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Comparing light transmission aggregometry and PFA100 for monitoring antiplatelet therapy in patients with coronary heart disease
Journal of Taibah University Medical Sciences (2016) 11(5), 478e484
Taibah University
Journal of Taibah University Medical Sciences www.sciencedirect.com
Original Article
Comparing light transmission aggregometry and PFA100 for monitoring antiplatelet therapy in patients with coronary heart disease Mostafa Q. Al Shamiri, MRCP a, Abelgalil M. Abdel Gader, PhD b, * and Nervana M. Bayoumy, PhD b a b
Coronary Care Unit, King Khalid University Hospital, College of Medicine, King Saud University, Riyadh, KSA Coagulation Research Laboratory, Physiology Department, College of Medicine, King Saud University, Riyadh, KSA
Received 25 April 2016; revised 4 August 2016; accepted 7 August 2016; Available online 7 September 2016
ﺍﻟﻤﻠﺨﺺ ﻻﻳﺰﺍﻝ ﻛﺜﻴﺮ ﻣﻦ ﺍﻟﻤﺮﺿﻰ ﺍﻟﺬﻳﻦ ﻳﺘﻨﺎﻭﻟﻮﻥ ﺍﻟﻌﻼﺟﺎﺕ ﺍﻟﻤﻀﺎﺩﺓ:ﺃﻫﺪﺍﻑ ﺍﻟﺒﺤﺚ ﻣﻤﺎ ﻳﺆﺩﻱ ﺇﻟﻰ ﻣﺎ،ﻟﻠﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﻳﻌﺎﻧﻮﻥ ﻣﻦ ﺣﺪﻭﺙ ﺍﻟﺠﻠﻄﺎﺕ ﻭﻓﺸﻞ ﺍﻟﻌﻼﺝ ﻛﺎﻥ ﻫﻨﺎﻙ ﺯﻳﺎﺩﺓ٬ ﻣﺆﺧﺮﺍ.ﻳﺴﻤﻰ “ﻣﻘﺎﻭﻣﺔ” ﺍﻟﻌﻼﺟﺎﺕ ﺍﻟﻤﻀﺎﺩﺓ ﻟﻠﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﻓﻲ ﺍﻟﻠﺠﻮﺀ ﺇﻟﻰ ﺇﺟﺮﺍﺀ ﺍﻻﺧﺘﺒﺎﺭﺍﺕ ﺍﻟﻤﺨﺒﺮﻳﺔ ﻟﻤﺮﺍﻗﺒﺔ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﺍﻟﺘﻲ ﺗﻬﺪﻑ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﻟﺘﻮﺛﻴﻖ ﺍﻧﺘﺸﺎﺭ ﺑﺮﻫﺎﻥ.ﺗﺒﺸﺮ ﺑﺘﻌﻴﻴﻦ ﻣﺜﻞ ﻫﺆﻻﺀ ﺍﻟﻤﺮﺿﻰ ﺍﻟﻤﺨﺘﺒﺮﺍﺕ ﻟﻤﻘﺎﻭﻣﺔ ﺍﻷﺳﺒﺮﻳﻦ ﻭﻋﻼﺝ ﻛﻠﻮﺑﻴﺪﻭﻗﺮﻳﻞ ﻓﻲ ﺍﻟﻤﺮﺿﻰ ﺍﻟﺴﻌﻮﺩﻳﻴﻦ ﺍﻟﺬﻳﻦ .ﻳﻌﺎﻧﻮﻥ ﻣﻦ ﺃﻣﺮﺍﺽ ﺍﻟﺸﺮﺍﻳﻴﻦ ﺍﻟﺘﺎﺟﻴﺔ ﺍﻟﻤﺴﺘﻘﺮﺓ ﺃﺟﺮﻱ ﻗﻴﺎﺱ ﺍﻟﺘﻜﺪﺱ ﺑﻮﺍﺳﻄﺔ ﻧﻘﻞ ﺍﻟﻀﻮﺀ ﻓﻲ ﺍﻟﺒﻼﺯﻣﺎ ﺍﻟﻐﻨﻴﺔ:ﻃﺮﻕ ﺍﻟﺒﺤﺚ ، ﻭﺣﺎﻣﺾ ﺍﻷﺭﺍﻛﻴﺪﻭﻧﻴﻚ،ﺑﺎﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﻛﺈﺳﺘﺠﺎﺑﺔ ﻷﺩﻳﻨﻮﺳﻴﻦ ﺛﻨﺎﺋﻲ ﺍﻟﻔﻮﺳﻔﺎﺕ ﺍﻟﺬﻯ، ﺑﺎﻹﺿﺎﻓﺔ ﺇﻟﻰ ﺗﺤﻠﻴﻞ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ.ﻭﺍﻟﻜﻮﻻﺟﻴﻦ ﻭﺍﻷﺩﺭﻳﻨﺎﻟﻴﻦ /ﺃﺩﻧﻴﻮﺳﻴﻦ ﺛﻨﺎﺋﻲ ﺍﻟﻔﻮﺳﻔﺎﺕ ﻭﺍﻟﻜﻮﻻﺟﻴﻦ/ﻳﻌﻤﻞ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺃﻃﻘﻢ ﻛﻼ ﻣﻦ ﺍﻟﻜﻮﻻﺟﻴﻦ .ﺃﺩﺭﻳﻨﺎﻟﻴﻦ ﺃﻇﻬﺮ ﻗﻴﺎﺱ ﺍﻟﺘﻜﺪﺱ ﺑﻮﺍﺳﻄﺔ ﻧﻘﻞ ﺍﻟﻀﻮﺀ ﻣﻘﺎﻭﻣﺔ ﻟﻸﺳﺒﺮﻳﻦ ﻭﻋﻼﺝ:ﺍﻟﻨﺘﺎﺋﺞ ﻓﻲ٬ ﻭﻓﻘﺎ ﻟﻼﺳﺘﻤﺮﺍﺭ ﻻﺳﺘﺠﺎﺑﺔ ﺍﻟﺘﻜﺪﺱ ﻟﺘﺠﻤﻊ ﺣﺎﻣﺾ ﺍﻷﻛﺮﺍﻛﻴﺪﻭﻧﻴﻚ٬ﻛﻠﻮﺑﻴﺪﻭﻗﺮﻳﻞ ﻭﺑﺎﺳﺘﺨﺪﺍﻡ ﺗﺤﻠﻴﻞ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﺃﻭﻗﺎﺕ. ﻋﻠﻰ ﺍﻟﺘﻮﺍﻟﻲ٪٢٦ ﻭ٪١٣ ﻣﺸﻴﺮﺍ ﺇﻟﻰ ﺗﻔﺎﻋﻞ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ٬ﺍﻹﻏﻼﻕ ﻓﻲ ﺣﺪﻭﺩ ﺍﻟﻘﻴﻢ ﺍﻟﻤﺮﺟﻌﻴﺔ ﻟﻠﻤﺨﺘﺒﺮ ﻛﻤﺎ ﺗﻢ ﺗﺴﺠﻴﻞ ﺍﻧﺘﺸﺎﺭ ﺍﻟﻤﻘﺎﻭﻣﺔ ﻟﻠﻌﻼﺟﺎﺕ ﺍﻟﻤﻀﺎﺩﺓ ﻟﻠﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﻋﻨﺪ٬ﺍﻟﻤﺘﺒﻘﻴﺔ ﺑﺎﺳﺘﻌﻤﺎﻝ٪ ٣٠.٧ ﺃﺩﻧﻴﻮﺳﻴﻦ ﺛﻨﺎﺋﻲ ﺍﻟﻔﻮﺳﻔﺎﺕ ﻭ/ ﺑﺎﺳﺘﻌﻤﺎﻝ ﺃﻃﻘﻢ ﺍﺍﻟﻜﻮﻻﺟﻴﻦ٪٣٣
* Corresponding address: Department of Basic Medical Science, College of Medicine King Saud bin Abdulaziz University for Health Sciences, Riyadh 11426, KSA. E-mail: [email protected] (A.M. Abdel Gader) Peer review under responsibility of Taibah University.
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ﻛﻤﺎ ﻇﻬﺮ ﺍﻟﺘﻮﺍﻓﻖ ﺑﻴﻦ ﺍﻟﺘﻜﺪﺱ ﺑﻮﺍﺳﻄﺔ ﻧﻘﻞ ﺍﻟﻀﻮﺀ. ﺃﺩﺭﻳﻨﺎﻟﻴﻦ/ﺃﻃﻘﻢ ﺍﻟﻜﻮﻻﺟﻴﻦ . ﻣﻦ ﺍﻟﻤﺮﺿﻰ٪٢٢.٦ ﻭﺗﺤﻠﻴﻞ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﻋﻨﺪ ﺃﻇﻬﺮﺕ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﺍﻻﻧﺘﺸﺎﺭ ﺍﻟﻮﺍﺳﻊ ﻟﺘﻔﺎﻋﻞ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ:ﺍﻻﺳﺘﻨﺘﺎﺟﺎﺕ ﻟﻠﻤﺮﺿﻰ ﺍﻟﺬﻳﻦ ﻳﻌﺎﻧﻮﻥ ﻣﻦ ﺃﻣﺮﺍﺽ ﺍﻟﺸﺮﺍﻳﻴﻦ ﺍﻟﺘﺎﺟﻴﺔ ﻭﻫﻢ ﺗﺤﺖ ﺍﻟﻌﻼﺝ ﺍﻟﺜﻨﺎﺋﻲ ﻳﻌﻄﻲ ﺍﺧﺘﺒﺎﺭ٬ ﺑﺎﻹﺿﺎﻓﺔ.(ﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ )ﺍﻷﺳﺒﺮﻳﻦ ﻭﻛﻠﻮﺑﻴﺪﻭﻗﺮﻳﻞ ﺍﻟﺪﻡ ﺍﻟﻜﺎﻣﻞ ﺍﻟﻌﺎﻟﻤﻲ ﻟﻮﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﺑﻮﺍﺳﻄﺔ ﺗﺤﻠﻴﻞ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﺍﺭﺗﻔﺎﻋﺎ ﺃﻋﻠﻰ ﺑﻜﺜﻴﺮ ﻟﻤﻌﺪﻝ ﺍﻧﺘﺸﺎﺭ ﺍﻟﻤﻘﺎﻭﻣﺔ ﻋﻦ ﺍﻟﺘﻜﺪﺱ ﺑﻮﺍﺳﻄﺔ ﻧﻘﻞ ﺍﻟﻀﻮﺀ ﻋﻨﺪ ﻣﻘﺎﺭﻧﺘﻪ ﺑﺎﻟﻤﻌﻴﺎﺭ ﺍﻟﺬﻫﺒﻲ ﻭﺍﻷﻛﺜﺮ ﺗﺤﺪﻳﺪﺍ ﻟﺘﺤﻠﻴﻞ ﺍﻟﺘﻜﺪﺱ ﺑﻮﺍﺳﻄﺔ ﻧﻘﻞ .ﺍﻟﻀﻮﺀ ﺍﻟﻌﻼﺟﺎﺕ ﺍﻟﻤﻀﺎﺩﺓ ﻟﻠﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ؛ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ:ﺍﻟﻜﻠﻤﺎﺕ ﺍﻟﻤﻔﺘﺎﺣﻴﺔ ﺍﻟﺪﻣﻮﻳﺔ؛ ﺍﻷﺳﺒﺮﻳﻦ؛ ﻛﻠﻮﺑﻴﺪﻭﻗﺮﻳﻞ؛ ﻣﻘﺎﻭﻣﺔ ﺍﻷﺳﺒﺮﻳﻦ؛ ﻣﺮﺽ ﺍﻟﻘﻠﺐ ﺍﻹﻗﻔﺎﺭﻱ؛ ﺗﻜﺪﺱ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ
Abstract Objectives: Many patients undergoing antiplatelet therapy continue to experience thrombotic events and failure of therapy leading to so-called ‘resistance’ to antiplatelet therapy. Recently, there has been an increasing focus on in vitro laboratory monitoring of platelet functions, which has the promise of identifying these patients. This study aimed to document the prevalence of laboratory evidence of resistance to aspirin and clopidogrel therapy in Saudi patients with stable coronary heart disease (CHD). Methods: Light transmission aggregometry in plateletrich plasma was performed in response to adenosine diphosphate (ADP), arachidonic acid (AA), collagen and adrenaline. In addition, a platelet function analyser (PFA100) was employed using both collagen/ADP and collagen/epinephrine cartridges.
1658-3612 Ó 2016 The Authors. Production and hosting by Elsevier Ltd on behalf of Taibah University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.jtumed.2016.08.002
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M.Q. Al Shamiri et al. Results: Light transmission aggregometry (LTA) identified the resistance to aspirin and clopidogrel therapy, according to the persistence of the aggregation response to AA aggregation, to be 13% and 26%, respectively. By PFA100 testing, closure times within the limits of laboratory reference values indicated residual platelet reactivity, and the prevalence of resistance to anti-platelet therapy was reported to be 33% for collagen/ADP cartridges and 30.7% for collagen/epinephrine. A concordance between LTA and PFA100 CT was noted in only 22.6% of patients. Conclusion: This study showed a wide prevalence of ontreatment platelet reactivity in patients with CHD on dual platelet therapy (aspirin and clopidogrel). In addition, the global whole blood test of platelet function by PFA100 estimated a much higher prevalence of resistance than LTA when compared to the gold standard and more specific LTA analysis. Keywords: Anti-platelet therapy; Aspirin resistance; Clopidogrel; PFA100; Platelet aggregation Ó 2016 The Authors. Production and hosting by Elsevier Ltd on behalf of Taibah University. This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
Introduction Recent reports from the KSA highlighted the magnitude of coronary heart disease,1,2 which is already proving to exert a high economic burden.3 In outlining the current diagnostic and treatment strategies for patients with acute coronary syndrome (ACS), it was shown that the most common medications used are the two antiplatelet drugs, aspirin (98%) and clopidogrel (73%)1; both drugs have proved their efficacy in the primary as well as the secondary prevention of cardiovascular and cerebrovascular disease.4 However, some patients taking aspirin and/or clopidogrel still develop thrombotic cardio- and/or cerebrovascular events, referred to as so-called ‘aspirin/clopidogrel resistance’ or ‘treatment failure’.5,6 Recently, there have been increasing efforts focused on in vitro laboratory monitoring of platelet function to try to identify these patients and determine whether they display normal in vitro platelet function (residual platelet activity) despite treatment with antiplatelet drugs. Of the numerous tests of platelet function, two tests have found wide popularity: light transmission aggregometry and the platelet function analyser (PFA100). To our knowledge, resistance to antiplatelet therapy has never been explored in the KSA; the available data on this topic have emerged mainly from Western countries. Our interest in this area was triggered by the reported racial variation in both CHD mortality7,8 as well as in haemostatic9 and platelet function testing.10 This encouraged us to undertake the laboratory evaluation of resistance to antiplatelet therapy to determine the scale of the problem in our population.
Therefore, the main aim of this study was to document the prevalence of aspirin and clopidogrel resistance in KSA patients with stable coronary heart disease, using the locally familiar light transmission aggregometry and the platelet function analyser (PFA100). Materials and Methods Selection of patients Patients (n ¼ 106; Table 1) were recruited consecutively and according to the following inclusion and exclusion criteria: Inclusion criteria Consecutive admissions to the Medical Outpatient Department, medical wards, Coronary Care Unit and Medical Intensive Care at King Khalid University Hospital, who had any of the following conditions: Saudis, older than 20 years. Patients, after receiving the standard loading dose of aspirin and clopidogrel, continued on maintenance dose of either aspirin (81 mg/day) and or clopidogrel (75 mg/day) for more than 7 days. All recruited patients (n ¼ 106) were on low-dose aspirin as well as clopidogrel. They had an established diagnosis of any the following: Acute coronary syndrome (ACS) Coronary heart disease (CHD) defined as follows: chronic stable angina, prior history of unstable angina or myocardial infarction, previous history of revascularization or disease of at least one coronary vessel on previous angiography or non-invasive imaging procedure. Exclusion criteria Non-Saudis Patients younger than 20 years of age. Persons with known bleeding or thrombotic disorders. This study has been approved by Institutional Review Board, the College of Medicine, King Saud University, Riyadh. Blood samples and laboratory techniques Samples of 10 ml blood were collected from an easily accessible vein in the antecubital fossa and with minimum of Table 1: Characteristics of the patients with stable ischaemic heart disease in whom both the light transmission aggregometry and the PFA100 test were performed (n [ 106). Age (years) Sex (M/F) Weight (kg) Diabetes mellitus Hypertension Dyslipidaemia Aspirin (81 mg) Clopidogrel (75 mg)
59.6 12.4 (28e90) 70/36 75.9 14.9 (52e117) 42/106 64/106 78/106 All patients
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Resistance to anti-platelet therapy
venous stasis or from arterial or central access, in two 5-ml tubes containing sodium citrate anticoagulant solution (3.8% w/v), to give a blood-to-citrate ratio of 9:1. Samples were transported within maximum of one hour, without delay, to the Coagulation Laboratory, Department of Physiology. Whole blood was centrifuged for 3 min at 1500 rpm, and supernatant plateletrich plasma (PRP) was removed carefully a using plasma pipette and put in a plastic tube. What remained of the blood samples was centrifuged further at 30,000 rpm for 15 min and the supernatant platelet poor plasma (PPP) was removed carefully and put in a separate plastic tube. Laboratory assays i. Platelet aggregation in platelet rich plasma (PRP) was assessed using a standard aggregometer [PAP-4, BioData Corporation, USA]. This machine has also been in use in the Coagulation Research Laboratory in many previous studies in health and disease and the aggregation technique has been detailed before.11,12
deviation. The chi-square test was used to determine the significance of the difference between different prevalences. A P value of <0.05 was considered significant. This study has been approved by Institutional Review Board, the College of Medicine, King Saud University, Riyadh. The study was conducted according to the Declaration of Helsinki and participants gave their consent before enrolling in the study. Results Resistance to antiplatelet therapy is defined as the detection of normal aggregation responses or normal PFA100 CT while the patient is on antiplatelet drugs (aspirin and clopidogrel). A summary of the prevalence of resistance to aspirin and clopidogrel is shown in Table 2. It is clear that the prevalence of resistance to antiplatelet therapy varied widely between the aggregation tests and the PFA100 closure times. Light transmission aggregometry
Summary of the platelet aggregation technique Platelet aggregation in response to adenosine diphosphate (ADP) and arachidonic acid (AA), collagen and epinephrine
Before performing the aggregometry studies, PRP was diluted using PPP to adjust the platelet count to the range of 150,000e200,000/mm3. Platelet aggregometry was undertaken in response to ADP (20 mmol/L) (Sigma Chemical Co., St. Louis, MO, USA), and AA (10 mg/ml), collagen and epinephrine (Bio/Data Corp. Horsham, PA, USA). The concentrations of agonists represent the final concentrations obtained by adding 20 ml of the aggregating agent to 180 ml of PRP. Laboratory conditions were carefully standardized and these include: time interval between venipuncture and the measurement of platelet aggregation (<2 h), temperature of testing (37 C) and the rate of PRP stirring (1000 rpm). The aggregometer registers the results of the aggregation responses as maximum aggregation (MA %) of the aggregation curves. ii. The PFA-100 platelet function analyser (Dade Behring, Miami, Florida, USA).11 This instrument provides an in vitro quantitative measurement of platelet adhesion and aggregation requires whole blood collected in 3.8% sodium citrate and utilizes cartridges coated with the aggregation agonists collagen/epinephrine and collagen/ ADP. It measures agonist-induced platelet plug formation as time in seconds (closure time) to occlude the aperture in these cartridges. Both cartridges were employed in this study. The local laboratory reference closure time for collagen/epinephrine cartridge ranges from 84 to 198 s (mean SD: 141.6 27 s) and for the collagen/ADP cartridge is 52e153 s (mean SD: 100.7 24.1 s; n ¼ 88).11 Any closure time less than the maximum in these two ranges is taken to represent residual platelet reactivity (or resistance to antiplatelet therapy). iii. Statistical analysis: Data were analysed using the SPSS statistical package and the results were expressed as the mean and standard
The resistance to aspirin therapy is specifically indicated by the presence of normal aggregation response to AA aggregation, which tests the integrity of the prostaglandin (thromboxane A2, TX2) pathway, was detected in 13% of patients. The resistance to clopidogrel, which blocks the ADP receptor P2Y12 and that is tested for by the aggregation responses to ADP, was 26%. The observed prevalence of the non-specific aggregation responses to collagen and epinephrine was 23% and 14%, respectively. The PFA100 closure time (CT) Using the PFA100 test, a CT that falls within the normal reference normal range of CT was taken to indicate residual Table 2: The prevalence of resistance to aspirin and clopidogrel in patients with stable ischaemic heart disease using both the platelet function analyser (PFA100) as well as optical aggregometry. Type of drug Laboratory method
Aspirin þ PFA100 clopidogrel Col-ADP cartridges Col-epinephrine cartridges Optical aggregometry Arachidonic acid ADP Collagen Adrenaline
Inhibited Normal response (on-treatment) response (resistance to antiplatelet therapy) 67%
33%
70%
30.7%
87.4% 74.9% 77% 86%
13% 26.1% 23% 14%
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M.Q. Al Shamiri et al. platelet reactivity (resistance to antiplatelet therapy). Accordingly, the prevalence of resistance for the collagen/ ADP cartridges was 33%, while for the collagen/epinephrine was 30.7%.
Concordance between the results of the PFA100 and light transmission aggregometry (LTA) In only 24 patients out of 106 (22.6%), aggregation responses to ADP and arachidonic acid as well as the PFA100 CT times of both cartridges indicated good response to antiplatelet therapy. For the rest of the patients, there were wide variations in the detection rate of individual tests.
Discussion Antiplatelet therapy plays a major role in the treatment of patients with acute coronary syndrome (ACS), resulting in a significant reduction in ischaemic events.4 This topic continues to receive extensive coverage in the literature, which is predominantly from developed countries; scant information comes from developing countries where coronary heart disease is proving to be an emerging health problem.4 The findings of recent nationwide studies in the KSA1e3 provided, for the first time, a comprehensive view of the local scale of the problem and current diagnostic and treatment strategies for patients with ACS and highlighted opportunities to improve the care of these patients. One major finding of these studies was that aspirin (acetylsalicylic acid) is consumed by 98% of patients with acute coronary syndromes; the corresponding figure for clopidogrel is 73%. Aspirin is usually combined with other anti-platelet agents in order to achieve a more effective inhibition of platelet aggregation. However, it has repeatedly been shown that in a significant number of patients on anti-platelet therapy, no therapeutic benefit could be gained whether by evidence of the laboratory detection of platelet function inhibition or the prevention of further complications such as recurrence of cardiac and or
cerebrovascular thrombotic events, so-called resistance to anti-platelet therapy.5,6 This has resulted in a very broad range in the reported prevalence of resistance to aspirin and clopidogrel, depending on how the term resistance is defined for aspirin resistance Table 313e24 and clopidogrel resistance Table 4,25e33 based on evidence of laboratory platelet testing. Recently, Cattaneo34 clarified the confusion between resistance and other definitions of failure of antiplatelet therapy. He noted that the inhibition of the two major pathways of platelet activation, the P2Y12 receptor and the prostaglandin (thromboxane 2, TX2) pathways will result in the inhibition of both thrombus formation and in vitro platelet functions. Accordingly, the use of a platelet function test that is specific for either of these two platelet activation pathways and that is targeted by the anti-platelet drug, will identify patients who display ‘on-treatment platelet reactivity’, sometimes referred as ‘high-on treatment platelet reactivity’ (HTPR)35 or ‘residual platelet reactivity’ (RPR),36 i.e., a clear reference to the laboratory definition of aspirin/clopidogrel resistance. This is analogous to the definition of bacterial resistance to a specific antimicrobial drug. In this way, light transmission aggregation responses to AA and ADP that employs platelet rich plasma, tests specifically the integrity of the TXA2 and P2Y12 pathways of platelet activation are most suited for identifying high on-treatment reactivity or resistance to antiplatelet therapy. On the other hand, the ‘so-called global tests of platelet function, such as PFA100, Veryflow, and Multiplate (also called point-of-care systems), fit the clinical definition of antiplatelet therapy (i.e., treatment failure).34 Therefore, a growing number of studies employing these diverse tests of platelet function have generated diverse reported prevalences of resistance to antiplatelet therapy. Disagreements exist even between the reported prevalence of resistance to antiplatelet therapy between studies employing the same laboratory test (whether LTA or global tests of platelet function). In the current study, agreement between the findings of the LTA and the PFA100 was noted in only 22.6% of the tested patients. Indeed, the marked lack of concordance
Table 3: List of studies reporting the prevalence of aspirin resistance. Study (year)
Aspirin dose
Aspirin resistance assay
Prevalence
Grotemeyer et al. (1993) Mueller et al. (1997)14 Eikelboom et al. (2002)15 Gum et al. (2003)16 Chen et al. (2004)17 Poston et al. (2006)18 Vivas et al. (2011)19 Rejendran et al. (2011)20 Neubauer et al. (2011)21 Ulehlova et al. (2011)22
500 mg TID 100 mg/day Unspecified 325 mr/day 80 to 325 mg 325 mg/day 100 mg/day Aspirin & clopidogrel 100 mg/day 100 mg/day
El-Atat et al. (2011)23 Postula et al. (2010)24
100 mg/day 75e150 mg/day
The current study
81 mg/day
Platelet reactivity test Corrected whole blood aggregometry Urinary 11-dehydro TXB2 Optical aggregometry Ultegra rapid platelet function assay Whole-blood aggregometry PFA100 VerifyNow Chronolog 590 Arachidonic acid aggregometry Multiplate VerifyNow PFA100; non-responders: Semi-responders: Arachidonic acid light transmission aggregation
33% 60% Quartile comparison 5.2% 19.2% 30% 50.7% 75% 19.4% 22.2% 21.2% 10.9% 29% 30% 12.6%
13
482
Resistance to anti-platelet therapy
Table 4: List of studies reporting the resistance to clopidogrel therapy. Study (year)
Clopidogrel dose
Clopidogrel resistance assay
Mobley et al. (2004)26
Clopidogrel 75 mg twice daily 75 mg daily
Matetzky et al. (2004)27 Awidi et al. (2010)28 Wang et al. (2010)29 Gurbel et al. (2007)30 Petterson et al. (2011)31 Neubauer et al. (2011)21 Maruyama et al. (2011)32 Fukuoda et al. (2011)33 The current study
75 75 75 75 75 75 75 75 75
Flow-cytometric assay VASP phosphorylation (% platelet reactivity) Optical aggregometry e ADP; <10% average platelet inhibition ADP optical aggregometry Multiplate ADP light aggregometry ADP light aggregometry VASP-PRU Chronolog 500 VeriyNow ADP optical aggregometry ADP light aggregometry
25
Barragan et al. (2003)
mg daily mg/day mg/day mg daily mg daily mg/day mg/day mg/day mg/day
between platelet function tests has been widely reported in the literature,19,21,22,24,36e44 which makes it difficult to compare the findings of different publications or compare between ethnic groups. These wide variations in prevalence can be due to many reasons. First and foremost, the platelet has multiple haemostatic mechanisms (adhesion, aggregation and release) and different tests monitor these mechanisms to different extents. Second, differences between the results of the same test that has been conducted in different laboratories can be related to technical factors including differences in the cut-off point determining the abnormal responses.19,34,36 Third, there are also differences in patient characteristics (both clinical and genetic), stage of the disease, associated medical conditions and additional consumed drugs.19,21,24,19,37e44 It should be noted that clopidogrel is a pro-drug and its conversion to the active drug by the liver differs between individuals; this could also account for its less active antiplatelet action when compared to aspirin.19,34 It should be noted that the LTA and the PFA100 differ in many ways; the former test employs platelet rich plasma while the latter employs whole blood. The PFA100 closure time, which employs whole blood, bears some similarity to the bleeding time test, which depends on the adhesion of platelets to collagen in the cartridges, which in turn is influenced by genetic predisposition or mild von Willebrand disease.36 Considering that the LTA is the widely recognized gold standard of platelet function testing34e36 and is the reference method in the current study, with the observed wide agreement between the results of the two tests (agreement between the results in only 22.6% of patients), we can conclude that the PFA100 has low sensitivity and specificity in the laboratory detection of RPR or resistance to antiplatelet therapy. One limitation of this study is that we employed only two tests of platelet function (LTA and the PFA100). While we could have used more tests, these two tests are widely available and are used frequently in routine hospital laboratories worldwide. We are aware that even the LTA has its disadvantages, and therefore, the search continues to find an ideal test of platelet function. We believe that this will remain an elusive goal, given the diverse and overlapping mechanisms of the functions of the blood platelet. Eventually, we may ultimately settle for multiple tests of platelet functions in
Prevalence
30% 25% 26% 20.1% 33% 32% 30.8% 29% 18% 25.1%
the investigation of patients, in the same fashion as liver function tests. However, it should also be emphasized that even with the currently available platelet function tests, it has repeatedly been demonstrated that CHD patients on antiplatelet therapy who show laboratory evidence of high on-treatment reactivity (or resistance to antiplatelet therapy) are at higher risk of developing clinical adverse cardio- or cerebrovascular events than those who respond to therapy.16,36,45 This necessitates the serious consideration of platelet function testing in monitoring antiplatelet therapy in CHD patients. This will no doubt be a strong indicator to the treating physician that the prescribed drug therapy is fulfilling its intended pharmaceutical action. In conclusion, laboratory testing of platelet function, which has historically been employed in the diagnosis of bleeding disorders, is increasingly being requested for monitoring antiplatelet therapy in cardio- and cerebrovascular disease. The findings of this study have demonstrated the wide prevalence of on-treatment residual platelet reactivity (RPR) in patients with CHD on dual platelet therapy (aspirin and clopidogrel). The prevalence compares favourably with the findings reported in the literature. It was also shown that the global whole blood test of platelet function (the PFA100) gives a much higher prevalence of resistance than the currently accepted gold standard test of platelet function, light transmission aggregometry.
Conflict of interest The authors have no conflict of interest to declare.
Authors’ contribution AGMAG contributed to the inception of the research idea, took responsibility for the laboratory assays and analysis of data as well writing and final approval of the manuscript. MQA responsible for recruiting patients, completing patients’ data sheets, supervising the collection of blood samples, and also participated in the writing and final approval of the manuscript. NMB took an active role in the laboratory assays, the entry of data into the computer,
M.Q. Al Shamiri et al. analysis of the data and writing and final approval of the manuscript. Acknowledgements The authors would like to thank M.A. Hamid and L.G. El-Sid for their technical assistance and F. Chatila for her secretarial work. They also thank M. Al-Dukhayil for her assistance during sample collection. This research was supported by grants from the College of Medicine Research Center (CMRC), King Saud University, Riyadh, as well as King Abdul Aziz City for Science and Technology (LGPe 14e55), Riyadh, KSA. References 1. AlHabib KF, Hersi A, AlFaleh H, Kurdi M, Arafah M, Youssef M, AlNemer K, Bakheet A, AlQarni A, Soomro T, Taraben A, Malik A, Ahmed WH. The Saudi Project for Assessment of Coronary Events [SPACE] registry: design and results of a phase I pilot study. Can J Cardiol 2009; 25: 255e 258. 2. AlHabib KF, Elasfar AA, AlBackr H, AlFaleh H, Hersi A, AlShaer F, Kashour T, AlNemer K, Hussein GA, Mimish L. Design and preliminary results of the heart function assessment registry trial in Saudi Arabia [HEARTS] in patients with acute and chronic heart failure. Eur J Heart Fail 2011; 13: 1178e1184. 3. Osman AM, Alsultan MS, Al-Mutairi MA. The burden of ischemic heart disease at a major cardiac center in Central Saudi Arabia. Saudi Med J 2011; 32: 1279e1284. 4. Antithrombotic Trialist Collaboration. Collaborative metaanalysis of randomized trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high-risk patients. BMJ 2002; 324: 71e86. 5. Shantsila E, Watson T, Lip GYH. Aspirin resistance: what, why and when? Editorial e Thromb Res 2007; 119: 551e554. 6. Qureshi Z, Hobson AR. Clopidogrel “Resistance”: where are we now? Cardiovasc Ther 2011 August 3rd: 1e9. 7. Gillum RF, Mehari A, Curry B, Obisesan TO. Racial and geographic variation in coronary heart disease mortality trends. BMC Public Health 2012 Jun 6; 12: 410. http://dx.doi.org/ 10.1186/1471-2458-12-410. 8. Freund KM, Jacobs AK, Pechacek JA, White HF, Ash AS. Disparities by race, ethnicity, and sex in treating acute coronary syndromes. J Womens Health (Larchmt) 2012; 21: 126e132. 9. Leck I, Thomson JM, Bocaz JA, Barja P, Bonnar J, Daly L, Carrol A, Coutinho E, Goncalves M, Tsakok MA. Multicentre study of coagulation and haemostatic variables during oral contraception: variations with geographical location and ethnicity. Int J Epidemiol 1991; 20: 913e920. 10. Gader AM, Bahakim HM, Malaika SS. Ethnic variations in platelet aggregation-comparison between Saudi Arabs, Westerners [Europeans and Americans], Asians and Africans. Platelets 1991; 2: 197e201. 11. Gader AMA, Ghumlas AK, Hussain MF, Al-Haidary AI. Platelet aggregation and platelet function analyser (PFA-100) closure time in camels e a comparative study with humans. Comp Clin Pathol 2006; 15: 31e37. 12. Gader AMA, Bahakim HM, Malaika SS. A study of the normal pattern of platelet aggregation in healthy Saudis: a populationbased study. Platelets 1990; 1: 139e143. 13. Groterneyer KH, Scharafinski HW, Husstedt IW. Two-year follow-up of aspirin responder and aspirin nonresponder. A pilot-study including 180 post-stroke patients. Thromb Res 1993; 71: 397e403.
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How to cite this article: Al Shamiri MQ, Abdel Gader AM, Bayoumy NM. Comparing light transmission aggregometry and PFA100 for monitoring antiplatelet therapy in patients with coronary heart disease. J Taibah Univ Med Sc 2016;11(5):478e484.
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Original Article
Comparing light transmission aggregometry and PFA100 for monitoring antiplatelet therapy in patients with coronary heart disease Mostafa Q. Al Shamiri, MRCP a, Abelgalil M. Abdel Gader, PhD b, * and Nervana M. Bayoumy, PhD b a b
Coronary Care Unit, King Khalid University Hospital, College of Medicine, King Saud University, Riyadh, KSA Coagulation Research Laboratory, Physiology Department, College of Medicine, King Saud University, Riyadh, KSA
Received 25 April 2016; revised 4 August 2016; accepted 7 August 2016; Available online 7 September 2016
ﺍﻟﻤﻠﺨﺺ ﻻﻳﺰﺍﻝ ﻛﺜﻴﺮ ﻣﻦ ﺍﻟﻤﺮﺿﻰ ﺍﻟﺬﻳﻦ ﻳﺘﻨﺎﻭﻟﻮﻥ ﺍﻟﻌﻼﺟﺎﺕ ﺍﻟﻤﻀﺎﺩﺓ:ﺃﻫﺪﺍﻑ ﺍﻟﺒﺤﺚ ﻣﻤﺎ ﻳﺆﺩﻱ ﺇﻟﻰ ﻣﺎ،ﻟﻠﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﻳﻌﺎﻧﻮﻥ ﻣﻦ ﺣﺪﻭﺙ ﺍﻟﺠﻠﻄﺎﺕ ﻭﻓﺸﻞ ﺍﻟﻌﻼﺝ ﻛﺎﻥ ﻫﻨﺎﻙ ﺯﻳﺎﺩﺓ٬ ﻣﺆﺧﺮﺍ.ﻳﺴﻤﻰ “ﻣﻘﺎﻭﻣﺔ” ﺍﻟﻌﻼﺟﺎﺕ ﺍﻟﻤﻀﺎﺩﺓ ﻟﻠﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﻓﻲ ﺍﻟﻠﺠﻮﺀ ﺇﻟﻰ ﺇﺟﺮﺍﺀ ﺍﻻﺧﺘﺒﺎﺭﺍﺕ ﺍﻟﻤﺨﺒﺮﻳﺔ ﻟﻤﺮﺍﻗﺒﺔ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﺍﻟﺘﻲ ﺗﻬﺪﻑ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﻟﺘﻮﺛﻴﻖ ﺍﻧﺘﺸﺎﺭ ﺑﺮﻫﺎﻥ.ﺗﺒﺸﺮ ﺑﺘﻌﻴﻴﻦ ﻣﺜﻞ ﻫﺆﻻﺀ ﺍﻟﻤﺮﺿﻰ ﺍﻟﻤﺨﺘﺒﺮﺍﺕ ﻟﻤﻘﺎﻭﻣﺔ ﺍﻷﺳﺒﺮﻳﻦ ﻭﻋﻼﺝ ﻛﻠﻮﺑﻴﺪﻭﻗﺮﻳﻞ ﻓﻲ ﺍﻟﻤﺮﺿﻰ ﺍﻟﺴﻌﻮﺩﻳﻴﻦ ﺍﻟﺬﻳﻦ .ﻳﻌﺎﻧﻮﻥ ﻣﻦ ﺃﻣﺮﺍﺽ ﺍﻟﺸﺮﺍﻳﻴﻦ ﺍﻟﺘﺎﺟﻴﺔ ﺍﻟﻤﺴﺘﻘﺮﺓ ﺃﺟﺮﻱ ﻗﻴﺎﺱ ﺍﻟﺘﻜﺪﺱ ﺑﻮﺍﺳﻄﺔ ﻧﻘﻞ ﺍﻟﻀﻮﺀ ﻓﻲ ﺍﻟﺒﻼﺯﻣﺎ ﺍﻟﻐﻨﻴﺔ:ﻃﺮﻕ ﺍﻟﺒﺤﺚ ، ﻭﺣﺎﻣﺾ ﺍﻷﺭﺍﻛﻴﺪﻭﻧﻴﻚ،ﺑﺎﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﻛﺈﺳﺘﺠﺎﺑﺔ ﻷﺩﻳﻨﻮﺳﻴﻦ ﺛﻨﺎﺋﻲ ﺍﻟﻔﻮﺳﻔﺎﺕ ﺍﻟﺬﻯ، ﺑﺎﻹﺿﺎﻓﺔ ﺇﻟﻰ ﺗﺤﻠﻴﻞ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ.ﻭﺍﻟﻜﻮﻻﺟﻴﻦ ﻭﺍﻷﺩﺭﻳﻨﺎﻟﻴﻦ /ﺃﺩﻧﻴﻮﺳﻴﻦ ﺛﻨﺎﺋﻲ ﺍﻟﻔﻮﺳﻔﺎﺕ ﻭﺍﻟﻜﻮﻻﺟﻴﻦ/ﻳﻌﻤﻞ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺃﻃﻘﻢ ﻛﻼ ﻣﻦ ﺍﻟﻜﻮﻻﺟﻴﻦ .ﺃﺩﺭﻳﻨﺎﻟﻴﻦ ﺃﻇﻬﺮ ﻗﻴﺎﺱ ﺍﻟﺘﻜﺪﺱ ﺑﻮﺍﺳﻄﺔ ﻧﻘﻞ ﺍﻟﻀﻮﺀ ﻣﻘﺎﻭﻣﺔ ﻟﻸﺳﺒﺮﻳﻦ ﻭﻋﻼﺝ:ﺍﻟﻨﺘﺎﺋﺞ ﻓﻲ٬ ﻭﻓﻘﺎ ﻟﻼﺳﺘﻤﺮﺍﺭ ﻻﺳﺘﺠﺎﺑﺔ ﺍﻟﺘﻜﺪﺱ ﻟﺘﺠﻤﻊ ﺣﺎﻣﺾ ﺍﻷﻛﺮﺍﻛﻴﺪﻭﻧﻴﻚ٬ﻛﻠﻮﺑﻴﺪﻭﻗﺮﻳﻞ ﻭﺑﺎﺳﺘﺨﺪﺍﻡ ﺗﺤﻠﻴﻞ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﺃﻭﻗﺎﺕ. ﻋﻠﻰ ﺍﻟﺘﻮﺍﻟﻲ٪٢٦ ﻭ٪١٣ ﻣﺸﻴﺮﺍ ﺇﻟﻰ ﺗﻔﺎﻋﻞ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ٬ﺍﻹﻏﻼﻕ ﻓﻲ ﺣﺪﻭﺩ ﺍﻟﻘﻴﻢ ﺍﻟﻤﺮﺟﻌﻴﺔ ﻟﻠﻤﺨﺘﺒﺮ ﻛﻤﺎ ﺗﻢ ﺗﺴﺠﻴﻞ ﺍﻧﺘﺸﺎﺭ ﺍﻟﻤﻘﺎﻭﻣﺔ ﻟﻠﻌﻼﺟﺎﺕ ﺍﻟﻤﻀﺎﺩﺓ ﻟﻠﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﻋﻨﺪ٬ﺍﻟﻤﺘﺒﻘﻴﺔ ﺑﺎﺳﺘﻌﻤﺎﻝ٪ ٣٠.٧ ﺃﺩﻧﻴﻮﺳﻴﻦ ﺛﻨﺎﺋﻲ ﺍﻟﻔﻮﺳﻔﺎﺕ ﻭ/ ﺑﺎﺳﺘﻌﻤﺎﻝ ﺃﻃﻘﻢ ﺍﺍﻟﻜﻮﻻﺟﻴﻦ٪٣٣
* Corresponding address: Department of Basic Medical Science, College of Medicine King Saud bin Abdulaziz University for Health Sciences, Riyadh 11426, KSA. E-mail: [email protected] (A.M. Abdel Gader) Peer review under responsibility of Taibah University.
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ﻛﻤﺎ ﻇﻬﺮ ﺍﻟﺘﻮﺍﻓﻖ ﺑﻴﻦ ﺍﻟﺘﻜﺪﺱ ﺑﻮﺍﺳﻄﺔ ﻧﻘﻞ ﺍﻟﻀﻮﺀ. ﺃﺩﺭﻳﻨﺎﻟﻴﻦ/ﺃﻃﻘﻢ ﺍﻟﻜﻮﻻﺟﻴﻦ . ﻣﻦ ﺍﻟﻤﺮﺿﻰ٪٢٢.٦ ﻭﺗﺤﻠﻴﻞ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﻋﻨﺪ ﺃﻇﻬﺮﺕ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﺍﻻﻧﺘﺸﺎﺭ ﺍﻟﻮﺍﺳﻊ ﻟﺘﻔﺎﻋﻞ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ:ﺍﻻﺳﺘﻨﺘﺎﺟﺎﺕ ﻟﻠﻤﺮﺿﻰ ﺍﻟﺬﻳﻦ ﻳﻌﺎﻧﻮﻥ ﻣﻦ ﺃﻣﺮﺍﺽ ﺍﻟﺸﺮﺍﻳﻴﻦ ﺍﻟﺘﺎﺟﻴﺔ ﻭﻫﻢ ﺗﺤﺖ ﺍﻟﻌﻼﺝ ﺍﻟﺜﻨﺎﺋﻲ ﻳﻌﻄﻲ ﺍﺧﺘﺒﺎﺭ٬ ﺑﺎﻹﺿﺎﻓﺔ.(ﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ )ﺍﻷﺳﺒﺮﻳﻦ ﻭﻛﻠﻮﺑﻴﺪﻭﻗﺮﻳﻞ ﺍﻟﺪﻡ ﺍﻟﻜﺎﻣﻞ ﺍﻟﻌﺎﻟﻤﻲ ﻟﻮﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﺑﻮﺍﺳﻄﺔ ﺗﺤﻠﻴﻞ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ ﺍﺭﺗﻔﺎﻋﺎ ﺃﻋﻠﻰ ﺑﻜﺜﻴﺮ ﻟﻤﻌﺪﻝ ﺍﻧﺘﺸﺎﺭ ﺍﻟﻤﻘﺎﻭﻣﺔ ﻋﻦ ﺍﻟﺘﻜﺪﺱ ﺑﻮﺍﺳﻄﺔ ﻧﻘﻞ ﺍﻟﻀﻮﺀ ﻋﻨﺪ ﻣﻘﺎﺭﻧﺘﻪ ﺑﺎﻟﻤﻌﻴﺎﺭ ﺍﻟﺬﻫﺒﻲ ﻭﺍﻷﻛﺜﺮ ﺗﺤﺪﻳﺪﺍ ﻟﺘﺤﻠﻴﻞ ﺍﻟﺘﻜﺪﺱ ﺑﻮﺍﺳﻄﺔ ﻧﻘﻞ .ﺍﻟﻀﻮﺀ ﺍﻟﻌﻼﺟﺎﺕ ﺍﻟﻤﻀﺎﺩﺓ ﻟﻠﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ؛ ﻭﻇﺎﺋﻒ ﺍﻟﺼﻔﺎﺋﺢ:ﺍﻟﻜﻠﻤﺎﺕ ﺍﻟﻤﻔﺘﺎﺣﻴﺔ ﺍﻟﺪﻣﻮﻳﺔ؛ ﺍﻷﺳﺒﺮﻳﻦ؛ ﻛﻠﻮﺑﻴﺪﻭﻗﺮﻳﻞ؛ ﻣﻘﺎﻭﻣﺔ ﺍﻷﺳﺒﺮﻳﻦ؛ ﻣﺮﺽ ﺍﻟﻘﻠﺐ ﺍﻹﻗﻔﺎﺭﻱ؛ ﺗﻜﺪﺱ ﺍﻟﺼﻔﺎﺋﺢ ﺍﻟﺪﻣﻮﻳﺔ
Abstract Objectives: Many patients undergoing antiplatelet therapy continue to experience thrombotic events and failure of therapy leading to so-called ‘resistance’ to antiplatelet therapy. Recently, there has been an increasing focus on in vitro laboratory monitoring of platelet functions, which has the promise of identifying these patients. This study aimed to document the prevalence of laboratory evidence of resistance to aspirin and clopidogrel therapy in Saudi patients with stable coronary heart disease (CHD). Methods: Light transmission aggregometry in plateletrich plasma was performed in response to adenosine diphosphate (ADP), arachidonic acid (AA), collagen and adrenaline. In addition, a platelet function analyser (PFA100) was employed using both collagen/ADP and collagen/epinephrine cartridges.
1658-3612 Ó 2016 The Authors. Production and hosting by Elsevier Ltd on behalf of Taibah University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.jtumed.2016.08.002
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M.Q. Al Shamiri et al. Results: Light transmission aggregometry (LTA) identified the resistance to aspirin and clopidogrel therapy, according to the persistence of the aggregation response to AA aggregation, to be 13% and 26%, respectively. By PFA100 testing, closure times within the limits of laboratory reference values indicated residual platelet reactivity, and the prevalence of resistance to anti-platelet therapy was reported to be 33% for collagen/ADP cartridges and 30.7% for collagen/epinephrine. A concordance between LTA and PFA100 CT was noted in only 22.6% of patients. Conclusion: This study showed a wide prevalence of ontreatment platelet reactivity in patients with CHD on dual platelet therapy (aspirin and clopidogrel). In addition, the global whole blood test of platelet function by PFA100 estimated a much higher prevalence of resistance than LTA when compared to the gold standard and more specific LTA analysis. Keywords: Anti-platelet therapy; Aspirin resistance; Clopidogrel; PFA100; Platelet aggregation Ó 2016 The Authors. Production and hosting by Elsevier Ltd on behalf of Taibah University. This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
Introduction Recent reports from the KSA highlighted the magnitude of coronary heart disease,1,2 which is already proving to exert a high economic burden.3 In outlining the current diagnostic and treatment strategies for patients with acute coronary syndrome (ACS), it was shown that the most common medications used are the two antiplatelet drugs, aspirin (98%) and clopidogrel (73%)1; both drugs have proved their efficacy in the primary as well as the secondary prevention of cardiovascular and cerebrovascular disease.4 However, some patients taking aspirin and/or clopidogrel still develop thrombotic cardio- and/or cerebrovascular events, referred to as so-called ‘aspirin/clopidogrel resistance’ or ‘treatment failure’.5,6 Recently, there have been increasing efforts focused on in vitro laboratory monitoring of platelet function to try to identify these patients and determine whether they display normal in vitro platelet function (residual platelet activity) despite treatment with antiplatelet drugs. Of the numerous tests of platelet function, two tests have found wide popularity: light transmission aggregometry and the platelet function analyser (PFA100). To our knowledge, resistance to antiplatelet therapy has never been explored in the KSA; the available data on this topic have emerged mainly from Western countries. Our interest in this area was triggered by the reported racial variation in both CHD mortality7,8 as well as in haemostatic9 and platelet function testing.10 This encouraged us to undertake the laboratory evaluation of resistance to antiplatelet therapy to determine the scale of the problem in our population.
Therefore, the main aim of this study was to document the prevalence of aspirin and clopidogrel resistance in KSA patients with stable coronary heart disease, using the locally familiar light transmission aggregometry and the platelet function analyser (PFA100). Materials and Methods Selection of patients Patients (n ¼ 106; Table 1) were recruited consecutively and according to the following inclusion and exclusion criteria: Inclusion criteria Consecutive admissions to the Medical Outpatient Department, medical wards, Coronary Care Unit and Medical Intensive Care at King Khalid University Hospital, who had any of the following conditions: Saudis, older than 20 years. Patients, after receiving the standard loading dose of aspirin and clopidogrel, continued on maintenance dose of either aspirin (81 mg/day) and or clopidogrel (75 mg/day) for more than 7 days. All recruited patients (n ¼ 106) were on low-dose aspirin as well as clopidogrel. They had an established diagnosis of any the following: Acute coronary syndrome (ACS) Coronary heart disease (CHD) defined as follows: chronic stable angina, prior history of unstable angina or myocardial infarction, previous history of revascularization or disease of at least one coronary vessel on previous angiography or non-invasive imaging procedure. Exclusion criteria Non-Saudis Patients younger than 20 years of age. Persons with known bleeding or thrombotic disorders. This study has been approved by Institutional Review Board, the College of Medicine, King Saud University, Riyadh. Blood samples and laboratory techniques Samples of 10 ml blood were collected from an easily accessible vein in the antecubital fossa and with minimum of Table 1: Characteristics of the patients with stable ischaemic heart disease in whom both the light transmission aggregometry and the PFA100 test were performed (n [ 106). Age (years) Sex (M/F) Weight (kg) Diabetes mellitus Hypertension Dyslipidaemia Aspirin (81 mg) Clopidogrel (75 mg)
59.6 12.4 (28e90) 70/36 75.9 14.9 (52e117) 42/106 64/106 78/106 All patients
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Resistance to anti-platelet therapy
venous stasis or from arterial or central access, in two 5-ml tubes containing sodium citrate anticoagulant solution (3.8% w/v), to give a blood-to-citrate ratio of 9:1. Samples were transported within maximum of one hour, without delay, to the Coagulation Laboratory, Department of Physiology. Whole blood was centrifuged for 3 min at 1500 rpm, and supernatant plateletrich plasma (PRP) was removed carefully a using plasma pipette and put in a plastic tube. What remained of the blood samples was centrifuged further at 30,000 rpm for 15 min and the supernatant platelet poor plasma (PPP) was removed carefully and put in a separate plastic tube. Laboratory assays i. Platelet aggregation in platelet rich plasma (PRP) was assessed using a standard aggregometer [PAP-4, BioData Corporation, USA]. This machine has also been in use in the Coagulation Research Laboratory in many previous studies in health and disease and the aggregation technique has been detailed before.11,12
deviation. The chi-square test was used to determine the significance of the difference between different prevalences. A P value of <0.05 was considered significant. This study has been approved by Institutional Review Board, the College of Medicine, King Saud University, Riyadh. The study was conducted according to the Declaration of Helsinki and participants gave their consent before enrolling in the study. Results Resistance to antiplatelet therapy is defined as the detection of normal aggregation responses or normal PFA100 CT while the patient is on antiplatelet drugs (aspirin and clopidogrel). A summary of the prevalence of resistance to aspirin and clopidogrel is shown in Table 2. It is clear that the prevalence of resistance to antiplatelet therapy varied widely between the aggregation tests and the PFA100 closure times. Light transmission aggregometry
Summary of the platelet aggregation technique Platelet aggregation in response to adenosine diphosphate (ADP) and arachidonic acid (AA), collagen and epinephrine
Before performing the aggregometry studies, PRP was diluted using PPP to adjust the platelet count to the range of 150,000e200,000/mm3. Platelet aggregometry was undertaken in response to ADP (20 mmol/L) (Sigma Chemical Co., St. Louis, MO, USA), and AA (10 mg/ml), collagen and epinephrine (Bio/Data Corp. Horsham, PA, USA). The concentrations of agonists represent the final concentrations obtained by adding 20 ml of the aggregating agent to 180 ml of PRP. Laboratory conditions were carefully standardized and these include: time interval between venipuncture and the measurement of platelet aggregation (<2 h), temperature of testing (37 C) and the rate of PRP stirring (1000 rpm). The aggregometer registers the results of the aggregation responses as maximum aggregation (MA %) of the aggregation curves. ii. The PFA-100 platelet function analyser (Dade Behring, Miami, Florida, USA).11 This instrument provides an in vitro quantitative measurement of platelet adhesion and aggregation requires whole blood collected in 3.8% sodium citrate and utilizes cartridges coated with the aggregation agonists collagen/epinephrine and collagen/ ADP. It measures agonist-induced platelet plug formation as time in seconds (closure time) to occlude the aperture in these cartridges. Both cartridges were employed in this study. The local laboratory reference closure time for collagen/epinephrine cartridge ranges from 84 to 198 s (mean SD: 141.6 27 s) and for the collagen/ADP cartridge is 52e153 s (mean SD: 100.7 24.1 s; n ¼ 88).11 Any closure time less than the maximum in these two ranges is taken to represent residual platelet reactivity (or resistance to antiplatelet therapy). iii. Statistical analysis: Data were analysed using the SPSS statistical package and the results were expressed as the mean and standard
The resistance to aspirin therapy is specifically indicated by the presence of normal aggregation response to AA aggregation, which tests the integrity of the prostaglandin (thromboxane A2, TX2) pathway, was detected in 13% of patients. The resistance to clopidogrel, which blocks the ADP receptor P2Y12 and that is tested for by the aggregation responses to ADP, was 26%. The observed prevalence of the non-specific aggregation responses to collagen and epinephrine was 23% and 14%, respectively. The PFA100 closure time (CT) Using the PFA100 test, a CT that falls within the normal reference normal range of CT was taken to indicate residual Table 2: The prevalence of resistance to aspirin and clopidogrel in patients with stable ischaemic heart disease using both the platelet function analyser (PFA100) as well as optical aggregometry. Type of drug Laboratory method
Aspirin þ PFA100 clopidogrel Col-ADP cartridges Col-epinephrine cartridges Optical aggregometry Arachidonic acid ADP Collagen Adrenaline
Inhibited Normal response (on-treatment) response (resistance to antiplatelet therapy) 67%
33%
70%
30.7%
87.4% 74.9% 77% 86%
13% 26.1% 23% 14%
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M.Q. Al Shamiri et al. platelet reactivity (resistance to antiplatelet therapy). Accordingly, the prevalence of resistance for the collagen/ ADP cartridges was 33%, while for the collagen/epinephrine was 30.7%.
Concordance between the results of the PFA100 and light transmission aggregometry (LTA) In only 24 patients out of 106 (22.6%), aggregation responses to ADP and arachidonic acid as well as the PFA100 CT times of both cartridges indicated good response to antiplatelet therapy. For the rest of the patients, there were wide variations in the detection rate of individual tests.
Discussion Antiplatelet therapy plays a major role in the treatment of patients with acute coronary syndrome (ACS), resulting in a significant reduction in ischaemic events.4 This topic continues to receive extensive coverage in the literature, which is predominantly from developed countries; scant information comes from developing countries where coronary heart disease is proving to be an emerging health problem.4 The findings of recent nationwide studies in the KSA1e3 provided, for the first time, a comprehensive view of the local scale of the problem and current diagnostic and treatment strategies for patients with ACS and highlighted opportunities to improve the care of these patients. One major finding of these studies was that aspirin (acetylsalicylic acid) is consumed by 98% of patients with acute coronary syndromes; the corresponding figure for clopidogrel is 73%. Aspirin is usually combined with other anti-platelet agents in order to achieve a more effective inhibition of platelet aggregation. However, it has repeatedly been shown that in a significant number of patients on anti-platelet therapy, no therapeutic benefit could be gained whether by evidence of the laboratory detection of platelet function inhibition or the prevention of further complications such as recurrence of cardiac and or
cerebrovascular thrombotic events, so-called resistance to anti-platelet therapy.5,6 This has resulted in a very broad range in the reported prevalence of resistance to aspirin and clopidogrel, depending on how the term resistance is defined for aspirin resistance Table 313e24 and clopidogrel resistance Table 4,25e33 based on evidence of laboratory platelet testing. Recently, Cattaneo34 clarified the confusion between resistance and other definitions of failure of antiplatelet therapy. He noted that the inhibition of the two major pathways of platelet activation, the P2Y12 receptor and the prostaglandin (thromboxane 2, TX2) pathways will result in the inhibition of both thrombus formation and in vitro platelet functions. Accordingly, the use of a platelet function test that is specific for either of these two platelet activation pathways and that is targeted by the anti-platelet drug, will identify patients who display ‘on-treatment platelet reactivity’, sometimes referred as ‘high-on treatment platelet reactivity’ (HTPR)35 or ‘residual platelet reactivity’ (RPR),36 i.e., a clear reference to the laboratory definition of aspirin/clopidogrel resistance. This is analogous to the definition of bacterial resistance to a specific antimicrobial drug. In this way, light transmission aggregation responses to AA and ADP that employs platelet rich plasma, tests specifically the integrity of the TXA2 and P2Y12 pathways of platelet activation are most suited for identifying high on-treatment reactivity or resistance to antiplatelet therapy. On the other hand, the ‘so-called global tests of platelet function, such as PFA100, Veryflow, and Multiplate (also called point-of-care systems), fit the clinical definition of antiplatelet therapy (i.e., treatment failure).34 Therefore, a growing number of studies employing these diverse tests of platelet function have generated diverse reported prevalences of resistance to antiplatelet therapy. Disagreements exist even between the reported prevalence of resistance to antiplatelet therapy between studies employing the same laboratory test (whether LTA or global tests of platelet function). In the current study, agreement between the findings of the LTA and the PFA100 was noted in only 22.6% of the tested patients. Indeed, the marked lack of concordance
Table 3: List of studies reporting the prevalence of aspirin resistance. Study (year)
Aspirin dose
Aspirin resistance assay
Prevalence
Grotemeyer et al. (1993) Mueller et al. (1997)14 Eikelboom et al. (2002)15 Gum et al. (2003)16 Chen et al. (2004)17 Poston et al. (2006)18 Vivas et al. (2011)19 Rejendran et al. (2011)20 Neubauer et al. (2011)21 Ulehlova et al. (2011)22
500 mg TID 100 mg/day Unspecified 325 mr/day 80 to 325 mg 325 mg/day 100 mg/day Aspirin & clopidogrel 100 mg/day 100 mg/day
El-Atat et al. (2011)23 Postula et al. (2010)24
100 mg/day 75e150 mg/day
The current study
81 mg/day
Platelet reactivity test Corrected whole blood aggregometry Urinary 11-dehydro TXB2 Optical aggregometry Ultegra rapid platelet function assay Whole-blood aggregometry PFA100 VerifyNow Chronolog 590 Arachidonic acid aggregometry Multiplate VerifyNow PFA100; non-responders: Semi-responders: Arachidonic acid light transmission aggregation
33% 60% Quartile comparison 5.2% 19.2% 30% 50.7% 75% 19.4% 22.2% 21.2% 10.9% 29% 30% 12.6%
13
482
Resistance to anti-platelet therapy
Table 4: List of studies reporting the resistance to clopidogrel therapy. Study (year)
Clopidogrel dose
Clopidogrel resistance assay
Mobley et al. (2004)26
Clopidogrel 75 mg twice daily 75 mg daily
Matetzky et al. (2004)27 Awidi et al. (2010)28 Wang et al. (2010)29 Gurbel et al. (2007)30 Petterson et al. (2011)31 Neubauer et al. (2011)21 Maruyama et al. (2011)32 Fukuoda et al. (2011)33 The current study
75 75 75 75 75 75 75 75 75
Flow-cytometric assay VASP phosphorylation (% platelet reactivity) Optical aggregometry e ADP; <10% average platelet inhibition ADP optical aggregometry Multiplate ADP light aggregometry ADP light aggregometry VASP-PRU Chronolog 500 VeriyNow ADP optical aggregometry ADP light aggregometry
25
Barragan et al. (2003)
mg daily mg/day mg/day mg daily mg daily mg/day mg/day mg/day mg/day
between platelet function tests has been widely reported in the literature,19,21,22,24,36e44 which makes it difficult to compare the findings of different publications or compare between ethnic groups. These wide variations in prevalence can be due to many reasons. First and foremost, the platelet has multiple haemostatic mechanisms (adhesion, aggregation and release) and different tests monitor these mechanisms to different extents. Second, differences between the results of the same test that has been conducted in different laboratories can be related to technical factors including differences in the cut-off point determining the abnormal responses.19,34,36 Third, there are also differences in patient characteristics (both clinical and genetic), stage of the disease, associated medical conditions and additional consumed drugs.19,21,24,19,37e44 It should be noted that clopidogrel is a pro-drug and its conversion to the active drug by the liver differs between individuals; this could also account for its less active antiplatelet action when compared to aspirin.19,34 It should be noted that the LTA and the PFA100 differ in many ways; the former test employs platelet rich plasma while the latter employs whole blood. The PFA100 closure time, which employs whole blood, bears some similarity to the bleeding time test, which depends on the adhesion of platelets to collagen in the cartridges, which in turn is influenced by genetic predisposition or mild von Willebrand disease.36 Considering that the LTA is the widely recognized gold standard of platelet function testing34e36 and is the reference method in the current study, with the observed wide agreement between the results of the two tests (agreement between the results in only 22.6% of patients), we can conclude that the PFA100 has low sensitivity and specificity in the laboratory detection of RPR or resistance to antiplatelet therapy. One limitation of this study is that we employed only two tests of platelet function (LTA and the PFA100). While we could have used more tests, these two tests are widely available and are used frequently in routine hospital laboratories worldwide. We are aware that even the LTA has its disadvantages, and therefore, the search continues to find an ideal test of platelet function. We believe that this will remain an elusive goal, given the diverse and overlapping mechanisms of the functions of the blood platelet. Eventually, we may ultimately settle for multiple tests of platelet functions in
Prevalence
30% 25% 26% 20.1% 33% 32% 30.8% 29% 18% 25.1%
the investigation of patients, in the same fashion as liver function tests. However, it should also be emphasized that even with the currently available platelet function tests, it has repeatedly been demonstrated that CHD patients on antiplatelet therapy who show laboratory evidence of high on-treatment reactivity (or resistance to antiplatelet therapy) are at higher risk of developing clinical adverse cardio- or cerebrovascular events than those who respond to therapy.16,36,45 This necessitates the serious consideration of platelet function testing in monitoring antiplatelet therapy in CHD patients. This will no doubt be a strong indicator to the treating physician that the prescribed drug therapy is fulfilling its intended pharmaceutical action. In conclusion, laboratory testing of platelet function, which has historically been employed in the diagnosis of bleeding disorders, is increasingly being requested for monitoring antiplatelet therapy in cardio- and cerebrovascular disease. The findings of this study have demonstrated the wide prevalence of on-treatment residual platelet reactivity (RPR) in patients with CHD on dual platelet therapy (aspirin and clopidogrel). The prevalence compares favourably with the findings reported in the literature. It was also shown that the global whole blood test of platelet function (the PFA100) gives a much higher prevalence of resistance than the currently accepted gold standard test of platelet function, light transmission aggregometry.
Conflict of interest The authors have no conflict of interest to declare.
Authors’ contribution AGMAG contributed to the inception of the research idea, took responsibility for the laboratory assays and analysis of data as well writing and final approval of the manuscript. MQA responsible for recruiting patients, completing patients’ data sheets, supervising the collection of blood samples, and also participated in the writing and final approval of the manuscript. NMB took an active role in the laboratory assays, the entry of data into the computer,
M.Q. Al Shamiri et al. analysis of the data and writing and final approval of the manuscript. Acknowledgements The authors would like to thank M.A. Hamid and L.G. El-Sid for their technical assistance and F. Chatila for her secretarial work. They also thank M. Al-Dukhayil for her assistance during sample collection. This research was supported by grants from the College of Medicine Research Center (CMRC), King Saud University, Riyadh, as well as King Abdul Aziz City for Science and Technology (LGPe 14e55), Riyadh, KSA. References 1. AlHabib KF, Hersi A, AlFaleh H, Kurdi M, Arafah M, Youssef M, AlNemer K, Bakheet A, AlQarni A, Soomro T, Taraben A, Malik A, Ahmed WH. The Saudi Project for Assessment of Coronary Events [SPACE] registry: design and results of a phase I pilot study. Can J Cardiol 2009; 25: 255e 258. 2. AlHabib KF, Elasfar AA, AlBackr H, AlFaleh H, Hersi A, AlShaer F, Kashour T, AlNemer K, Hussein GA, Mimish L. Design and preliminary results of the heart function assessment registry trial in Saudi Arabia [HEARTS] in patients with acute and chronic heart failure. Eur J Heart Fail 2011; 13: 1178e1184. 3. Osman AM, Alsultan MS, Al-Mutairi MA. The burden of ischemic heart disease at a major cardiac center in Central Saudi Arabia. Saudi Med J 2011; 32: 1279e1284. 4. Antithrombotic Trialist Collaboration. Collaborative metaanalysis of randomized trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high-risk patients. BMJ 2002; 324: 71e86. 5. Shantsila E, Watson T, Lip GYH. Aspirin resistance: what, why and when? Editorial e Thromb Res 2007; 119: 551e554. 6. Qureshi Z, Hobson AR. Clopidogrel “Resistance”: where are we now? Cardiovasc Ther 2011 August 3rd: 1e9. 7. Gillum RF, Mehari A, Curry B, Obisesan TO. Racial and geographic variation in coronary heart disease mortality trends. BMC Public Health 2012 Jun 6; 12: 410. http://dx.doi.org/ 10.1186/1471-2458-12-410. 8. Freund KM, Jacobs AK, Pechacek JA, White HF, Ash AS. Disparities by race, ethnicity, and sex in treating acute coronary syndromes. J Womens Health (Larchmt) 2012; 21: 126e132. 9. Leck I, Thomson JM, Bocaz JA, Barja P, Bonnar J, Daly L, Carrol A, Coutinho E, Goncalves M, Tsakok MA. Multicentre study of coagulation and haemostatic variables during oral contraception: variations with geographical location and ethnicity. Int J Epidemiol 1991; 20: 913e920. 10. Gader AM, Bahakim HM, Malaika SS. Ethnic variations in platelet aggregation-comparison between Saudi Arabs, Westerners [Europeans and Americans], Asians and Africans. Platelets 1991; 2: 197e201. 11. Gader AMA, Ghumlas AK, Hussain MF, Al-Haidary AI. Platelet aggregation and platelet function analyser (PFA-100) closure time in camels e a comparative study with humans. Comp Clin Pathol 2006; 15: 31e37. 12. Gader AMA, Bahakim HM, Malaika SS. A study of the normal pattern of platelet aggregation in healthy Saudis: a populationbased study. Platelets 1990; 1: 139e143. 13. Groterneyer KH, Scharafinski HW, Husstedt IW. Two-year follow-up of aspirin responder and aspirin nonresponder. A pilot-study including 180 post-stroke patients. Thromb Res 1993; 71: 397e403.
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How to cite this article: Al Shamiri MQ, Abdel Gader AM, Bayoumy NM. Comparing light transmission aggregometry and PFA100 for monitoring antiplatelet therapy in patients with coronary heart disease. J Taibah Univ Med Sc 2016;11(5):478e484.