid-triggered aggregation but not in TRAP-triggered aggregation compared with pre-bypass testing. There was no relationship between the post-bypass tests and bleeding. & 2015 Elsevier Inc. All rights reserved.

P

thrombin receptor activating peptide (TRAP-6 test), and arachidonic acid (ASPI test). The Multiplate has been validated against both flow cytometry and optical light aggregation in studies looking at platelet inhibition by clopidogrel.9 In the only thus far published study, an ADP-induced aggregation of less than 31 u was found to be associated independently with an increased risk of bleeding and transfusion in cardiac surgery.1 The Thromboelastograph (TEG) PlateletMapping (PM) Device (Haemonetics, IL) is a point-of-care coagulation monitor that has been used for more than 20 years in both cardiac and hepatic surgery as a guide to transfusion. Its use has been associated with a reduction in inappropriate transfusion.10 While the TEG readily demonstrates certain platelet aggregation defects, including post-cardiopulmonary bypassinduced platelet dysfunction related to reduced or dysfunctional GP IIb/IIIa receptors, the conventional TEG, because of overwhelming thrombin generation, is not able to detect the platelet defects that occur with aspirin or ADP-receptor blockade. A modification of the TEG assay generates clot without thrombin generation using reptilase and factor XIIIa. The addition of platelet agonists (arachidonic acid or ADP) enables the measurement of the degree of platelet inhibition resulting from aspirin or ADP-receptor antagonists, respectively. This technique has been shown to have good agreement with light transmission aggregometry.11 It is unknown whether post-bypass testing can identify patients with drug-induced receptor inhibition and whether the platelet function at this time has a relationship with bleedingrelated outcomes. The aim of this study was to investigate the changes in platelet function as shown by MEA and PM before and after bypass and the relationship between the post-bypass testing and bleeding-related outcomes. The null hypothesis was that, after cardiopulmonary bypass, there would be no difference in platelet function between patients receiving ADPreceptor antagonists and those who did not receive them and that these tests, therefore, would not correlate with clinical outcomes of bleeding or resternotomy.

OINT-OF-CARE PLATELET FUNCTION testing increasingly is being used, usually in combination with a viscoelastic measure of coagulation, in the management of cardiac patients. Pre-bypass testing has been shown to identify patients at risk of bleeding perioperatively,1,2 and both multiple electrode aggregometry (MEA) and TEG PlateletMapping (PM) performed preoperatively have been shown to reduce blood loss and transfusions when used in algorithms guiding therapy.3–5 However, it is well known that during cardiopulmonary bypass platelets suffer with bypass-induced platelet activation, loss of receptors for fibrinogen (GpIIbIIIa) and von Willebrand factor (GpIb), and reduced aggregation due to heparin as well as the presence of a dilutional thrombocytopenia.6–8 The Multiplate Instrument (MEA; Verum Diagnostica, Roche, Munich, Germany) is a multiple electrode aggregometer that analyzes whole blood that has been anticoagulated with hirudin or heparin at 371C. The blood is mixed with an activator that causes the platelets to adhere to the electrode surface. The attachment of platelets onto the sensor electrodes leads to a change in electrical conductivity, which is recorded continuously. This increase in impedance is expressed as “aggregation units.” A variety of agonists can be used as the activator for platelet aggregation including adenosine diphosphate (ADP test),

From the Liverpool Heart and Chest Hospital, Liverpool, UK. Disposables and reagents for Multiplate analysis were received from Verum Diagnostica. Disposables and reagents for TEG and PlateletMapping were received from Haemonetics. There was no direct financial aid and the authors did not receive honoraria from either manufacturer. Address reprint requests to Seema Agarwal, BM, BS, MA, FRCA, Liverpool Heart and Chest Hospital, Thomas Drive, Liverpool, UK L143PE. E-mail: [email protected] © 2015 Elsevier Inc. All rights reserved. 1053-0770/2601-0001$36.00/0 http://dx.doi.org/10.1053/j.jvca.2015.01.028 1272

KEY WORDS: platelet function tests, thromboelastography, cardiac surgery

Journal of Cardiothoracic and Vascular Anesthesia, Vol 29, No 5 (October), 2015: pp 1272–1276

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METHODS

This prospective study was approved by the hospital review board and regional ethics committee (NREC number 12/NW/ 0127). Written informed consent was obtained for all participants by investigators trained in Good Clinical Practice and following the Declaration of Helsinki guidelines. Participants All adult patients presenting for coronary artery surgery (with or without valve surgery) were included in this study, including emergent and urgent procedures and those taking antiplatelet and anticoagulant medication. Exclusion criteria were valve-only patients (to exclude those not routinely taking antiplatelet medications), significant hepatic disease (ALT 2 normal), renal disease requiring dialysis, and those requiring preoperative inotrope support. In total, 54 patients, in whom preoperative and postoperative platelet function testing was performed, were recruited to the study. Outcome Measures The primary outcome measure was platelet function parameters after cardiopulmonary bypass. Estimated blood loss (EBL) also was assessed at 4 and 12 hours after closure of the sternum and resternotomy requirements.

All instruments had quality control performed daily, and the tests performed were following manufacturer specification. All tests were performed by staff fully trained in their use, with regular assessments of their ability to perform the tests. Statistical Analyses Data were analyzed using JMP 11.0 for Mac (SAS Institute Inc, Cary, NC). Results were compared using χ2 for categoric data, t test for normally distributed continuous data, and Wilcoxon rank sum for non-parametric data, using paired techniques where appropriate. Between-group analysis was performed using simple repeated measures after paired comparison. Normality and skew were assessed by visual inspection. Regression analyses were performed using stepwise selection based on the minimum corrected Akaike Information Criterion (AICc). Continuous variables were not dichotomized to maintain maximal diagnostic information. All candidate predictor variables (demographic, preoperative hematologic tests and preoperative platelet function results) were included in a non-parsimonious fashion for model development. Blood product usage and postoperative point-of-care test results were included in post hoc analyses. Model discrimination was assessed using the c-statistic, equivalent to the area under the receiver operating characteristic curve (AUC), for resternotomy and R2 for the remaining, continuous variables.

Conduct of Study Baseline laboratory tests were conducted on all patients, including full blood count, prothrombin time, activated partial thromboplastin time, and von Clauss fibrinogen. Baseline point-of-care tests also were conducted on all patients; these included baseline platelet function analysis (both multiple electrode aggregometry [MEA] and TEG PlateletMapping [PM] using different agonist assays as described below); and other coagulation point-of-care tests including a kaolin-activated TEG, and a functional fibrinogen TEG. The benefits and limitations of these tests have been described elsewhere.12 Operative technique was according to standard local protocol. General anesthesia was established and maintained using propofol and volatile agents. At the onset of surgery, all patients received tranexamic acid, 5 mg/kg bolus, followed by an infusion of 5 mg/kg until the end of surgery, as per usual practice. Cardiopulmonary bypass (CPB) was conducted in the standard fashion at 321C to 341C according to the surgery, with anticoagulation accomplished using heparin sulphate, 300 u/kg, with additional aliquots as required to maintain an ACT above 400 seconds. Protamine reversal of heparin was performed after CPB decannulation at a dose of 1 mg per 100 u of total heparin dose. Ten minutes after the administration of protamine, pointof-care tests were performed, prior to any transfusion being administered, if required. These included TEG kaolin and kaolin heparinase, functional fibrinogen TEG, and platelet function testing (MEA). Transfusions were administered according to protocol, based on TEG and platelet function test results (Appendix 1). Resternotomy was performed as per institutional practice—for hemodynamic instability, chest tube drainage of greater than 500 mL in the first hour or greater than 1000 mL within 24 hours.

RESULTS

The demographics of the study population of 54 patients are outlined in Table 1 and show an unremarkable distribution. Antiplatelet use in the 54 patients who had pre- and postbypass point-of-care testing is shown in Table 2. The majority of patients continued taking aspirin until surgery, with almost half stopping thienopyridines several days before. Blood loss at 4 hours was 338.5 ⫾ 138.2 mL and at 12 hours was 560.4 ⫾ 189.0 mL. There was no blood product use. Results of hematologic and point-of-care tests are summarized in Table 3. There was a statistically significant fall in serum hemoglobin and platelet count after CPB. Platelet function testing with both MEA and PM for ADP and ASPI were significantly different after bypass, with both types of test demonstrating a reduction in platelet function. The PM, which reports a percentage platelet inhibition, showed a platelet inhibition of 37% increasing to 88% with arachidonic acid stimulation (p o 0.01) and of 13% increasing to 66% with ADP stimulation (p o 0.01). The MEA, which reports an area under the curve (AUC) where a greater area indicates better platelet function, showed an AUC of 90 decreasing to 61 with Table 1. Preoperative Demographic Data Age, yr ⫾ SD BMI, kg/m2 ⫾ SD CPB, min ⫾ SD AXC, min ⫾ SD Male gender, n (%) Urea, mmol/L ⫾ SD Creatinine, mmol/L ⫾ SD

68.2 ⫾ 10.5 28.0 ⫾ 4.3 98.8 ⫾ 57.1 67.3 ⫾ 43.2 42 (77.7%) 6.3 ⫾ 2.8 96.0 ⫾ 58.6

Abbreviations: SD, standard deviation; BMI, body mass index; CPB, cardiopulmonary bypass time; AXC, aortic cross-clamp time.

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Table 2. Antiplatelet Use in Patients with POCT Results Before and After Bypass Aspirin within 1 week, n (%) Days since last aspirin, median (IQR) Thienopyridine use, n (%) Days since last thienopyridine, median (IQR)

44 0 24 5

(81.4) (0-5) (44.4) (1.25-5)

Abbreviation: POCT, point-of-care testing; IQR, interquartile range.

ADP (p o 0.01), and of 60 decreasing to 35 with ASPI (p o 0.01). Of note is that there was no significant difference in the pre- and post-bypass TRAP values. Resternotomy No patients required re-exploration for bleeding or tamponade in this study group. Consequently, planned statistical analyses were not performed. Estimated Blood Loss at 4 Hours

DISCUSSION

Stepwise regression could not identify a robust model to predict postoperative blood loss at 4 hours. Using the minimum AICc criteria to identify the best model gave a maximum R2 ¼ 0.15. Postoperative platelet function tests were not predictive. There was no correlation between either TEG PM ADP and EBL at 4 hours (R2 ¼ 0.20, Fig 1) or MEA ADP and EBL at 4 hours (R2 ¼ 0.07, Fig 2). Subgroup analysis of patients who had been taking ADP-inhibitors did not identify correlations (patients with ADP-receptor blockers indicated by a plus sign (þ) in Figs 1 and 2). Estimated Blood Loss at 12 Hours Statistical analysis of the factors affecting blood loss at 12 hours also failed to find a relationship between postoperative platelet function tests and blood loss. There was no correlation between either post-bypass TEG PM ADP and EBL at 12 hours (R2 = 0.06) or post-bypass MEA ADP and EBL at 12 hours (R2 = 0.07). Table 3. Results of Pre- and Post-Bypass POCT Pre-CPB

Fig 1. Post-bypass TEG ADP % inhibition versus estimated blood loss at 4 hours postoperatively (þ denotes recent ADP-receptor antagonists; ○ denotes none). Abbreviations: EBL, estimated blood loss; Postop, postoperative; TEG, Thromboelastograph; ADP, adenosine.

Post-CPB

p Value

Hb, g/dL ⫾ SD 13.1 ⫾ 1.5 9.6 ⫾ 1.3 o0.0001* 9 Platelet count, 10 /L ⫾ SD 220.8 ⫾ 73.0 154.2 ⫾ 64.0 o0.0001* TEG R, min ⫾ SD 7.2 ⫾ 5.6 7.8 ⫾ 3.9 0.2597 TEG K, min ⫾ SD 1.9 ⫾ 0.9 2.3 ⫾ 2.6 0.1180 TEG angle, degrees ⫾ SD 63.4 ⫾ 10.6 59.9 ⫾ 13.7 0.0121* TEG MA, mm ⫾ SD 67.9 ⫾ 8.3 65.2 ⫾ 8.6 0.0037* LAB Clauss, g/L ⫾ SD 3.3 ⫾ 1.0 2.9 ⫾ 1.0 o0.0001* TEG FF, g/L ⫾ SD 5.8 ⫾ 3.7 4.9 ⫾ 2.0 o0.0001* MEA TRAP – AUC (IQR) 143 (116-168) 132 (83-168.5) 0.7917 MEA ADP – AUC (IQR) 90 (64.5-107.5) 61 (38.5-78.5) 0.0078* MEA ASPI – AUC (IQR) 60 (26.5-100.5) 35.0 (22.5-80.5) 0.0958* PM AA, % (IQR) 37 (2-81) 88 (42-100) 0.0081* PM ADP, % (IQR) 13 (1.3-31.5) 66 (53-94) o0.0001* Abbreviations: POCT, point-of-care testing; CPB, cardiopulmonary bypass; Hb, hemoglobin; SD, standard deviation; TEG, thromboelastograph; R, reaction time; K, kinetic time; MA, maximal amplitude; FF, functional fibrinogen; MEA, multiple electrode aggregometry; TRAP, thrombin-receptor-activating peptide; AUC, area under the curve; IQR, interquartile range; ADP, adenosine phosphate; ASPI, aspirin MEA test; PM, platelet mapping; AA, arachidonic acid. *denotes statistically significant results

The main finding of this study was that MEA and PM showed a reduction in platelet function after bypass, both using arachidonic acid and ADP as stimulators. Neither test correlated with postoperative bleeding. Postoperatively, the tests did not appear to show only the effects of drugs, such as aspirin and clopidogrel, but instead a complete inhibition regardless of antiplatelet therapy or assay. Platelet function tests can be affected by low platelet count, transport tubes, heparin, protamine, and other factors.3,13,14 In the course of the surgery, the mean platelet count did fall from 220 to 154 109/L, but this was still within the normal range. Additionally, although the reduction in platelet count may have led to a decreased aggregation signal, it could be expected for this to be consistent for all MEA tests. This was not the case with the TRAP test and the decreases in aggregation in ADP and ASPI were far greater than the proportional reduction in platelet count. The post-bypass reductions in ADP and ASPI stimulated aggregation could be explained by degradation of receptors that occurs with extracorporeal circulation.15 Mechanical shear forces involved in cardiopulmonary bypass may cause platelet and erythrocyte lysis, releasing ADP and promoting platelet

Fig 2. Post-bypass MEA ADP area under the curve versus estimated blood loss at 4 hours postoperatively (þ denotes recent ADP receptor antagonists; ○ denotes none). Abbreviations: EBL, estimated blood loss; Postop, postoperative; PM, platelet mapping; ADP, adenosine.

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aggregation.16 This might lead to consumption of platelets or transient ADP-receptor desensitization that has been shown to improve with time. There were, however, no statistically significant changes in the preoperative and postoperative TRAP MEA values, which runs counter to previous experience.17,18 It may be that the thrombin-receptor activating peptide-6 (TRAP-6) used in the test overwhelmed the PAR-1 platelet membrane thrombin receptor in functioning platelets. It is known that shear forces can overstimulate the TRAP-mediated microvesicule formation that accompanies aggregation.19 Furthermore, platelet cell surface receptor expression can be reduced by 50% without any effect on TRAP-stimulated aggregation.20 Carroll et al examined the relationship between postoperative TEG PM and platelet function tests using glass bead adhesion and, similarly, found that there was significant platelet dysfunction after cardiopulmonary bypass but that this was not associated with postoperative bleeding.21 Forestier et al found that platelet function, as shown by the PFA-100 and HemoSTATUS devices, was deranged in those who bled after bypass but did not correlate with postoperative bleeding or laboratory measured platelet aggregation.22 Ranucci et al recently reported a correlation between preoperative MEA ADP and postoperative bleeding. Their study had the advantage of a homogenous group who were all recent ADP antagonist users.1 There was no relationship between postoperative tests and estimated blood loss. This lack of correlation suggested that postoperative testing does not identify those at a higher risk of bleeding. This would be consistent with the global effects of cardiopulmonary bypass on platelet function. The sensitivity,

specificity, and potential confounders of point-of-care tests is complex and reflects the different surrogate markers used to determine platelet function.12 Although recent clopidogrel use has been shown to affect postoperative bleeding,23 point-ofcare tests conducted after surgery presently lack the discrimination required to identify drug-related inhibition from CPB-induced dysfunction. Limitations Despite its prospective nature, the present data suffered from the inherent limitations of a small-volume, single-center study. Potential improvements for further study would be a larger study population with greater numbers in the subgroups of patients with different antiplatelet exposure. Repeated measures at fixed intervals from the end of cardiopulmonary bypass also might help to establish the rate of recovery of platelet function. The normal ranges for platelet function preoperatively do not apply after cardiopulmonary bypass. CONCLUSION

It would appear that platelet function testing postbypass to assess drug inhibition is ineffective and that this should be performed before bypass to get meaningful results. Post-bypass testing shows enhanced platelet inhibition when stimulated by ADP or arachidonic acid, regardless of aspirin or P2Y12-inhibitor use, while TRAP testing remained unchanged. Post-bypass point-of-care platelet function tests currently do not help predict outcome or guide management.

REFERENCES 1. Ranucci M, Colella D, Baryshnikova E, et al: Effect of preoperative P2Y12 and thrombin platelet receptor inhibition on bleeding after cardiac surgery. Br J Anaesth 113:970-976, 2014 2. Ranucci M, Baryshnikova E, Soro G, et al: Multiple electrode whole-blood aggregometry and bleeding in cardiac surgery patients receiving thienopyridines. Ann Thorac Surg 91:123-129, 2011 3. Weber CF, Görlinger K, Meininger D, et al: Point-of-care testing: A prospective, randomized clinical trial of efficacy in coagulopathic cardiac surgery patients. Anesthesiology 117:531-547, 2012 4. Görlinger K, Dirkmann D, Hanke AA, et al: First-line therapy with coagulation factor concentrates combined with point-of-care coagulation testing is associated with decreased allogeneic blood transfusion in cardiovascular surgery: A retrospective, single-center cohort study. Anesthesiology 115:1179-1191, 2011 5. Agarwal S, Johnson RI, Shaw M: Preoperative point-of-care platelet function testing in cardiac surgery. J Cardiothorac Vasc Anesth : [Epub ahead of print], 2014 Nov 1 6. Harker LA, Malpass TW, Branson HE, et al: Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass: Acquired transient platelet dysfunction associated with selective alphagranule release. Blood 56:824-834, 1980 7. Rinder CS, Mathew JP, Rinder HM, et al: Modulation of platelet surface adhesion receptors during cardiopulmonary bypass. Anesthesiology 75:563-570, 1991 8. Woodman RC, Harker LA: Bleeding complications associated with cardiopulmonary bypass. Blood 76:1680-1697, 1990 9. Velik-Salchner C, Maier S, Innerhofer P, et al: An assessment of cardiopulmonary bypass-induced changes in platelet function using

whole blood and classical light transmission aggregometry: The results of a pilot study. Anesth Analg 108:1747-1754, 2009 10. Shore-Lesserson L, Manspeizer HE, DePerio M, et al: Thromboelastography-guided transfusion algorithm reduces transfusions in complex cardiac surgery. Anesth Analg 88:312-319, 1999 11. Agarwal S, Coakley M, Reddy K, et al: Quantifying the effect of antiplatelet therapy: a comparison of the platelet function analyzer (PFA-100) and modified thromboelastography (mTEG) with light transmission platelet aggregometry. Anesthesiology 105:676-683, 2006 12. Sambu N, Curzen N: Monitoring the effectiveness of antiplatelet therapy: Opportunities and limitations. Br J Clin Pharmacol 72: 683-696, 2011 13. Bolliger D, Seeberger MD, Tanaka KA, et al: Pre-analytical effects of pneumatic tube transport on impedance platelet aggregometry. Platelets 20:458-465, 2009 14. Hanke AA, Roberg K, Monaca E, et al: Impact of platelet count on results obtained from multiple electrode platelet aggregometry (Multiplate). Eur J Med Res 15:214-219, 2010 15. Wahba A, Rothe G, Lodes H, et al: Effects of extracorporeal circulation and heparin on the phenotype of platelet surface antigens following heart surgery. Thromb Res 97:379-386, 2000 16. Krajewski S, Kurz J, Neumann B, et al: Short-acting P2Y12 blockade to reduce platelet dysfunction and coagulopathy during experimental extracorporeal circulation and hypothermia. Br J Anaesth 108:912-921, 2012 17. Ferraris VA, Ferraris SP, Singh A, et al: The platelet thrombin receptor and postoperative bleeding. Ann Thorac Surg 65:352-358, 1998

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18. Ferraris VA, Ferraris SP, Reich H, et al: Thrombin receptorrelated hemostatic defect after cardiopulmonary bypass. Semin Thromb Hemost 22:351-356, 1996 19. Chow TW, Hellums JD, Thiagarajan P: Thrombin receptor activating peptide (SFLLRN) potentiates shear-induced platelet microvesiculation. J Lab Clin Med 135:66-72, 2000 20. van Zanten GH, Heijnen HF, Wu Y, et al: A fifty percent reduction of platelet surface glycoprotein Ib does not affect platelet adhesion under flow conditions. Blood 91:2353-2359, 1998

21. Carroll RC, Chavez JJ, Snider CC, et al: Correlation of perioperative platelet function and coagulation tests with bleeding after cardiopulmonary bypass surgery. J Lab Clin Med 147:197-204, 2006 22. Forestier F, Coiffic A, Mouton C, et al: Platelet function point-ofcare tests in post-bypass cardiac surgery: Are they relevant? Br J Anaesth 89:715-721, 2002 23. Blais DM, Zukkoor SM, Hayes C, et al: Bleeding outcomes associated with coronary artery bypass graft surgery and recent clopidogrel exposure. Heart Surg Forum 16:E70-E77, 2013

APPENDIX 1

Figure E1

Fig E1.

Pre- and Post-Bypass Platelet Function Testing With Multiple Electrode Aggregometry and TEG Platelet Mapping in Cardiac Surgery.

To investigate the differences between pre- and post-cardiopulmonary bypass platelet function tests and to examine whether post-bypass tests could pre...
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