Detection and quantification of circulating immature platelets: agreement between flow cytometric and automated detection.

Immature platelets-also termed reticulated platelets (RP)-are platelets newly released into the circulation, and have been associated with a variety of pathological thrombotic events. They can be assessed by flow cytometry after staining with thiazole orange (TO) or by using a module added to a fully automated analyzer that is currently in wide clinical use and expressed as a fraction of the total platelet count (IPF). We sought to assess the correlation and agreement between these two methods. IPF was measured using Sysmex XE 2100-and at the same time point- we used TO staining and flow cytometry to measure RP levels. Two different gates were used for the flow cytometry method, 1 and 0.5 %. Measurements from the automated analyzer were then compared separately to measurements performed using each gate. Agreement between methods was assessed using Bland-Altman method. Pearson's correlation coefficient was also calculated. 129 subjects were enrolled and stratified into 5 groups: (1) Healthy subjects, (2) End stage renal disease, (3) Chronic stable coronary artery disease, (4) Post Coronary artery bypass surgery, (5) Peripheral thrombocytopenia. Median IPF levels were increased for patients in groups 2, 3, 4 and 5 (4.0, 4.7, 4.3, and 8.3 % respectively) compared to healthy subjects (2.5 %) p = 0.0001. Although the observed correlation between the two methods tended to be good in patients with high IPF values (i.e., group 5), the overall observed correlation was poor (Pearson's correlation coefficient r = 0.27). Furthermore, there was poor agreement between the two methods in all groups. Despite the good correlation that was observed between the two methods at higher IPF values, the lack of agreement was significant.

Association of immature platelets with adverse cardiovascular outcomes.

BACKGROUND: Immature platelets are less responsive to the effects of antiplatelet drugs and contain messenger ribonucleic acid that is translationally active. They can be measured easily using an automated hematoanalyzer and reported as part of the complete blood count. OBJECTIVES: The purpose of this study was to determine the prognostic significance of elevated immature platelet count (IPC) in patients with coronary artery disease (CAD). METHODS: In this prospective cohort study in patients with CAD, patients underwent IPC measurement and were then followed up for the composite endpoint of major adverse cardiovascular events (MACE), defined as a composite of all-cause mortality, myocardial infarction, unplanned revascularization, or hospitalization for angina. For the purposes of analysis, patients were stratified into tertiles of IPC. RESULTS: Eighty-nine patients were followed up for a median of 31 months. Stratification to the high IPC tertile was associated with higher rates of MACE compared with the intermediate and low tertiles (60% vs. 24% vs. 16%, respectively; p < 0.001). Time-dependent receiver-operating characteristic analysis revealed that an IPC level ≥7,632 platelets/µl was 70.7% sensitive and 82.1% specific for MACE. After adjustment for age, admission diagnosis, index revascularization, heart failure, smoking, hematocrit, and baseline platelet count, patients with an IPC level ≥7,632 platelets/µl were more likely to experience a MACE (hazard ratio: 4.65; 95% confidence interval: 1.78 to 12.16; p < 0.002). CONCLUSIONS: IPC is a novel biomarker for MACE risk stratification in patients with CAD. Future studies should focus on the utilization of this marker for individualized antiplatelet therapy.

Immature platelet fraction (IPF) determined with an automated method predicts clopidogrel hyporesponsiveness.

The mechanisms for the variability in antiplatelet effects of clopidogrel are not elucidated entirely. Immature (reticulated) platelets may modulate the antiplatelet effects of clopidogrel but must be measured using flow cytometry. Whether new automated detection techniques yield similar results is not known. The objectives of the study to evaluate the role of immature platelets assessed by an automated method in response to the antiplatelet effects of clopidogrel. Twenty-nine healthy volunteers had platelet studies performed before and 1 week after 75 mg daily dosing of clopidogrel. Immature platelet fraction (IPF) was determined using an automated particle counter. Subjects were stratified into tertiles based on the IPF. Platelet studies included light transmission aggregometry (LTA), and vasodilator stimulated phosphoprotein phosphorylation (VASP-P) determined by platelet reactivity index (PRI). Baseline platelet aggregation responses to 2, 5 and 20 µM ADP, were similar in all three tertiles, however they were greater in the upper than in the lower tertile of immature platelets after clopidogrel in response to 5 µM ADP (54% vs. 23%, P = 0.02), with concordant trends for the other two concentrations. PRI was also greater in the upper tertile after clopidogrel (71.2% vs. 57.8%, P = 0.04). The frequency of clopidogrel hyporesponsiveness (aggregation >50% in response to 5 µM of ADP) was also higher in the upper tertile when compared to lower tertile, (60%) versus (10%) respectively (P = 0.02). [corrected]. Immature platelets measured using an automated method, are associated with impaired response to antiplatelet effects of clopidogrel.

Platelet reactivity and response to aspirin in subjects with the metabolic syndrome.

BACKGROUND: Metabolic syndrome (MS) is associated with a prothrombotic state and predicts the subsequent development of type 2 diabetes mellitus. We hypothesized that similar to diabetes, subjects with MS may have increased platelet reactivity, and reduced response to aspirin. We, therefore, compared platelet reactivity and response to aspirin among subjects with MS and healthy volunteers. METHODS: Fifty subjects with MS, defined by Adult Treatment Panel III criteria (age 44+/-9 years, 80% women, body mass index 35+/-8 kg/m2) were compared to 50 healthy controls who met none of the MS criteria (age 40+/-7 years, 80% women, body mass index: 24+/-3 kg/m2). Blood samples were taken before and 24 hours after 325 mg aspirin (single dose). Platelet function was evaluated by aggregation in response to 1.5 mmol/L arachidonic acid, 1 microg/mL collagen, and 5 and 20 micromol/L adenosine diphosphate; the VerifyNow Aspirin assay (Accumetrics Inc, San Diego, CA); Impact-R Cone and Plate(let) Analyzer (shear-dependent test) (DiaMed, Cresier, Switzerland) and flow cytometric determination of P-selectin expression and activated glycoprotein IIb/IIIa expression; and reticulated platelets (reflecting platelet turnover). RESULTS: Subjects with MS had higher baseline P-selectin levels (14.5+/-5 vs 11.3+/-4 mean fluorescence intensity, P=.002), reticulated platelets (2.8%+/-3% vs 1.2%+/-1%, P=.04) and platelet deposition under flow (Impact-R 7.5%+/-2% vs 5.9%+/-2%, P=.003). Subjects with MS also had lower response to aspirin, as evaluated by the change in all platelet aggregation assays and the VerifyNow score. CONCLUSIONS: Subjects with MS appear to have increased baseline platelet reactivity and turnover and a lower antiplatelet response to aspirin. Further research is required to elucidate platelet properties in subjects with MS and find ways to modify them.

Role of reticulated platelets and platelet size heterogeneity on platelet activity after dual antiplatelet therapy with aspirin and clopidogrel in patients with stable coronary artery disease.

OBJECTIVES: The aim of this study was to evaluate the relationship between reticulated platelets (RPs), platelet size, and platelet function in patients with stable coronary artery disease (CAD) taking aspirin and clopidogrel. BACKGROUND: Reticulated platelets are young platelets that are larger and possibly more active than non-RPs. METHODS: Flow cytometry was used to measure RPs after staining with thiazole orange and to define the upper 20% and lower 20% of platelets by size. Platelet aggregation was measured with light transmission aggregometry (LTA); platelet activation was assessed by measuring activated platelet surface expression of P-selectin and glycoprotein (GP) IIb/IIIa. RESULTS: Ninety patients were recruited and stratified into tertiles of %RPs. Patients in the upper tertile displayed greater platelet aggregation to 5-mumol/l adenosine diphosphate (ADP) (50.7 +/- 16.4% vs. 34.2 +/- 17.3%, p < 0.001), 1.5-mmol/l arachidonic acid (AA) (27.3 +/- 16.9% vs. 11.7 +/- 9.3%, p < 0.001), and 1-mug/ml collagen (18 +/- 11.6% vs. 12.1 +/- 8.7%, p < 0.05) and greater expression of GP IIb/IIIa (4.7 +/- 1.8% vs. 3.1 +/- 2.2%, p < 0.001). Frequency of low response to aspirin (AA LTA >20%) was higher in the upper tertile (53% vs. 17%, p < 0.001) compared with the lower tertile; low response to clopidogrel (ADP LTA >50%) was also elevated in the upper tertile (50% vs. 13%, p = 0.003). The larger platelet gate had a higher % of RPs compared with the smaller gate (15.4 +/- 16.7% vs. 1.7 +/- 2.3%, p < 0.001) and greater GP IIb/IIIa (5.7 +/- 3.1 vs. 2.1 +/- 1.2, p < 0.001) and P-selectin expression (7.8 +/- 4.9 vs. 4.6 +/- 2.7, p < 0.001). CONCLUSIONS: The proportion of circulating RPs strongly correlates with response to antiplatelet therapy in patients with stable CAD. Large platelets exhibit increased reactivity despite dual antiplatelet therapy, compared with smaller platelets.

Increased platelet sensitivity among individuals with aspirin resistance - platelet aggregation to submaximal concentration of arachidonic acid predicts response to antiplatelet therapy.

Aspirin 'resistance' (AR) is a phenomenon of uncertain etiology describing decreased platelet inhibition by aspirin. We studied whether (i) platelets in AR demonstrate increased basal sensitivity to a lower degree of stimulation and (ii) platelet aggregation with submaximal stimulation could predict responses to aspirin. Serum thromboxane B(2) (TxB(2)) levels and platelet aggregation with light transmission aggregometry (LTA) were measured at baseline and 24 hours after 325 mg aspirin administration in 58 healthy subjects. AR was defined as the upper sixth of LTA (> or = 12%) to 1.5 mM AA. Baseline platelet aggregation with submaximal concentrations of agonists [ADP 2 microM, arachidonic acid (AA) 0.75 mM, collagen 0.375 and 0.5 microg/ml] was greater in AR subjects compared with non-AR subjects, but not with higher concentrations (ADP 5 microM and 20 microM, AA 1.5 mM and collagen 1 microg/ml). Post-aspirin platelet aggregation was elevated in AR subjects with both submaximal and maximal stimulation. Baseline and post-aspirin serum TxB(2) were higher in AR subjects and decreased further with ex-vivo COX-1 inhibition, suggesting incompletely suppressed COX-1 activity. Pre-aspirin platelet aggregation to 0.75 AA demonstrated a dichotomous response with 29/58 subjects having aggregation < or = 15% and 29/58 subjects having aggregation > or = 75%. In the high aggregation group 28% had AR compared to 6% in the non-AR group (p = 0.04). In conclusion, platelets in AR subjects demonstrate increased basal sensitivity to submaximal stimulation, which could predict responses to antiplatelet therapy.

Effect of ranitidine on the antiplatelet effects of aspirin in healthy human subjects.

Aspirin is often taken with H2-receptor antagonists. In vitro data suggest that certain antagonists, such as ranitidine, have inhibitory effects on platelet function. There are no reports on the combined effect of aspirin and H2-receptor antagonists on platelet function in humans. Therefore, this study's aim was to evaluate the effects of aspirin, ranitidine, and their combination on platelet function in humans. Ten healthy men aged 34.7 +/- 2 years received aspirin 325 mg/day for 4 days followed by a 9-day washout period, 3 days of ranitidine treatment (150 mg twice daily), and 4 days of dual-drug treatment. Blood samples were drawn at baseline and on the last days of aspirin monotherapy, the washout period, ranitidine monotherapy, and dual-drug treatment. Platelet aggregation was measured in response to 0.5 mg/ml arachidonic acid, 5 and 10 mumol/L adenosine diphosphate, and 1 micro g/ml collagen. The Platelet Function Analyzer 100 test was performed, and blood salicylate levels were measured in 6 subjects. Aspirin caused a marked reduction in platelet aggregation and prolongation of Platelet Function Analyzer 100 closure time. Ranitidine caused a modest decrease in platelet aggregation. Unexpectedly, the combination of aspirin and ranitidine caused less inhibition of platelet aggregation and prolongation of Platelet Function Analyzer 100 time than aspirin alone (p = 0.02 to 0.07 compared with aspirin alone). Blood salicylate levels were lower when subjects took aspirin with ranitidine than when they took aspirin alone (1 +/- 0.8 vs 1.6 +/- 0.7 mg/dl, p = 0.005). In conclusion, ranitidine appears to attenuate the antiplatelet effects of aspirin in healthy volunteers. The most likely mechanism for these findings is a change in the absorption conditions of aspirin in the presence of ranitidine.

Platelet reactivity in patients with subacute stent thrombosis compared with non-stent-related acute myocardial infarction.

BACKGROUND: Recent case control studies suggest that patients with subacute stent thrombosis (SAT) have increased platelet reactivity. However, SAT often presents as acute myocardial infarction (AMI), which is also associated with augmented platelet activation. We therefore compared platelet reactivity in patients with SAT and patients with AMI unrelated to stenting. METHODS: We identified 20 patients with SAT, 20 patients with ST-elevation AMI who underwent primary percutaneous coronary intervention (PCI), and 20 patients who underwent stenting without SAT occurrence (stable control group). Platelet function was measured > or = 3 days after PCI in the SAT (repeat procedure) and AMI groups and > or = 3 months after stenting in the stable group. All patients received aspirin and clopidogrel. Platelet reactivity was evaluated by aggregation in response to 5 and 20 micromol/L of adenosine diphosphate and 1.5 mmol/L arachidonic acid, and by flow cytometric determination of P-selectin and glycoprotein IIb/IIIa activation. RESULTS: Clinical characteristics were similar among the groups. Platelet testing was performed 4.9 +/- 1.7, 3.1 +/- 0.3, and 108 +/- 22 days after PCI in the SAT, AMI, and stable groups, respectively. The SAT group had higher platelet aggregation and activation markers than the stable group. However, platelet aggregation and activation was very similar in the SAT and AMI groups. CONCLUSIONS: Patients with SAT have increased platelet reactivity, compared with patients who do not develop SAT following stenting. However, the augmented platelet reactivity does not appear to differ from patients with AMI unrelated to stenting. This study highlights the need for large prospective studies to determine whether platelet hyperreactivity increases the risk of SAT.

Aspirin and clopidogrel drug response in patients undergoing percutaneous coronary intervention: the role of dual drug resistance.

UNLABELLED: We evaluated the response to clopidogrel among aspirin-resistant versus aspirin-sensitive patients undergoing elective coronary stenting. Patients (n = 150) treated with aspirin but not clopidogrel had blood samples drawn at baseline and 24 h after clopidogrel loading. Depending on the definition used, 9% to 15% were resistant to aspirin and 24% to clopidogrel. About half of the aspirin-resistant patients were also resistant to clopidogrel. As a group, aspirin-resistant patients had lower response to clopidogrel (assessed by platelet aggregation and activation markers) than aspirin-sensitive patients. Both aspirin- and clopidogrel-resistant patients had higher incidence of creatine kinase-MB elevation than the respective sensitive patients. OBJECTIVES: We sought to evaluate the response to clopidogrel among aspirin-resistant versus aspirin-sensitive patients undergoing percutaneous coronary intervention (PCI). BACKGROUND: Wide variability has been reported in response to aspirin and clopidogrel. There are limited data on the simultaneous responses to both drugs. METHODS: Elective PCI patients (n = 150) who received aspirin for > or = 1 week but not clopidogrel were included. All patients received bivalirudin during PCI. Blood samples were drawn at baseline and 20 to 24 h after a 300-mg clopidogrel dose. Aspirin resistance was defined by > or = 2 of 3 criteria: rapid platelet function analyzer-ASA score > or = 550, 5 micromol/l adenosine diphosphate (ADP)-induced aggregation > or = 70%, and 0.5 mg/ml arachidonic acid-induced aggregation > or = 20%. Clopidogrel resistance was defined as baseline minus post-treatment aggregation < or = 10% in response to 5 and 20 micromol/l ADP. RESULTS: Nineteen (12.7%) patients were resistant to aspirin and 36 (24%) to clopidogrel. Nine (47.4%) of the aspirin-resistant patients were also clopidogrel resistant. Aspirin-resistant patients were more likely to be women and have diabetes than were aspirin-sensitive patients. They also had lower response to clopidogrel, assessed by platelet aggregation and activation markers (flow cytometry-determined PAC-1 binding and P-selectin expression). Elevation of creatine kinase-myocardial band after stenting occurred more frequently in aspirin-resistant versus aspirin-sensitive patients (38.9% vs. 18.3%; p = 0.04) and in clopidogrel-resistant versus clopidogrel-sensitive patients (32.4% vs. 17.3%; p = 0.06). CONCLUSIONS: Aspirin-resistant patients as a group have reduced response to clopidogrel. Furthermore, we have identified a unique group of dual drug-resistant patients who may be at increased risk for thrombotic complications after PCI.