Flow Cytometry Based Platelet Reactivity Testing to Predict the Occurrence of Per-operative Solid Microemboli During Carotid Endarterectomy.

OBJECTIVE: Peri-operative antiplatelet therapy (APT) aims to prevent thrombotic events such as stroke. High platelet reactivity ,despite the use use of APT, increases the risk of thrombotic events. Transcranial Doppler imaging (TCD) is used to detect peri-operative microembolic signals (MES) during carotid endarterectomy (CEA). Peri-operative MES are associated with an increased risk of procedural stroke and new silent lesions on diffusion weighted magnetic resonance imaging following surgery. The main components of TCD detected MES are platelet aggregates, and therefore patients displaying multiple MES during surgery could have benefited from more stringent APT. This study investigated whether the use of flow cytometry based platelet reactivity measurements were correlated with the incidence of pre-operative MES and thereby in the future suitable to predict patients at increased risk of peri-operative thrombotic events. METHODS: Bilateral TCD with MES detection was performed in 197 patients undergoing CEA. Platelet reactivity was assessed with a flow cytometry based platelet reactivity assay measuring platelet response in whole blood. High on treatment platelet reactivity status was assessed for all patients. The secondary outcome was major adverse cardiovascular events (MACE) within one year. RESULTS: In total, 197 patients were included, 49 had peri-operative MES. The platelet response to adenosine diphosphate (ADP) correlated with MES (p = .021), and high on treatment platelet reactivity after adenosine diphosphate stimulation was associated with MACE (OR 2.34, 95% confidence interval 1.126 - 4.890, p = .023). CONCLUSION: Pre-operative platelet reactivity determined by flow cytometry after ADP stimulation correlated with the occurrence of intra-operative MES and post-operative MACE. Clopidogrel treatment showed the most substantial effect on reducing MES frequency and platelet reactivity measured by flow cytometry.

Reticulated Platelets as Predictor of Myocardial Injury and 30 Day Mortality After Non-cardiac Surgery.

OBJECTIVE: A pre-operative marker for identification of patients at risk of peri-operative adverse events and 30 day mortality might be the percentage of young, reticulated platelets (pRP). This study aimed to determine the predictive value of pre-operative pRP on post-operative myocardial injury (PMI) and 30 day mortality, in patients aged ≥ 60 years undergoing moderate to high risk non-cardiac surgery. METHODS: The incidence of PMI (troponin I > 0.06 µg/L) and 30 day mortality was compared for patients with normal and high pRP (≥2.82%) obtained from The Utrecht Patient Orientated Database. The predictive pRP value was assessed using logistic regression. A prediction model for PMI or 30 day mortality with known risk factors was compared with a model including increased pRP using the area under the receiving operator characteristics curve (AUROC). RESULTS: In total, 26.5% (607/2289) patients showed pre-operative increased pRP. Increased pRP was associated with more PMI and 30 day mortality compared with normal pRP (36.1% vs. 28.3%, p < .001 and 8.6% vs. 3.6%, p < .001). The median pRP was higher in patients suffering PMI and 30 day mortality compared with not (2.21 [IQR: 1.57-3.11] vs. 2.07 [IQR: 1.52-1.78], p = .002, and 2.63 [IQR: 1.76-4.15] vs. 2.09 [IQR: 1.52-3.98], p < .001). pRP was independently related to PMI (OR: 1.28 [95% CI: 1.04-1.59], p = .02) and 30 day mortality (OR: 2.35 [95% CI: 1.56-3.55], p < .001). Adding increased pRP to the predictive model of PMI or 30 day mortality did not increase the AUROC 0.71 vs. 0.72, and 0.80 vs. 0.81. CONCLUSION: In patients undergoing major non-cardiac surgery, increased pre-operative pRP is related to 30 day mortality and PMI.

Validation of the Automated Electronic Microemboli Detection System in Patients Undergoing Carotid Endarterectomy.

PURPOSE: To assess the diagnostic value of automatic embolus detection software (AEDS) in transcranial Doppler (TCD) monitoring for the detection of solid microemboli in patients at risk for perioperative stroke during carotid endarterectomy (CEA). MATERIALS AND METHODS: In 50 patients undergoing CEA, perioperative TCD registration was recorded. All recorded events, identified and saved by the AEDS, were analyzed off-line doubly by two human experts (HEs) within a time frame of > 4 months. The inter- and intraobserver variability was assessed. The overall agreement with the HEs, the sensitivity, specificity, negative and positive predictive values (NPV and PPV) of the AEDS were computed for different cut-offs (patient displaying perioperative 5, 10, 20, 25, or 50 microemboli). RESULTS: 77 233 events were analyzed. The inter- and intraobserver variability was good (min κ = 0.72, max κ = 0.79). AEDS and the HEs identified 760 and 470 solid emboli, respectively. The agreement between AEDS and the HEs for solid emboli detection was poor (κ = 0.24, SE = 0.016). The specificity and NPV were high (99.2 % and 99.6 %) but the sensitivity and PPV were low (30.6 % and 19.8 %). Applying a threshold of > 20 microemboli resulted in the best sensitivity (100.0 %), specificity (84.4 %), PPV (42.7 %), NPV (100.0 %) and area under the curve (0.898). However, 58.3 % of the patients were false positive as classified by AEDS. CONCLUSION: In this validation cohort, AEDS has insufficient agreement with HEs in the identification of solid emboli. AEDS and HEs disagree with respect to the identification of specific patients at risk. Therefore, AEDS cannot be used as a standalone system to identify patients at risk for perioperative stroke during CEA.

The effect of P2Y12 inhibition on platelet activation assessed with aggregation- and flow cytometry-based assays.

Patients on P2Y12 inhibitors may still develop thrombosis or bleeding complications. Tailored antiplatelet therapy, based on platelet reactivity testing, might reduce these complications. Several tests have been used, but failed to show a benefit of tailored antiplatelet therapy. This could be due to the narrowness of current platelet reactivity tests, which are limited to analysis of platelet aggregation after stimulation of the adenosine diphosphate (ADP)-pathway. However, the response to ADP does not necessarily reflect the effect of P2Y12 inhibition on platelet function in vivo. Therefore, we investigated whether measuring platelet reactivity toward other physiologically relevant agonists could provide more insight in the efficacy of P2Y12 inhibitors. The effect of in vitro and in vivo P2Y12 inhibition on αIIbß3-activation, P-selectin and CD63-expression, aggregate formation, release of alpha, and dense granules content was assessed after stimulation of different platelet activation pathways. Platelet reactivity measured with flow cytometry in 72 patients on P2Y12 inhibitors was compared to VerifyNow results. P2Y12 inhibitors caused strongly attenuated platelet fibrinogen binding after stimulation with peptide agonists for protease activated receptor (PAR)-1 and -4, or glycoprotein VI ligand crosslinked collagen-related peptide (CRP-xl), while aggregation was normal at high agonist concentration. P2Y12 inhibitors decreased PAR-agonist and CRP-induced dense granule secretion, but not alpha granule secretion. A proportion of P2Y12-inhibitor responsive patients according to VerifyNow, displayed normal fibrinogen binding assessed with flow cytometry after stimulation with PAR-agonists or CRP despite full inhibition of the response to ADP, indicating suboptimal platelet inhibition. Concluding, measurement of platelet fibrinogen binding with flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response identifies different patients as nonresponders to P2Y12 inhibitors, compared to only ADP-induced aggregation-based assays. Future studies should investigate the value of both assays for monitoring on-treatment platelet reactivity.

Clinical Risk Factors and Plaque Characteristics Associated with New Development of Contralateral Stenosis in Patients Undergoing Carotid Endarterectomy.

BACKGROUND: Following carotid endarterectomy (CEA), cerebrovascular hemodynamic may be hampered by ipsilateral restenosis or development of contralateral stenosis. It remains to be clarified if these patients need follow-up for identifying development of contralateral stenosis. Identification of risk factors contributing to development of contralateral stenosis could allow more specific follow-up. In this current study, we assessed clinical risk factors and plaque characteristics of patients undergoing CEA with development of new contralateral stenosis during mid-term follow-up. METHODS: Seven hundred and sixty patients undergoing CEA between 2003 and 2011 at UMC Utrecht were included. Atherosclerotic plaques were excised and analyzed for smooth muscle cells (SMCs), collagen, macrophages, lipid core, plaque hemorrhage and vessel density. Patients underwent clinical and duplex ultrasound follow-up at 3 and 12 months and yearly thereafter. Association between plaque- and patient characteristics with development of contralateral stenosis ≥50% was assessed with univariate and multivariate analysis. Clinical outcome during follow-up was associated with development of new contralateral stenosis. RESULTS: After a median follow-up time of 2.5 years, development of contralateral stenosis was observed in 108 patients (20%). Presence of high collagen (p = 0.025) and high SMC (p = 0.027) was associated with development of new contralateral stenosis, whereas large lipid core was negatively associated with new development of contralateral stenosis (p = 0.034). The same plaque characteristics were related to contralateral occlusion. History of coronary artery disease (p = 0.031) and asymptomatic presentation (p = 0.000) were univariably associated with development of contralateral stenosis. Multiple regression analysis indicated that asymptomatic status was independently associated with contralateral stenosis (p = 0.001). Patients with new development of contralateral stenosis more often showed symptoms during follow-up (p = 0.049). CONCLUSION: Dissection of a lipid-poor, collagen-rich or SMC-rich plaque yielded an association with development of new contralateral stenosis during mid-term follow-up after CEA. Asymptomatic patients had a significantly higher risk for development of contralateral stenosis. New contralateral stenosis was related to the presence of new cerebral symptoms. These findings may help to develop individual treatment algorithms for patients with cerebrovascular atherosclerotic burden.

The use of platelet reactivity testing in patients on antiplatelet therapy for prediction of bleeding events after cardiac surgery.

Many patients are treated with platelet inhibitors such as aspirin and clopidogrel for prevention of thrombotic cardiovascular events. However, the inhibitory effect of antiplatelet therapy is variable between patients; in some, the platelets are hardly inhibited, while in others, the platelets are excessively inhibited. The newer and more potent platelet inhibitors, prasugrel and ticagrelor, often lead to low platelet reactivity, which potentially leads to bleeding events. Preoperative measurement of platelet reactivity in patients receiving platelet inhibitors who undergo cardiac surgery, could be useful to identify those with low platelet reactivity and thus have an increased risk of bleeding during or after surgery. In this review, we discuss the most commonly used platelet inhibitors and platelet function tests. Furthermore, we will provide an overview of the evidence for the prediction of post-operative bleeding at the operation site with preoperative platelet reactivity testing in patients undergoing cardiac surgery.

Lower Platelet Reactivity Is Associated with Presentation of Unstable Coronary Artery Disease.

In patients with acute coronary syndrome, high platelet reactivity (PR) is associated with an increased risk of secondary thrombotic events. However, in patients undergoing elective percutaneous coronary intervention (PCI), no association between high PR and outcome has been demonstrated. At present, the relation of PR and clinical symptoms is unknown. To examine the association of PR with clinical indication for diagnostic angiography (stable or unstable coronary artery disease [CAD]), taking into account the influence of P2Y12 inhibitors. A platelet function score (PFS) was determined in 195 patients by quantifying fibrinogen binding and P-selectin expression with flow cytometry. We evaluated the PFS with clinical presentation of stable or unstable CAD, angiographic severity of CAD, and the incidence of cardiovascular events during 2 years of follow-up. All data were analyzed stratified by P2Y12 inhibitor use (long-term and preprocedural versus none). Surprisingly, among non-P2Y12 inhibitor users, the PFS was lower in patients with unstable CAD compared with stable CAD (5.6 ± 1.8 vs. 7.4 ± 1.6; p = 0.001). Angiographic CAD severity showed no relation with PFS. The SYNTAX score tended to be inversely related with PFS: low PFS, 13.2 (IQR, 11.9-19.1); median PFS, 10.0 (IQR, 5.0-14.0); and high PFS, 8.0 (IQR, 5.0-13.0), without significance (p = 0.304). Patients with low PFSs required more re-PCIs than those with median and high PFSs (11.1 vs. 4.7 vs. 0.0%, p = 0.004). This association was modified for patients using P2Y12 inhibitors. Among patients without P2Y12 inhibitors undergoing coronary angiography, presentation of unstable CAD is independently associated with lower PR.