Original Article
Monitoring Residual Platelet Activity Among Patients With Acute Coronary Syndrome Post-PCI by Modified Impedance Whole Blood Platelet Aggregation and Release Method
Clinical and Applied Thrombosis/Hemostasis 2016, Vol. 22(4) 366-371 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1076029615587354 cat.sagepub.com
Chanjuan Cui, MD1, Rui Qiao, MD1, and Jie Zhang, MD1
Abstract Antiplatelet medicines have been one of the cornerstones in the treatment of patients with acute coronary syndrome (ACS). However, adverse cardiovascular events still occur in some patients on standard antiplatelet therapy. Therefore, a reliable laboratory test to monitor the residual platelet activity (RPA) is urgent. We aim to modify the impedance whole blood platelet aggregation (WBA) assay associated with release assay to monitor RPA, despite antiplatelet therapy and assess their relationship with clinical ischemic events. In this study, RPA was tested in 133 patients with ACS postpercutaneous coronary intervention between 24 and 36 hours after a 300-mg clopidogrel loading dose by modified assay. Then, these patients were followed up for 3 months for clinical ischemic events. Meanwhile, platelet activity of 58 healthy volunteers was also tested by modified assay. Results showed that in modified assay the point of platelet magnified activation time (MAT) and maximal platelet adenosine triphosphate release values (RV) have significant differences between healthy volunteers and patients ([90.86 + 27.60 seconds] vs [206.44 + 58.97 seconds] and [2.07 + 0.64 nmol] vs [0.98 + 0.49 nmol]; P < .001 and P < .001, respectively). During follow-up, 5 patients present ischemic events. Receiver-operator characteristic curve showed that the cutoff values for MAT and RV were 156.5 seconds and 1.05 nmol, respectively, with the sensitivity and specificity of 60.00% and 83.30% and 80.00% and 67.50%, respectively; when MAT combined with RV, the sensitivity can be increased to 100%. Therefore, modified impedance WBA and release assay may be a potentially recommended reliable laboratory assays for monitoring the RPA. Keywords acute coronary syndrome, residual platelet activity, antiplatelet drug, platelet aggregation, platelet release, modified assay
Introduction Dual antiplatelet treatment (DAPT) is the cornerstone in the management of atherothrombotic diseases, especially for patients with acute coronary syndromes (ACSs) and/or undergoing percutaneous coronary interventions (PCIs).1,2 Among the antiplatelet drugs, clopidogrel which is an adenosine diphosphate (ADP) receptor inhibitor, alone or in combination with aspirin, has proved significant clinical benefits. However, recently several studies have described that many patients experience recurrent ischemic events, despite recommended antiplatelet therapy and good responsiveness to antiplatelet agents by laboratory assessments.3,4 Might laboratory tests of platelet function have limitations and can’t detect all high on-treatment platelet reactivity? Nowadays several laboratory methods (such as impedance whole blood platelet aggregation [WBA], turbidimetric light transmittance aggregometry) have been used to assess clopidogrel-induced antiplatelet effects. These methods are all performed on citrate-anticoagulated
blood. Citrate can bind all extracellular calcium (Ca2þ) that plays an important role in the platelet activation. Therefore, these methods may provide misleading information on platelet reactivity and antiplatelet effects in vivo.5,6 Due to the absence of calcium in citrate-anticoagulated blood, these methods can’t detect a strong effect of thrombin on platelet activation. Meanwhile, these methods are unable to reflect platelet-amplified activation and release function actually. Therefore, it puts forward the urgent request to improve laboratory testing method of platelet function. In our study, in order to overcome
1
The Department of Laboratory Medicine, Peking University Third Hospital, Haidian, Beijing, China
Corresponding Author: Jie Zhang, The Department of Laboratory Medicine, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China. Email: [email protected]
Downloaded from cat.sagepub.com at PENNSYLVANIA STATE UNIV on June 4, 2016
Cui et al
367
the problems created by citrate, we adjust in vitro citrateanticoagulated blood Ca2þ concentration to physiological levels before testing platelet function by modified impedance WBA and release method.
Methods Study Population Fifty-eight healthy volunteers of Chinese Han ethnicity were recruited. Individuals without a history of cardiovascular disease, hematology disease, cerebrovascular disease, cancers, autoimmune disease, and not taking any drugs were qualified for inclusion. A total of 157 patients with ACS were enrolled, while 133 patients with PCI met the inclusion criteria. Some patients, who didn’t take clopidogrel for at least 7 days before PCI, received a 300-mg loading dose of clopidogrel followed by a maintenance dose of 75 mg daily. Other patients, who had taken clopidogrel for over 7 days before PCI, continued to receive a maintenance dose of 75 mg daily. All patients received aspirin at a maintenance dose of 100 mg daily. Patients were tested for platelet function between 24 and 36 hours after a 300-mg clopidogrel loading dose. Patients were excluded mostly due to ACS without PCI, patients with acute inflammation, and patients’ treatment with any platelet inhibitor expect aspirin and clopidogrel. The study was reviewed and approved by the Hospital Research and Ethical Committee. All study participants voluntarily provided informed consent personally before any study procedures were undertaken.
Blood Samples Whole blood samples for analysis were drawn by venipuncture of an antecubital vein and collected into vacuum tubes (Becton Dickinson Medical Devices Co Ltd, Franklin Lakes, New Jersey) containing 0.3 mL of 3.2% sodium citrate. The initial 3 mL of blood drawn was discarded to avoid inclusion of activated platelets. Either impedance WBA or platelet release function was performed on Choron-Log lumi-aggregometer instruments (CHRONO-LOG Model 560-CA, Chrono-Log Co, Havertown, Pennsylvania). The vasodilator stimulated phosphoprotein (VASP) phosphorylation analysis was performed on a flow cytometer (Beckman Coulter, Margency, France). Impedance WBA and release assay. An electrode probe assembly was inserted into 450 mL of blood diluted with 450 mL of saline which was incubated at 37 C for 5 minutes before; then, 100 mL Chronolume (Chrono-Log Co, Havertown, Pennsylvania), which was used to measure the adenosine triphosphate (ATP) released from platelets,7,8 and 10 mL ADP (Chrono-Log Co) at 10 mmol/L were added. The maximal increased electrical resistance (O) values and the maximal platelet ATP release values (RV) were recorded. Modified impedance WBA and release assay. An electrode probe assembly was inserted into 450 mL of blood diluted with
450 mL of saline which was incubated at 37 C for 5 minutes before; 100 mL Chronolume (Chrono-Log Co, Havertown, Pennsylvania) was added and incubated at 37 C for 2 minutes; and then, CaCl2 (at a final concentration of 2.5 mmol/L9) and ADP (at 10 mmol/L) were added. The point of platelet MAT and the maximal platelet ATP RVs were recorded. Flow cytometric VASP phosphorylation assay. Blood samples were incubated with prostaglandin E1 (PGE1) or with PGE1 and ADP 10 mmol/L at room temperature for 10 minutes and then fixed with paraformaldehyde, after which the platelets were permeabilized with nonionic detergent. Subsequently, cells were labeled with a monoclonal antibody against serine 239-phosphorylated VASP (16C2) or a negative isotypic control antibody using fluorescein isothiocyanate (FITC) staining and a CD61 phycoerythrin-labeled platelet-specific antibody. Analyses were performed on a flow cytometer (Beckman Coulter, Margency, France). A platelet reactivity index (PRI) VASP was calculated from the mean fluorescence intensity (MFI) of samples incubated with PGE1 or PGE1 and ADP according to the following formula: PRIVASP ¼ MFIðPGE1Þ MFIðPGE1þADPÞ =MFIðPGE1Þ 100%: Precision evaluation. Ten specimens from healthy volunteers were tested 5 times by modified impedance WBA and release method. Genotyping. Blood samples of patients with recurrent ischemic events were collected in tubes containing EDTA for genetic testing. The genomic DNA was extracted from whole blood using a commercially available DNA isolation kit (Tiangen Biotech Co, Ltd, Beijing, China) according to manufacturer’s instruction. CYP2C19 polymorphisms were analyzed for the loss of functional alleles *2 and *3 by identifying 2 polymorphic positions, 681G > A in exon 5 and 636G > A in exon 4, respectively. The region including the polymorphisms was amplified by polymerase chain reaction (PCR), and genotyping was performed using a commercially available validated drug metabolism genotyping assay (Sky Molecular Biotechnology, Suzhou, China).
Follow-Up Patients were followed by telephone interviews and review of medical records after 3 months of PCI (96 + 11 days). End points were clinical ischemic events. Clinical ischemic events were defined as myocardial infarction (MI; excluding in-stent thrombosis), in-stent thrombosis, cardiac death, and stroke.10
Statistical Analysis Data were checked for normality. Normally distributed continuous data are expressed as mean and standard deviation (SD); otherwise, median (M) and quartile ranges from the lower quartile Q1 to the upper quartile Q3 (Q1-Q3) were used. Coefficient of
Downloaded from cat.sagepub.com at PENNSYLVANIA STATE UNIV on June 4, 2016
368
Clinical and Applied Thrombosis/Hemostasis 22(4) Table 1. Precision and Reference Intervals for Modified Impedance Whole Blood Platelet Aggregation (WBA) and Release Assay.
MAT RV
CV
Reference Intervals
9.31% 6.13%
39-144 s 1.03-3.43 nmol
Abbreviations: MAT, magnified activation time; RV, release value; CV, coefficient of variation.
Figure 1. Modified impedance whole blood platelet aggregation (WBA) and release curves. The upper curve: impedance WBA curve; the lower curve: platelet ATP release curve. MAT indicates magnified activation time; ATP, adenosine triphosphate.
variation (CV) was calculated from the ratio of SD to the mean. Reference intervals were estimated as the 2.5th and 97.5th percentile of the distribution. Statistical comparisons were tested with t test in the case of normally distributed data or with Mann-Whitney U test when data distribution was asymmetrical. Receiver–operator characteristic (ROC) curves were used to generate clinical cutoff values for predicting patients’ outcome. A 2-tailed P value
Monitoring Residual Platelet Activity Among Patients With Acute Coronary Syndrome Post-PCI by Modified Impedance Whole Blood Platelet Aggregation and Release Method
Clinical and Applied Thrombosis/Hemostasis 2016, Vol. 22(4) 366-371 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1076029615587354 cat.sagepub.com
Chanjuan Cui, MD1, Rui Qiao, MD1, and Jie Zhang, MD1
Abstract Antiplatelet medicines have been one of the cornerstones in the treatment of patients with acute coronary syndrome (ACS). However, adverse cardiovascular events still occur in some patients on standard antiplatelet therapy. Therefore, a reliable laboratory test to monitor the residual platelet activity (RPA) is urgent. We aim to modify the impedance whole blood platelet aggregation (WBA) assay associated with release assay to monitor RPA, despite antiplatelet therapy and assess their relationship with clinical ischemic events. In this study, RPA was tested in 133 patients with ACS postpercutaneous coronary intervention between 24 and 36 hours after a 300-mg clopidogrel loading dose by modified assay. Then, these patients were followed up for 3 months for clinical ischemic events. Meanwhile, platelet activity of 58 healthy volunteers was also tested by modified assay. Results showed that in modified assay the point of platelet magnified activation time (MAT) and maximal platelet adenosine triphosphate release values (RV) have significant differences between healthy volunteers and patients ([90.86 + 27.60 seconds] vs [206.44 + 58.97 seconds] and [2.07 + 0.64 nmol] vs [0.98 + 0.49 nmol]; P < .001 and P < .001, respectively). During follow-up, 5 patients present ischemic events. Receiver-operator characteristic curve showed that the cutoff values for MAT and RV were 156.5 seconds and 1.05 nmol, respectively, with the sensitivity and specificity of 60.00% and 83.30% and 80.00% and 67.50%, respectively; when MAT combined with RV, the sensitivity can be increased to 100%. Therefore, modified impedance WBA and release assay may be a potentially recommended reliable laboratory assays for monitoring the RPA. Keywords acute coronary syndrome, residual platelet activity, antiplatelet drug, platelet aggregation, platelet release, modified assay
Introduction Dual antiplatelet treatment (DAPT) is the cornerstone in the management of atherothrombotic diseases, especially for patients with acute coronary syndromes (ACSs) and/or undergoing percutaneous coronary interventions (PCIs).1,2 Among the antiplatelet drugs, clopidogrel which is an adenosine diphosphate (ADP) receptor inhibitor, alone or in combination with aspirin, has proved significant clinical benefits. However, recently several studies have described that many patients experience recurrent ischemic events, despite recommended antiplatelet therapy and good responsiveness to antiplatelet agents by laboratory assessments.3,4 Might laboratory tests of platelet function have limitations and can’t detect all high on-treatment platelet reactivity? Nowadays several laboratory methods (such as impedance whole blood platelet aggregation [WBA], turbidimetric light transmittance aggregometry) have been used to assess clopidogrel-induced antiplatelet effects. These methods are all performed on citrate-anticoagulated
blood. Citrate can bind all extracellular calcium (Ca2þ) that plays an important role in the platelet activation. Therefore, these methods may provide misleading information on platelet reactivity and antiplatelet effects in vivo.5,6 Due to the absence of calcium in citrate-anticoagulated blood, these methods can’t detect a strong effect of thrombin on platelet activation. Meanwhile, these methods are unable to reflect platelet-amplified activation and release function actually. Therefore, it puts forward the urgent request to improve laboratory testing method of platelet function. In our study, in order to overcome
1
The Department of Laboratory Medicine, Peking University Third Hospital, Haidian, Beijing, China
Corresponding Author: Jie Zhang, The Department of Laboratory Medicine, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China. Email: [email protected]
Downloaded from cat.sagepub.com at PENNSYLVANIA STATE UNIV on June 4, 2016
Cui et al
367
the problems created by citrate, we adjust in vitro citrateanticoagulated blood Ca2þ concentration to physiological levels before testing platelet function by modified impedance WBA and release method.
Methods Study Population Fifty-eight healthy volunteers of Chinese Han ethnicity were recruited. Individuals without a history of cardiovascular disease, hematology disease, cerebrovascular disease, cancers, autoimmune disease, and not taking any drugs were qualified for inclusion. A total of 157 patients with ACS were enrolled, while 133 patients with PCI met the inclusion criteria. Some patients, who didn’t take clopidogrel for at least 7 days before PCI, received a 300-mg loading dose of clopidogrel followed by a maintenance dose of 75 mg daily. Other patients, who had taken clopidogrel for over 7 days before PCI, continued to receive a maintenance dose of 75 mg daily. All patients received aspirin at a maintenance dose of 100 mg daily. Patients were tested for platelet function between 24 and 36 hours after a 300-mg clopidogrel loading dose. Patients were excluded mostly due to ACS without PCI, patients with acute inflammation, and patients’ treatment with any platelet inhibitor expect aspirin and clopidogrel. The study was reviewed and approved by the Hospital Research and Ethical Committee. All study participants voluntarily provided informed consent personally before any study procedures were undertaken.
Blood Samples Whole blood samples for analysis were drawn by venipuncture of an antecubital vein and collected into vacuum tubes (Becton Dickinson Medical Devices Co Ltd, Franklin Lakes, New Jersey) containing 0.3 mL of 3.2% sodium citrate. The initial 3 mL of blood drawn was discarded to avoid inclusion of activated platelets. Either impedance WBA or platelet release function was performed on Choron-Log lumi-aggregometer instruments (CHRONO-LOG Model 560-CA, Chrono-Log Co, Havertown, Pennsylvania). The vasodilator stimulated phosphoprotein (VASP) phosphorylation analysis was performed on a flow cytometer (Beckman Coulter, Margency, France). Impedance WBA and release assay. An electrode probe assembly was inserted into 450 mL of blood diluted with 450 mL of saline which was incubated at 37 C for 5 minutes before; then, 100 mL Chronolume (Chrono-Log Co, Havertown, Pennsylvania), which was used to measure the adenosine triphosphate (ATP) released from platelets,7,8 and 10 mL ADP (Chrono-Log Co) at 10 mmol/L were added. The maximal increased electrical resistance (O) values and the maximal platelet ATP release values (RV) were recorded. Modified impedance WBA and release assay. An electrode probe assembly was inserted into 450 mL of blood diluted with
450 mL of saline which was incubated at 37 C for 5 minutes before; 100 mL Chronolume (Chrono-Log Co, Havertown, Pennsylvania) was added and incubated at 37 C for 2 minutes; and then, CaCl2 (at a final concentration of 2.5 mmol/L9) and ADP (at 10 mmol/L) were added. The point of platelet MAT and the maximal platelet ATP RVs were recorded. Flow cytometric VASP phosphorylation assay. Blood samples were incubated with prostaglandin E1 (PGE1) or with PGE1 and ADP 10 mmol/L at room temperature for 10 minutes and then fixed with paraformaldehyde, after which the platelets were permeabilized with nonionic detergent. Subsequently, cells were labeled with a monoclonal antibody against serine 239-phosphorylated VASP (16C2) or a negative isotypic control antibody using fluorescein isothiocyanate (FITC) staining and a CD61 phycoerythrin-labeled platelet-specific antibody. Analyses were performed on a flow cytometer (Beckman Coulter, Margency, France). A platelet reactivity index (PRI) VASP was calculated from the mean fluorescence intensity (MFI) of samples incubated with PGE1 or PGE1 and ADP according to the following formula: PRIVASP ¼ MFIðPGE1Þ MFIðPGE1þADPÞ =MFIðPGE1Þ 100%: Precision evaluation. Ten specimens from healthy volunteers were tested 5 times by modified impedance WBA and release method. Genotyping. Blood samples of patients with recurrent ischemic events were collected in tubes containing EDTA for genetic testing. The genomic DNA was extracted from whole blood using a commercially available DNA isolation kit (Tiangen Biotech Co, Ltd, Beijing, China) according to manufacturer’s instruction. CYP2C19 polymorphisms were analyzed for the loss of functional alleles *2 and *3 by identifying 2 polymorphic positions, 681G > A in exon 5 and 636G > A in exon 4, respectively. The region including the polymorphisms was amplified by polymerase chain reaction (PCR), and genotyping was performed using a commercially available validated drug metabolism genotyping assay (Sky Molecular Biotechnology, Suzhou, China).
Follow-Up Patients were followed by telephone interviews and review of medical records after 3 months of PCI (96 + 11 days). End points were clinical ischemic events. Clinical ischemic events were defined as myocardial infarction (MI; excluding in-stent thrombosis), in-stent thrombosis, cardiac death, and stroke.10
Statistical Analysis Data were checked for normality. Normally distributed continuous data are expressed as mean and standard deviation (SD); otherwise, median (M) and quartile ranges from the lower quartile Q1 to the upper quartile Q3 (Q1-Q3) were used. Coefficient of
Downloaded from cat.sagepub.com at PENNSYLVANIA STATE UNIV on June 4, 2016
368
Clinical and Applied Thrombosis/Hemostasis 22(4) Table 1. Precision and Reference Intervals for Modified Impedance Whole Blood Platelet Aggregation (WBA) and Release Assay.
MAT RV
CV
Reference Intervals
9.31% 6.13%
39-144 s 1.03-3.43 nmol
Abbreviations: MAT, magnified activation time; RV, release value; CV, coefficient of variation.
Figure 1. Modified impedance whole blood platelet aggregation (WBA) and release curves. The upper curve: impedance WBA curve; the lower curve: platelet ATP release curve. MAT indicates magnified activation time; ATP, adenosine triphosphate.
variation (CV) was calculated from the ratio of SD to the mean. Reference intervals were estimated as the 2.5th and 97.5th percentile of the distribution. Statistical comparisons were tested with t test in the case of normally distributed data or with Mann-Whitney U test when data distribution was asymmetrical. Receiver–operator characteristic (ROC) curves were used to generate clinical cutoff values for predicting patients’ outcome. A 2-tailed P value
