European Journal of Haematology 94 (138–144)
ORIGINAL ARTICLE
The clinical utility of Multiplate analyser measurement in platelet function testing following stroke and transient ischaemic attack Gurdeep S. Mannu1,2, Alistair Macartney2, Jonathan R. A. Lambert3, Joao H. Bettencourt-Silva2, Mark Lawn2, Hamish Lyall2, Anthony Kneale Metcalf2, John F. Potter2,4, John Wood4, Allan Clark4, Trevor Baglin5, Phyo Kyaw Myint6*, Kristian M. Bowles2,4* 1
Oxford University Hospitals NHS Trust, Oxford; 2Norfolk and Norwich University Hospital, Norfolk; 3UCL Institute of Child Health, London; University of East Anglia, Norwich; 5Addenbrooke’s Hospital, Cambridge; 6University of Aberdeen, Aberdeen, UK
4
Abstract Background: Platelet responsiveness to aspirin in people with cerebrovascular disease is poorly understood. Objectives: To determine: (i) normal reference range, imprecision and reproducibility of the Multiplate instrument in healthy volunteers naive to aspirin; (ii) imprecision and reproducibility of the Multiplate instrument in acute stroke and transient ischaemic attack (TIA); (iii) the relationship between aspirin responsiveness and clinical outcome. Materials and Methods: We evaluated platelet function response to three agonists [Adenosine Diphosphate (ADP), Arachidonic Acid (AA), Collagen (Col)] using the Mulitplate platelet function analyser in a two-phase pilot study. In phase 1, we recruited healthy volunteers to determine the normal reference range and imprecision of the Multiplate instrument. In phase 2, we assessed platelet function in acute stroke or TIA patients presenting to hospital. These patients were bled within 24 h of presentation and between 12 and 24 h after ≥75 mg dose of Aspirin. Patients were followed up to 1 yr to assess mortality and recurrent cardiovascular event. Results: Overall, 29 healthy volunteers and 81 stroke/TIA patients were recruited. On assessing components of variance, Multiplate testing is reproducible and precise in volunteers and stroke/TIA patients. In stroke patients receiving aspirin, Bland–Altman plots show initial day 1 measurement provided a reliable measure of continuing response to aspirin at day 3. We defined one-third of patients as aspirin resistant [31.8% (95% CI: 22.1%– 42.8%)] using cut off mean aggregation of ≥23.08% for AA and mean aggregation of ≥80.76% for ADP. Conclusion: The Multiplate device gives reproducible, precise results in volunteers and stroke/TIA patients. Key words aspirin resistance; platelet function testing; stroke; transient ischaemic attack; prognosis Correspondence Gurdeep S. Mannu, Norfolk and Norwich University Hospital, Colney Lane, Norwich NR4 7UY, UK. Tel: 01603 289979; Fax: 01603 286918; e-mail: [email protected] *These authors contributed equally to this study. Trial registry ID: ISRCTN09053426. Accepted for publication 25 June 2014
Antiplatelet drugs are the mainstay of treatment in acute ischaemic stroke for the majority of patients and for secondary prevention of further vascular events in patients with cerebral infarct or transient ischaemic attack (TIA). However, a large number of patients while on anti-platelet therapy still develop a vascular event, a meta-analysis of 20 studies in cardiovascular disease totalling 2930 patients estimated aspirin ‘resistance’ in ~28% of patients (1). The responsiveness of platelets
138
doi:10.1111/ejh.12406
to aspirin, a commonly used antiplatelet agent, in the stroke/ TIA population is poorly understood (2). Platelet function testing has traditionally been difficult to do with variable results. A new generation of platelet function analysers provide the potential for widespread standardised platelet function analysis in routine clinical practice. The hope is that this type of technology may in future allow the prospective identification of stroke/TIA patients who are resistant to anti-platelet drugs.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mannu et al.
The Multiplate analysis device uses impedance aggregometry to assess platelet function. The results are in the form of area under a time-aggregation curve generated by the machine, which can measure platelet aggregation in response to a number of agonists, including those affected by Aspirin [Adenosine Diphosphate (ADP), Arachidonic Acid (AA) and Collagen (Col)] (3). It is semi-automated and uses whole blood, hence negating the pre-analytical requirement for preparing platelet rich plasma. As a result, it lends itself potentially to widespread use in routine clinical practice but its functionality in the acute stroke/TIA setting has yet to be defined. We installed the Multiplate device into a busy, fully Clinical Pathology Accreditation [CPA - www.cpa-uk.co.uk/] compliant NHS laboratory and aimed to determine the following: (i) the normal reference range, imprecision and reproducibility of the Multiplate instrument in healthy volunteers who are naive to aspirin and compare it to published data, (ii) the imprecision and reproducibility of the Multiplate instrument in acute stroke and TIA patients to establish the optimal interval for analysing platelet function in these patients following aspirin therapy. Finally in the patients presenting with stroke/TIA, we explored (iii) the relationship between in-vitro aspirin responsiveness and clinical outcome during 1-year follow-up. Methods
The analysis method of the Multiplate impedance aggregometer device has been previously described (4). Activated platelets adhere on the surfaces of the sensors of the Multiplate device. As the platelets aggregate an insulation layer is formed and the impedance between the device’s electrodes rises. This process is measured and presented as an aggregation (AU) over time (minutes) graph and the primary output is the area under the curve (AUC). The lower the AUC, the less platelet aggregation has occurred in the assay. This methodology is termed impedance platelet aggregation (IPA). We used the Multiplate device to measure three agonists; Adenosine Diphosphate (ADP), Arachidonic Acid (AA) and Collagen (Col), the effect of which may be related to the risk of thrombosis (5, 6). To address our objectives, the study was conducted in two phases. Phase 1 of the study aimed to establish the reference ranges of these three agonists and to assess the Multiplate device’s precision and reproducibility we recruited healthy volunteers for platelet function testing. Phase 2 aimed to determine the accuracy and reproducibility of the Multiplate instrument in acute stroke/TIA patients who were on long-term aspirin and those who were aspirin naive before receiving anti-platelet therapy on admission to establish the optimal interval for analysing platelet function in these patients. We further aimed to collect patients’ clinical data to assess mortality and cardiovascular events within 1 yr of follow up (Figure S1).
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Norfolk platelet and stroke study
Recruitment
During phase 1, volunteers not taking Aspirin or non-steroidal anti-inflammatory drugs (NSAIDs) were recruited. In phase 2, we recruited patients admitted to the Norfolk and Norwich University Hospital with an acute cerebrovascular event (either TIA or stroke) and having received a dose of Aspirin within the proceeding 12–24 h. Aspirin ingestion was witnessed by a healthcare professional in all cases to ensure compliance. Entry into the study required informed and signed patient consent. Ethical approval was obtained from the Norfolk Ethics Committee (Ref: 09/H0310/92). Inclusion and exclusion criteria
For phase 1 of the study, volunteers were aged over 18 yr old, being excluded if they were (i) unfit for procedure (phlebotomy) or (ii) had taken Aspirin/non-steroidal antiinflammatory drugs or any other agents with anti-platelet effects (such as Clopidogrel, Ticlopidine, Dipyridamole) in the 2 wk prior to testing. For phase 2, the inclusion criteria specified the following: (i) patients over 18 yr old, (ii) admitted to the Acute Stroke Unit at the NNUH with an ischaemic stroke or TIA within 72 h of symptom onset, (iv) must have received 1 dose of at least 75 mg of Aspirin orally 12–24 h before first blood sample. Patients on longterm Aspirin prior to admission were included in phase 2. Patients were excluded if they were (i) unfit for procedure (phlebotomy), or (ii) not given Aspirin (e.g. use of an alternative antiplatelet agent). Sample collection
During phase 1, volunteers had an initial blood test with a single repeat blood test 7 d later; 3 mL of venous blood was taken using collection tubes containing 25 lg/mL hirudin anticoagulant as per the manufacturer’s instructions (Instrumentation Laboratory (UK) Ltd, Rotkreuz, Switzerland). During phase 2 patients presenting with a cerebrovascular event meeting the inclusion criteria had an initial blood test between 12 and 24 h after the first dose of aspirin. As it was unclear as to when the aspirin effect could be detected after initiation of therapy we repeated the platelet function analysis at least 48 h after the initial test which also allowed us to assess test reproducibility in the patients who were still alive and in hospital 2 d after study entry. Phlebotomy was performed by an appropriately trained member of staff. Sample analysis
All samples were analysed for platelet aggregation at 30 and 90 min post-phlebotomy, using the Multiplate impedance aggregometer in accordance with manufacturer’s instructions: 0.3 mL NaCl 0.9%w/v was added to 0.3 mL of
139
Norfolk platelet and stroke study
hirudin blood sample and incubated at 37°C for 3 min; 20 lL platelet agonist was added and aggregation monitored by impedance over 6 min. Platelet response was tested: ADP (0.2 mM); AA (15 mM); Collagen; Collagen (100 lg/mL). In phase 1 volunteers, comparison was made between the 30 and 90 min analysis on day 1 and at the further test on repeat sampling 7 d later. In phase 2, comparison was made between the 30 and 90 min analysis on day 1 and where applicable those with repeat testing at 48 h. Results were presented as AU 9 minutes. On the basis of published literature, we defined aspirin resistance as the higher tertile of our phase II cohort’s AA with an average aggregation above the mean for our cohort (7).
Mannu et al.
Results
Clinical data were also obtained, including age, sex, stroke type and severity [Oxfordshire Community Stroke Project Classification and National Institute of Health Stroke Scale (NIHSS) respectively], medications, ethnicity, past medical history, pre-morbid functional status, assessed by modified Rankin score, and vascular risk factors including hypertension, diabetes, smoking habit, hypercholesterolemia and atrial fibrillation. One-year follow-up was conducted by the research team using patients’ records for incidence of CV events and checked using the Patient Administrative System (PAS) to record mortality as described previously (8). Therefore, we had 100% follow-up.
Thirty-two healthy volunteers were recruited for phase 1, and three subjects were excluded since they were taking an antiplatelet medication. Of the 29 included, complete data were available for twenty-five subjects. Four had one or more agonist reading missing due to either technical device based issues or insufficient blood in the sample. In phase 2, a total of 93 ischaemic stroke patients were recruited. Three patients had no AA data and one patient had no ADP data available. As a result, 89 patients were included in the analysis. Of these, a total of 33 individuals had taken aspirin prior to admission with cerebrovascular event. There were no patients who received an alternative anti-platelet or dual anti-platelet medical on admission. The patient characteristics of this cohort are summarised in Table 1. The height of the area under the aggregation curve (AUC) directly demonstrated platelet aggregation. The mean area under curve (AUC) values phase 1 volunteers for ADP, AA, Collagen agonists at 30 min and at 90 min both on initial blood sample and repeated blood sample 7 d later were 82, 103 and 95 respectively. In order to assess the reproducibility of Multiplate device components of variance (AUC2) within occasions for each agonist were calculated and were ADP: 92, AA: 161 and Collagen: 82 and those between occasions were ADP: 63, AA: 43, Collagen: 33. Components of variance (AUC2) between subjects were ADP: 114, AA: 237, Collagen: 168. The 95% reference ranges in
Data analysis
Table 1 Patient characteristics
For phase 1, we established 95% reference ranges for healthy volunteers for each agonist. The components of variance were estimated using analysis of variance of technical replicate measurements taken one hour apart and repeated 5– 7 d later. For phase 2, the data were subject to analysis of variance, distinguishing between those patients previously on Aspirin and those initiated on Aspirin post-admittance. It was assumed that the patients previously on Aspirin should have readings that are stable between the two occasions of testing (which was verified using a Student’s paired-samples t-test), and so these data can be used to estimate the between-occasion component of variance in this population. Comparison between the means of the volunteer and patient groups, along with consideration of the relative sizes of the between-patient and between-occasion components of variance of the measurements, would allow a preliminary assessment of the potential utility of the Multiplate assay for distinguishing between individual patients in terms of their platelet function under aspirin treatment. We arbitrarily defined aspirin resistance as the highest third of AA and ADP measurements resulting in a mean aggregation of ≥23.08% for AA and a mean aggregation of ≥80.76% for ADP to be consistent with previous literature in the field (7, 9).
Factor
N
Mean (SD)
Age (years) NIHSS
87 45
72.64 (10.37) 3.84 (4.01)
N
%
64 23
73.6 26.4
36 36 13 2
41.4 41.4 14.9 2.3
1 1 21 1 35 17 2
1.3 1.3 26.9 1.3 44.9 21.8 2.6
Clinical data and follow up
140
Type of cerebrovascular event Infarct TIA OCSP classification* LACS PACS POCS TACS Discharge destination Nursing home Rehabilitation Home alone Home with care package Home with family Stroke inpatient rehab Transfer to other hospital
*OCSP, Oxfordshire Community Stroke Project; LACS, lacunar stroke; PACS, partial anterior circulation stroke; POCS, posterior circulation stroke; TACS, total anterior circulation stroke.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mannu et al.
Norfolk platelet and stroke study
healthy volunteers were ADP 49–114, AA 61–114 and Collagen 61–128. These results showed that the Multiplate device produced repeatable and accurate ADP, AA and Collagen readings. Phase 2 results were analysed separately. Bland–Altman plots of the difference between day 1 and day 3 sample results using the different agonists for individuals on longterm aspirin therapy were constructed and analysed for phase 2. The distribution of platelet response on day 1 at the two time points and by agonist is given in Fig. 1 which shows that there is no significant difference between readings taken at 30 min and at 90 min for AA, ADP or Collagen. The change from 30 to 90 min, with a positive value indicating a reduction, is given graphically in Fig. 2A.
There was no significant difference between readings taken at 30 min and at 90 min for AA, ADP or Collagen in acute stroke and TIA patients 12–24 h following aspirin (Fig. 2A). Although ADP and Collagen had slight temporal change in readings, there is no substantial change in agonist measurements over the measured time period, these findings are summarised in Table 2 as the limits of agreement. The distribution of the change in AUC from day 1 to day 3 is summarised graphically in Fig. 2B which shows the difference measured between day 1–30 and day 3–30 min AUC as the difference measured between day 1–90 min AUC and day 3–90 min AUC. It shows there was no significant difference in the readings of each of the three agonists between day 1 and day 3 and in the 30–90 min window post phlebotomy.
Figure 1 Distribution of resistance on day 1, time point (30 or 90 min) and by agonist. Density describes the frequency of individuals per change in resistance. ADP, Adenosine Diphosphate; AA, Arachidonic Acid; Col, Collagen.
A
B Figure 2 Distribution of the change in resistance over time. Panel A (top graph); distribution of the change in resistance from 30 to 90 min. Panel B (bottom graph); distribution of the change in resistance from day 1 to day 3. Density describes the frequency of individuals per change in resistance. ADP, Adenosine Diphosphate; AA, Arachidonic Acid; Col, Collagen.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
141
Norfolk platelet and stroke study
Mannu et al.
Table 2 Summary statistics of resistance by time and by agonist. Summary statistics of resistance on day 1 at 30 and 90 min and change, by agonist with Bland–Altman agreement limits between resistance at 30 and 90 min on day 1. All units for the mean and mean difference are in aggregation (AU) over time (minutes) and are presented as area under the curve (AUC) Agonist AA N
ADP Mean
SD
N
Resistance at 30 min 85 23.15 14.04 84 Resistance at 90 min 81 21.10 11.99 78 Change in resistance from 30 to 90 min 78 2.59 8.60 76 Bland–Altman agreement limits between resistance at 30mins and 90mins on day 1 Mean difference 2.59 18.82 95% limits of agreement 14.61 to 19.79 16.88 to
Event No
Yes (%)
Total
17 15 15 16 15
4 0 1 1 2
21 15 16 17 17
(19) (0) (6) (6) (12)
Where comparing the summary statistics of AUC after aspirin consumption on day 1 and day 3 by agonist, there were mean changes in AUC of 0.29 in AA (SD 15.13, n = 42), +1.43 in ADP (SD 17.44, n = 40), +1.61 in Collagen (SD 21.55, n = 41). However, on comparison of Bland– Altman agreement limits between AUC on day 1 and day 3 it is apparent that these differences were not significant with 95% limits of agreement ranging between AA ( 29.94 to 29.36), ADP ( 32.75 to 35.60) and Collagen ( 40.63 to 43.85). Therefore, initial day 1 measurement provided a reliable measure of a patient’s continuing response to aspirin therapy. During the follow-up period eight adverse events (six cerebrovascular events and two non-CV deaths) occurred. The AUC was split into quintiles as tabulated against if an event occurred within 1 year (Table 3). We used the highest tertile value in our definition of aspirin resistance. Using our definition of aspirin resistance, 27 of the 85 patients recruited were defined as aspirin resistant, giving a prevalence rate of 31.8% (95% CI: 22.1% to 42.8%). Table 3 demonstrates that in this cohort we cannot report or discount an association between the laboratory results and the clinical outcome. A comparison of the characteristics of the aspirin resistant and aspirin non-resistant patients is shown in Table S1. When comparing the phase I cohort to the phase II cohort, the median AA agonist AUC value for the healthy volunteer group was 105.85 compared to 21 for the stroke patients (P-value is
ORIGINAL ARTICLE
The clinical utility of Multiplate analyser measurement in platelet function testing following stroke and transient ischaemic attack Gurdeep S. Mannu1,2, Alistair Macartney2, Jonathan R. A. Lambert3, Joao H. Bettencourt-Silva2, Mark Lawn2, Hamish Lyall2, Anthony Kneale Metcalf2, John F. Potter2,4, John Wood4, Allan Clark4, Trevor Baglin5, Phyo Kyaw Myint6*, Kristian M. Bowles2,4* 1
Oxford University Hospitals NHS Trust, Oxford; 2Norfolk and Norwich University Hospital, Norfolk; 3UCL Institute of Child Health, London; University of East Anglia, Norwich; 5Addenbrooke’s Hospital, Cambridge; 6University of Aberdeen, Aberdeen, UK
4
Abstract Background: Platelet responsiveness to aspirin in people with cerebrovascular disease is poorly understood. Objectives: To determine: (i) normal reference range, imprecision and reproducibility of the Multiplate instrument in healthy volunteers naive to aspirin; (ii) imprecision and reproducibility of the Multiplate instrument in acute stroke and transient ischaemic attack (TIA); (iii) the relationship between aspirin responsiveness and clinical outcome. Materials and Methods: We evaluated platelet function response to three agonists [Adenosine Diphosphate (ADP), Arachidonic Acid (AA), Collagen (Col)] using the Mulitplate platelet function analyser in a two-phase pilot study. In phase 1, we recruited healthy volunteers to determine the normal reference range and imprecision of the Multiplate instrument. In phase 2, we assessed platelet function in acute stroke or TIA patients presenting to hospital. These patients were bled within 24 h of presentation and between 12 and 24 h after ≥75 mg dose of Aspirin. Patients were followed up to 1 yr to assess mortality and recurrent cardiovascular event. Results: Overall, 29 healthy volunteers and 81 stroke/TIA patients were recruited. On assessing components of variance, Multiplate testing is reproducible and precise in volunteers and stroke/TIA patients. In stroke patients receiving aspirin, Bland–Altman plots show initial day 1 measurement provided a reliable measure of continuing response to aspirin at day 3. We defined one-third of patients as aspirin resistant [31.8% (95% CI: 22.1%– 42.8%)] using cut off mean aggregation of ≥23.08% for AA and mean aggregation of ≥80.76% for ADP. Conclusion: The Multiplate device gives reproducible, precise results in volunteers and stroke/TIA patients. Key words aspirin resistance; platelet function testing; stroke; transient ischaemic attack; prognosis Correspondence Gurdeep S. Mannu, Norfolk and Norwich University Hospital, Colney Lane, Norwich NR4 7UY, UK. Tel: 01603 289979; Fax: 01603 286918; e-mail: [email protected] *These authors contributed equally to this study. Trial registry ID: ISRCTN09053426. Accepted for publication 25 June 2014
Antiplatelet drugs are the mainstay of treatment in acute ischaemic stroke for the majority of patients and for secondary prevention of further vascular events in patients with cerebral infarct or transient ischaemic attack (TIA). However, a large number of patients while on anti-platelet therapy still develop a vascular event, a meta-analysis of 20 studies in cardiovascular disease totalling 2930 patients estimated aspirin ‘resistance’ in ~28% of patients (1). The responsiveness of platelets
138
doi:10.1111/ejh.12406
to aspirin, a commonly used antiplatelet agent, in the stroke/ TIA population is poorly understood (2). Platelet function testing has traditionally been difficult to do with variable results. A new generation of platelet function analysers provide the potential for widespread standardised platelet function analysis in routine clinical practice. The hope is that this type of technology may in future allow the prospective identification of stroke/TIA patients who are resistant to anti-platelet drugs.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mannu et al.
The Multiplate analysis device uses impedance aggregometry to assess platelet function. The results are in the form of area under a time-aggregation curve generated by the machine, which can measure platelet aggregation in response to a number of agonists, including those affected by Aspirin [Adenosine Diphosphate (ADP), Arachidonic Acid (AA) and Collagen (Col)] (3). It is semi-automated and uses whole blood, hence negating the pre-analytical requirement for preparing platelet rich plasma. As a result, it lends itself potentially to widespread use in routine clinical practice but its functionality in the acute stroke/TIA setting has yet to be defined. We installed the Multiplate device into a busy, fully Clinical Pathology Accreditation [CPA - www.cpa-uk.co.uk/] compliant NHS laboratory and aimed to determine the following: (i) the normal reference range, imprecision and reproducibility of the Multiplate instrument in healthy volunteers who are naive to aspirin and compare it to published data, (ii) the imprecision and reproducibility of the Multiplate instrument in acute stroke and TIA patients to establish the optimal interval for analysing platelet function in these patients following aspirin therapy. Finally in the patients presenting with stroke/TIA, we explored (iii) the relationship between in-vitro aspirin responsiveness and clinical outcome during 1-year follow-up. Methods
The analysis method of the Multiplate impedance aggregometer device has been previously described (4). Activated platelets adhere on the surfaces of the sensors of the Multiplate device. As the platelets aggregate an insulation layer is formed and the impedance between the device’s electrodes rises. This process is measured and presented as an aggregation (AU) over time (minutes) graph and the primary output is the area under the curve (AUC). The lower the AUC, the less platelet aggregation has occurred in the assay. This methodology is termed impedance platelet aggregation (IPA). We used the Multiplate device to measure three agonists; Adenosine Diphosphate (ADP), Arachidonic Acid (AA) and Collagen (Col), the effect of which may be related to the risk of thrombosis (5, 6). To address our objectives, the study was conducted in two phases. Phase 1 of the study aimed to establish the reference ranges of these three agonists and to assess the Multiplate device’s precision and reproducibility we recruited healthy volunteers for platelet function testing. Phase 2 aimed to determine the accuracy and reproducibility of the Multiplate instrument in acute stroke/TIA patients who were on long-term aspirin and those who were aspirin naive before receiving anti-platelet therapy on admission to establish the optimal interval for analysing platelet function in these patients. We further aimed to collect patients’ clinical data to assess mortality and cardiovascular events within 1 yr of follow up (Figure S1).
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Norfolk platelet and stroke study
Recruitment
During phase 1, volunteers not taking Aspirin or non-steroidal anti-inflammatory drugs (NSAIDs) were recruited. In phase 2, we recruited patients admitted to the Norfolk and Norwich University Hospital with an acute cerebrovascular event (either TIA or stroke) and having received a dose of Aspirin within the proceeding 12–24 h. Aspirin ingestion was witnessed by a healthcare professional in all cases to ensure compliance. Entry into the study required informed and signed patient consent. Ethical approval was obtained from the Norfolk Ethics Committee (Ref: 09/H0310/92). Inclusion and exclusion criteria
For phase 1 of the study, volunteers were aged over 18 yr old, being excluded if they were (i) unfit for procedure (phlebotomy) or (ii) had taken Aspirin/non-steroidal antiinflammatory drugs or any other agents with anti-platelet effects (such as Clopidogrel, Ticlopidine, Dipyridamole) in the 2 wk prior to testing. For phase 2, the inclusion criteria specified the following: (i) patients over 18 yr old, (ii) admitted to the Acute Stroke Unit at the NNUH with an ischaemic stroke or TIA within 72 h of symptom onset, (iv) must have received 1 dose of at least 75 mg of Aspirin orally 12–24 h before first blood sample. Patients on longterm Aspirin prior to admission were included in phase 2. Patients were excluded if they were (i) unfit for procedure (phlebotomy), or (ii) not given Aspirin (e.g. use of an alternative antiplatelet agent). Sample collection
During phase 1, volunteers had an initial blood test with a single repeat blood test 7 d later; 3 mL of venous blood was taken using collection tubes containing 25 lg/mL hirudin anticoagulant as per the manufacturer’s instructions (Instrumentation Laboratory (UK) Ltd, Rotkreuz, Switzerland). During phase 2 patients presenting with a cerebrovascular event meeting the inclusion criteria had an initial blood test between 12 and 24 h after the first dose of aspirin. As it was unclear as to when the aspirin effect could be detected after initiation of therapy we repeated the platelet function analysis at least 48 h after the initial test which also allowed us to assess test reproducibility in the patients who were still alive and in hospital 2 d after study entry. Phlebotomy was performed by an appropriately trained member of staff. Sample analysis
All samples were analysed for platelet aggregation at 30 and 90 min post-phlebotomy, using the Multiplate impedance aggregometer in accordance with manufacturer’s instructions: 0.3 mL NaCl 0.9%w/v was added to 0.3 mL of
139
Norfolk platelet and stroke study
hirudin blood sample and incubated at 37°C for 3 min; 20 lL platelet agonist was added and aggregation monitored by impedance over 6 min. Platelet response was tested: ADP (0.2 mM); AA (15 mM); Collagen; Collagen (100 lg/mL). In phase 1 volunteers, comparison was made between the 30 and 90 min analysis on day 1 and at the further test on repeat sampling 7 d later. In phase 2, comparison was made between the 30 and 90 min analysis on day 1 and where applicable those with repeat testing at 48 h. Results were presented as AU 9 minutes. On the basis of published literature, we defined aspirin resistance as the higher tertile of our phase II cohort’s AA with an average aggregation above the mean for our cohort (7).
Mannu et al.
Results
Clinical data were also obtained, including age, sex, stroke type and severity [Oxfordshire Community Stroke Project Classification and National Institute of Health Stroke Scale (NIHSS) respectively], medications, ethnicity, past medical history, pre-morbid functional status, assessed by modified Rankin score, and vascular risk factors including hypertension, diabetes, smoking habit, hypercholesterolemia and atrial fibrillation. One-year follow-up was conducted by the research team using patients’ records for incidence of CV events and checked using the Patient Administrative System (PAS) to record mortality as described previously (8). Therefore, we had 100% follow-up.
Thirty-two healthy volunteers were recruited for phase 1, and three subjects were excluded since they were taking an antiplatelet medication. Of the 29 included, complete data were available for twenty-five subjects. Four had one or more agonist reading missing due to either technical device based issues or insufficient blood in the sample. In phase 2, a total of 93 ischaemic stroke patients were recruited. Three patients had no AA data and one patient had no ADP data available. As a result, 89 patients were included in the analysis. Of these, a total of 33 individuals had taken aspirin prior to admission with cerebrovascular event. There were no patients who received an alternative anti-platelet or dual anti-platelet medical on admission. The patient characteristics of this cohort are summarised in Table 1. The height of the area under the aggregation curve (AUC) directly demonstrated platelet aggregation. The mean area under curve (AUC) values phase 1 volunteers for ADP, AA, Collagen agonists at 30 min and at 90 min both on initial blood sample and repeated blood sample 7 d later were 82, 103 and 95 respectively. In order to assess the reproducibility of Multiplate device components of variance (AUC2) within occasions for each agonist were calculated and were ADP: 92, AA: 161 and Collagen: 82 and those between occasions were ADP: 63, AA: 43, Collagen: 33. Components of variance (AUC2) between subjects were ADP: 114, AA: 237, Collagen: 168. The 95% reference ranges in
Data analysis
Table 1 Patient characteristics
For phase 1, we established 95% reference ranges for healthy volunteers for each agonist. The components of variance were estimated using analysis of variance of technical replicate measurements taken one hour apart and repeated 5– 7 d later. For phase 2, the data were subject to analysis of variance, distinguishing between those patients previously on Aspirin and those initiated on Aspirin post-admittance. It was assumed that the patients previously on Aspirin should have readings that are stable between the two occasions of testing (which was verified using a Student’s paired-samples t-test), and so these data can be used to estimate the between-occasion component of variance in this population. Comparison between the means of the volunteer and patient groups, along with consideration of the relative sizes of the between-patient and between-occasion components of variance of the measurements, would allow a preliminary assessment of the potential utility of the Multiplate assay for distinguishing between individual patients in terms of their platelet function under aspirin treatment. We arbitrarily defined aspirin resistance as the highest third of AA and ADP measurements resulting in a mean aggregation of ≥23.08% for AA and a mean aggregation of ≥80.76% for ADP to be consistent with previous literature in the field (7, 9).
Factor
N
Mean (SD)
Age (years) NIHSS
87 45
72.64 (10.37) 3.84 (4.01)
N
%
64 23
73.6 26.4
36 36 13 2
41.4 41.4 14.9 2.3
1 1 21 1 35 17 2
1.3 1.3 26.9 1.3 44.9 21.8 2.6
Clinical data and follow up
140
Type of cerebrovascular event Infarct TIA OCSP classification* LACS PACS POCS TACS Discharge destination Nursing home Rehabilitation Home alone Home with care package Home with family Stroke inpatient rehab Transfer to other hospital
*OCSP, Oxfordshire Community Stroke Project; LACS, lacunar stroke; PACS, partial anterior circulation stroke; POCS, posterior circulation stroke; TACS, total anterior circulation stroke.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mannu et al.
Norfolk platelet and stroke study
healthy volunteers were ADP 49–114, AA 61–114 and Collagen 61–128. These results showed that the Multiplate device produced repeatable and accurate ADP, AA and Collagen readings. Phase 2 results were analysed separately. Bland–Altman plots of the difference between day 1 and day 3 sample results using the different agonists for individuals on longterm aspirin therapy were constructed and analysed for phase 2. The distribution of platelet response on day 1 at the two time points and by agonist is given in Fig. 1 which shows that there is no significant difference between readings taken at 30 min and at 90 min for AA, ADP or Collagen. The change from 30 to 90 min, with a positive value indicating a reduction, is given graphically in Fig. 2A.
There was no significant difference between readings taken at 30 min and at 90 min for AA, ADP or Collagen in acute stroke and TIA patients 12–24 h following aspirin (Fig. 2A). Although ADP and Collagen had slight temporal change in readings, there is no substantial change in agonist measurements over the measured time period, these findings are summarised in Table 2 as the limits of agreement. The distribution of the change in AUC from day 1 to day 3 is summarised graphically in Fig. 2B which shows the difference measured between day 1–30 and day 3–30 min AUC as the difference measured between day 1–90 min AUC and day 3–90 min AUC. It shows there was no significant difference in the readings of each of the three agonists between day 1 and day 3 and in the 30–90 min window post phlebotomy.
Figure 1 Distribution of resistance on day 1, time point (30 or 90 min) and by agonist. Density describes the frequency of individuals per change in resistance. ADP, Adenosine Diphosphate; AA, Arachidonic Acid; Col, Collagen.
A
B Figure 2 Distribution of the change in resistance over time. Panel A (top graph); distribution of the change in resistance from 30 to 90 min. Panel B (bottom graph); distribution of the change in resistance from day 1 to day 3. Density describes the frequency of individuals per change in resistance. ADP, Adenosine Diphosphate; AA, Arachidonic Acid; Col, Collagen.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
141
Norfolk platelet and stroke study
Mannu et al.
Table 2 Summary statistics of resistance by time and by agonist. Summary statistics of resistance on day 1 at 30 and 90 min and change, by agonist with Bland–Altman agreement limits between resistance at 30 and 90 min on day 1. All units for the mean and mean difference are in aggregation (AU) over time (minutes) and are presented as area under the curve (AUC) Agonist AA N
ADP Mean
SD
N
Resistance at 30 min 85 23.15 14.04 84 Resistance at 90 min 81 21.10 11.99 78 Change in resistance from 30 to 90 min 78 2.59 8.60 76 Bland–Altman agreement limits between resistance at 30mins and 90mins on day 1 Mean difference 2.59 18.82 95% limits of agreement 14.61 to 19.79 16.88 to
Event No
Yes (%)
Total
17 15 15 16 15
4 0 1 1 2
21 15 16 17 17
(19) (0) (6) (6) (12)
Where comparing the summary statistics of AUC after aspirin consumption on day 1 and day 3 by agonist, there were mean changes in AUC of 0.29 in AA (SD 15.13, n = 42), +1.43 in ADP (SD 17.44, n = 40), +1.61 in Collagen (SD 21.55, n = 41). However, on comparison of Bland– Altman agreement limits between AUC on day 1 and day 3 it is apparent that these differences were not significant with 95% limits of agreement ranging between AA ( 29.94 to 29.36), ADP ( 32.75 to 35.60) and Collagen ( 40.63 to 43.85). Therefore, initial day 1 measurement provided a reliable measure of a patient’s continuing response to aspirin therapy. During the follow-up period eight adverse events (six cerebrovascular events and two non-CV deaths) occurred. The AUC was split into quintiles as tabulated against if an event occurred within 1 year (Table 3). We used the highest tertile value in our definition of aspirin resistance. Using our definition of aspirin resistance, 27 of the 85 patients recruited were defined as aspirin resistant, giving a prevalence rate of 31.8% (95% CI: 22.1% to 42.8%). Table 3 demonstrates that in this cohort we cannot report or discount an association between the laboratory results and the clinical outcome. A comparison of the characteristics of the aspirin resistant and aspirin non-resistant patients is shown in Table S1. When comparing the phase I cohort to the phase II cohort, the median AA agonist AUC value for the healthy volunteer group was 105.85 compared to 21 for the stroke patients (P-value is
