Assessing the effect of PRP addition to facial micro-lipofilling for patients suffering from Scleroderma: A prospective routine care analysis.

BACKGROUND: Combining fat graft with platelet derived products is now common practice in regenerative surgery. We proposed to assess the safety and efficacy of Platelet-Rich Plasma (PRP) addition to a micro-lipofilling protocol for facial treatment of patients suffering from Systemic Sclerosis (SSc). OBJECTIVE: Main objective was to evaluate the improvement of the Mouth Handicap In Systemic Sclerosis (MHISS) scale score at 6 months post-therapy. METHOD: Included SSc patients had a MHISS score equal or up to 20. Surgery was performed under general anesthesia. Micro-fat and PRP (CCA-NA from DEPA Classification) were mixed in a 70/30 ratio, before injection in peri-oral sites according to a specific protocol. Efficacy criteria were recorded at baseline, 3 and 6 months. Moreover, we compared this cohort (current study) to a former (2015) non-enriched micro-lipofilling cohort in the same indication, using the same protocol. RESULTS: Thirteen women patients with mean age of 53.2 years (±14.3) have been included. At baseline, mean MHISS score was 29.5 (±8.7) and significantly decreased to 22.5 (±7.8) at 6 months (P=0.016), corresponding to a 22.0% of improvement from baseline, with a mean decrease of 6.5 points (±7.5) at 6 months. Patients received a mean volume of 30.8ml PRP-micro-fat (±8.1ml). CONCLUSION: PRP addition appeared beneficial, however, controlled studies are required to determine its superiority to facial micro-lipofilling.

Prototyping Trastuzumab Docetaxel Immunoliposomes with a New FCM-Based Method to Quantify Optimal Antibody Density on Nanoparticles.

Developing targeted nanoparticles is a rising strategy to improve drug delivery in oncology. Antibodies are the most commonly used targeting agents. However, determination of their optimal number at the surface remains a challenging issue, mainly due to the difficulties in measuring precisely surface coating levels when prototyping nanoparticles. We developed an original quantitative assay to measure the exact number of coated antibodies per nanoparticle. Using flow cytometry optimized for submicron particle analysis and beads covered with known amounts of human IgG-kappa mimicking various amounts of antibodies, this new method was tested as part of the prototyping of docetaxel liposomes coated with trastuzumab against Her2+ breast cancer. This quantification method allowed to discriminate various batches of immunoliposomes depending on their trastuzumab density on nanoparticle surface (i.e., 330 (Immunoliposome-1), 480 (Immunoliposome-2) and 690 (Immunoliposome-3), p = 0.004, One-way ANOVA). Here we showed that optimal number of grafted antibodies on nanoparticles should be finely tuned and highest density of targeting agent is not necessarily associated with highest efficacy. Overall, this new method should help to better prototype third generation nanoparticles.

Endothelial dysfunction in individuals born after fetal growth restriction: cardiovascular and renal consequences and preventive approaches.

Individuals born after intrauterine growth restriction (IUGR) have an increased risk of perinatal morbidity/mortality, and those who survive face long-term consequences such as cardiovascular-related diseases, including systemic hypertension, atherosclerosis, coronary heart disease and chronic kidney disease. In addition to the demonstrated long-term effects of decreased nephron endowment and hyperactivity of the hypothalamic-pituitary-adrenal axis, individuals born after IUGR also exhibit early alterations in vascular structure and function, which have been identified as key factors of the development of cardiovascular-related diseases. The endothelium plays a major role in maintaining vascular function and homeostasis. Therefore, it is not surprising that impaired endothelial function can lead to the long-term development of vascular-related diseases. Endothelial dysfunction, particularly impaired endothelium-dependent vasodilation and vascular remodeling, involves decreased nitric oxide (NO) bioavailability, impaired endothelial NO synthase functionality, increased oxidative stress, endothelial progenitor cells dysfunction and accelerated vascular senescence. Preventive approaches such as breastfeeding, supplementation with folate, vitamins, antioxidants, L-citrulline, L-arginine and treatment with NO modulators represent promising strategies for improving endothelial function, mitigating long-term outcomes and possibly preventing IUGR of vascular origin. Moreover, the identification of early biomarkers of endothelial dysfunction, especially epigenetic biomarkers, could allow early screening and follow-up of individuals at risk of developing cardiovascular and renal diseases, thus contributing to the development of preventive and therapeutic strategies to avert the long-term effects of endothelial dysfunction in infants born after IUGR.

Long-term follow-up after autologous adipose-derived stromal vascular fraction injection into fingers in systemic sclerosis patients.

INTRODUCTION: Hand involvement confers a substantial handicap in work and daily activities in patients with Systemic sclerosis (SSc). Autologous adipose-derived stromal vascular fraction is as an easily accessible source of cells with regenerative effects. We previously performed a phase I open-label clinical trial (NTC01813279) assessing the safety of subcutaneous injection of autologous adipose-derived stromal vascular fraction. Six and 12-month data have been reported. As patients were followed in our medical centre, we report their longer-term outcome beyond the end of the trial. PATIENTS AND METHOD: Twelve females, mean age 54.5±10.3 years, initially enrolled in the clinical trial were assessed during a scheduled medical care, which took place between 22 and 30months after treatment. RESULTS: Multiple patient-reported outcomes showed sustained improvement, in comparison with the assessment performed just before surgery: 62.5% in the Cochin Hand Function Scale, 51.1% in the Scleroderma Health Assessment Questionnaire, 33.1% in hand pain, and 88.3% in the Raynaud Condition Score. A decrease in the number of digital ulcers number was noted. Mobility, strength and fibrosis of the hand also showed improvement. None of the 8 patients who had previously received iloprost infusion required new infusion. CONCLUSION: Despite the limits of an open label study, the data are in favour of the long-term safety of the adipose-derived stromal vascular fraction injection. Two randomized double blind, placebo-controlled trials of this therapeutic agent are ongoing in the USA (NCT02396238) and in France (NCT02558543) and will help determine the place of this innovative therapy for SSc patients.

Increased mean corpuscular haemoglobin concentration: artefact or pathological condition?

INTRODUCTION: In daily practice in haematology laboratories, spurious increased MCHC induces an analytical alarm and needs prompt corrective action to ensure delivery of the right results to the clinicians. The aim of this study was to establish a 'decision tree' using the new parameters red blood cells (RBC-O) and haemoglobin (HGB-O) from the Sysmex XN-10 RET obtained by flow cytometry to deliver appropriate results. METHODS: From 128 unknown patients with MCHC > 365 g/L, all erythrocyte parameters including reticulocyte parameters were measured and analysed in parallel with blood smears, chemistry index and osmolarity. Differences between optical parameters (RBC-O, HGB-O) and usual parameters (RBC, HGB) obtained by impedance and photometry were reported also. RESULTS: Four groups were defined from observations: -RBC agglutination (n = 22); -optical interference (n = 17); -RBC disease (n = 18); and -others (n = 71). The use of RBC-O and HGB-O permitted efficient correction of the abnormalities when RBC agglutination and/or optical interference were present in 36 of 39 patients. Reticulocyte parameters permitted to elaborate an RBC score that allowed a highly sensitive detection of RBC disease patients (17/18). CONCLUSION: Based on new parameters, we propose a 'decision tree' that delivers time savings and supports biological interpretation in case of elevated MCHC.

Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop.

Essentials The clinical enumeration of microparticles (MPs) is hampered by a lack of standardization. A new strategy to standardize MP counts by flow cytometry was evaluated in a multicenter study. No difference was found between instruments using forward or side scatter as the trigger parameter. This study demonstrated that beads can be used as a standardization tool for MPs. Click to hear the ISTH Academy's webinar on microvesicles SUMMARY: Background Microparticles (MPs) are extracellular vesicles resulting from the budding of cellular membranes that have a high potential as emergent biomarkers; however, their clinical relevance is hampered by methodological enumeration concerns and a lack of standardization. Flow cytometry (FCM) remains the most commonly used technique with the best capability to determine the cellular origin of single MPs. However, instruments behave variably depending on which scatter parameter (forward (FSC) or side scatter (SSC)) provides the best resolution to discriminate submicron particles. To overcome this problem, a new approach, based on two sets of selected beads adapted to FSC or SSC-optimized instruments, was recently proposed to reproducibly enumerate platelet-derived MP counts among instruments with different optical systems. Objective The objective was to evaluate this strategy in an international workshop that included 44 laboratories accounting for 52 cytometers of 14 types. Methods/Results Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) of instruments. All instruments correctly ranked the platelet MP (PMP) levels of two platelet-free plasma samples. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side-scattered light as the relative sizing parameter. Conclusions Despite remaining limitations, this study is the first to demonstrate a real potential of bead-based strategies for standardization of MP enumeration across different FCM platforms. Additional standardization efforts are still mandatory to evaluate MPs' clinical relevance at a multicenter level.

Targeting soluble CD146 with a neutralizing antibody inhibits vascularization, growth and survival of CD146-positive tumors.

CD146 (MUC-18, MCAM) expression on cancer cells correlates with cancer progression and a bad prognosis in several tumors, including melanoma and pancreatic tumors. Deciphering the mechanism mediating the CD146 role in cancer is essential for generating new therapeutic strategies. We found that CD146 expression in cancer cells is associated with a secretion of soluble CD146 (sCD146) that constitutes an active player in tumor development. Indeed, sCD146 induces the overexpression of its binding protein, angiomotin, on both endothelial and cancer cells and promotes both paracrine effects on angiogenesis and autocrine effects on cancer cells proliferation and survival. These last effects are mediated in part through the induction and phosphorylation of c-myc in cancer cells. In mice models xenografted with human CD146-positive melanoma or pancreatic cancer cells, administration of a novel monoclonal antibody specifically targeting sCD146, but not its membrane form, successfully suppresses tumor vascularization and growth. Our findings demonstrate that sCD146 secreted by CD146-positive tumors mediates important pro-angiogenic and pro-tumoral effects. Targeting sCD146 with a novel neutralizing antibody could thus constitute an innovative therapeutic strategy for the treatment of CD146-positive tumors.

Standardized counting of circulating platelet microparticles using currently available flow cytometers and scatter-based triggering: Forward or side scatter?

Clinical determination of MP counts using flow cytometry has not been fully accepted yet due to the lack of standardization protocols. In the past 5 years, we have proposed two versions of a method with reproducible PMP counts in plasma samples. Both methods use forward scatter (FSC)-based threshold set with reference beads of appropriate sizes; first using 0.5 µm beads and later with 0.3 µm beads. Both systems provide reproducible PMP counts. However, this technique works only with some of currently used commercial flow cytometers. Instruments with limited resolution or generating heterogeneous FSC signals are excluded. Such performances are incompatible with the required interinstrument standardization. Here we show that (i) flow cytometers with sub-optimal FSC capabilities generally have higher SSC resolution and background rejection capacity, and (ii) that the same biological entities, "dim and bright PMP," both can be counted using alternative strategies, either as previously described, based on FSC measurements, or as presented here, based on SSC detection. The critical element in the standardization protocol is the use of different sizes of reference beads. This study was designed to permit simultaneous access to both FSC- and SSC-optimized platforms. A new range of about 0.17-0.6 µm eq. (µm-equivalents) is proposed for an alternative SSC-based MP gate generating the same PMP counts as those obtained in the previously proposed 0.3-1 µm eq. FSC-based MP gate. The two equivalent standardization options reconcile intrinsically different scattering behaviors between SSC- and FCS--triggered instruments and open the opportunity for multicenter studies in the future.

Circulating endothelial cells and progenitors as prognostic factors during autoimmune thrombotic thrombocytopenic purpura: results of a prospective multicenter French study.

BACKGROUND: Autoimmune thrombotic thrombocytopenic purpura (AI-TTP) is characterized by an excess of circulating ultralarge von Willebrand factor (VWF) caused by anti-ADAMTS-13 autoantibodies. Animal studies, however, have shown that endothelial cell activation may also be an important trigger of AI-TTP. OBJECTIVES: To prospectively study circulating biomarkers of endothelial lesion and activation, such as circulating endothelial cells (CECs), soluble P-selectin (sP-selectin), or VWF, and of endothelial repair, such as circulating progenitor cells (CPCs) and endothelial progenitor cells (EPCs), in AI-TTP, in relation to disease severity and prognosis. RESULTS: Twenty-two patients were included in this study. CEC (P < 0.01), VWF (P < 0.05) and sP-selectin (P < 0.01) levels were significantly increased during crisis, and returned to baseline levels during remission. Both CEC (P < 0.05) and sP-selectin (P < 0.05) levels were significantly higher in patients who died or developed neurologic sequelae. CPC levels were substantially increased during the acute phase of the disease (P < 0.001), and returned to baseline levels during remission. Among CPCs, EPC levels were also increased during crisis (P < 0.05) and significantly decreased during remission. Patients who received < 16 plasma exchanges (PEs) had significantly higher EPC counts (P < 0.05) than those who needed more numerous PEs to obtain remission, suggesting that initial EPC counts may be associated with faster endothelial repair. CONCLUSION: The profile of circulating endothelial markers shows massive endothelial activation and repair/remodeling during AI-TTP, and suggests that CECs and EPCs may be promising prognostic biomarkers of the disease.

Neutrophils recruit and activate human endothelial colony-forming cells at the site of vessel injury via P-selectin glycoprotein ligand-1 and L-selectin.

BACKGROUND: Endothelial colony-forming cells (ECFCs) represent a subpopulation of circulating endothelial progenitor cells that have been implicated in vascular repair. However, no study has evaluated the role of ECFCs in endothelial injury leading to thrombus formation. OBJECTIVE: We investigated the kinetics, mechanisms and role of ECFC recruitment in the dynamics of thrombus formation and stabilization. METHODS AND RESULTS: Using digital intravital microscopy in living mice, we show that ECFCs, but not mature endothelial cells, adhere to sites of laser-induced injury and do not affect the kinetics of thrombus formation. This interaction occurs once the platelet thrombus has been stabilized, and is dependent on the presence of neutrophils but not platelets or fibrin. In vitro, the interaction of the activated neutrophils with activated endothelial cells is a prerequisite for the capture of ECFCs. Neutrophils activate ECFCs and increase their angiogenic properties, such as their ability to migrate and to form pseudocapillaries. This newly identified interaction of ECFCs with the neutrophils is mediated by the P-selectin glycoprotein ligand-1 (PSGL-1)/L-selectin axis both in vitro and in vivo. CONCLUSIONS: This study is the first demonstration that neutrophils present at the site of injury recruit ECFCs via PSGL-1/L-selectin. This interaction between neutrophils and ECFCs could play a key role in the regeneration of injured vessels in pathophysiologic conditions.

Involvement of neutrophils in thrombus formation in living mice.

Thrombosis is one of the major causes of human death worldwide. Identification of the cellular and molecular mechanisms leading to thrombus formation is thus crucial for the understanding of the thrombotic process. To examine thrombus formation in a living mouse, new technologies have been developed. Digital intravital microscopy allows to visualize the development of thrombosis and generation of fibrin in real-time within living animal in a physiological context. This specific system allowed the identification of new cellular partners involved in platelet adhesion and activation. Furthermore, it improved, especially, the knowledge of the early phase of thrombus formation and fibrin generation in vivo. Until now, platelets used to be considered the sole central player in thrombus generation. However, recently, it has been demonstrated that leukocytes, particularly neutrophils, play a crucial role in the activation of the blood coagulation cascade leading to thrombosis. In this review, we summarized the mechanisms leading to thrombus formation in the microcirculation according to the method of injury in mice with a special focus on the new identified roles of neutrophils in this process.

Revisited role of microparticles in arterial and venous thrombosis.

Microparticles (MPs) represent a heterogeneous population of submicronic vesicles that are released in response to cell activation or apoptosis. MPs harbor a large repertoire of cell surface receptors and mRNA and biological activities representative of their parent cells and related to their involvement in many biological functions. Although MP generation is a physiological response, a dramatic increase in circulating MPs is detectable in a variety of thrombosis-associated disorders compared with healthy individuals. In this review, we will discuss a new vision of MPs as complex and ambivalent structures that express both activators and inhibitors of coagulation but also convey fibrinolytic properties. After summarizing our current knowledge about the role of MPs in venous and arterial thrombosis, this review will explore how this new vision of MPs influences their definition as emergent biomarkers in thrombotic diseases. Among the studies that have aimed to establish a link between thrombosis and MPs, a few studies have demonstrated a predictive value of MPs. So far, it is unclear whether this limited causative association is the result of current technical concerns and limited standardization or has to be integrated into a more complex vision of the role of MPs as key systems for regulating the balance between coagulation and fibrinolysis.

Standardization of pre-analytical variables in plasma microparticle determination: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop.

Microparticles (MP) are sub-micron sized vesicles released by activated or apoptotic cells. They are generally defined as 0.1 to 1 µm membrane particles that expose the anionic phospholipid phosphatidylserine (PS) and membrane antigens representative of their cellular origin [1]. It is now well recognized that MP behave as vectors of bioactive molecules, playing a role in blood coagulation, inflammation, cell activation and cancer metastasis. In clinical practice, circulating MP originating from blood and vascular cells are elevated in a variety of prothrombotic and inflammatory disorders, cardiovascular diseases, autoimmune conditions, infectious diseases and cancer [1-3]. © 2013 International Society on Thrombosis and Haemostasis.

Relationship between post-treatment platelet reactivity and ischemic and bleeding events at 1-year follow-up in patients receiving prasugrel.

BACKGROUND: Post-treatment platelet reactivity (PR) is associated with ischemic and bleeding events in patients receiving P2Y12 receptor antagonists. OBJECTIVES: We aimed to study the relationship between post-treatment PR after a 60-mg loading dose (LD) of prasugrel and 1-year thrombotic and bleeding events. METHOD: Patients were prospectively included in this multicenter study if they had a successful percutaneous coronary intervention (PCI) for acute coronary syndrome (ACS) and received prasugrel. The platelet reactivity index (PRI) was measured using the Vasodilator-Stimulated Phosphoprotein index (VASP) after a prasugrel LD. Endpoints included the rate of thrombotic events and bleeding events at 1 year. RESULTS: Among the 301 patients enrolled, 9 (3%) were lost to follow-up at 1 year. The rates of thrombotic and bleeding events at 1 year were of 7.5% and 6.8%, respectively. Receiver-operating curve (ROC) analysis demonstrated an optimal cut-off value of 53.5% of PRI to predict thrombotic events at 1 year. Using this cut-off value we observed that patients exhibiting high on-treatment platelet reactivity (HTPR) had a higher rate of thrombotic events (22.4% vs. 2.9%; P < 0.001). In parallel the optimal cut-off value of PRI to predict bleeding was 16%. Patients with a PRI ≤ 16% had a higher rate of bleeding events compared with those with a PRI > 16% (15.6% vs. 3.3%; P < 0.001). In multivariate analysis, the PRI predicted both thrombotic and bleeding events (OR: 1.44, 95% confidence interval [CI]: 1.2-1.72; P < 0.001 and OR: 0.75, 95% CI: 0.59-0.96; P = 0.024 [respectively, per 10% increase]). CONCLUSION: Platelet reactivity measurement after a prasugrel LD predicts both ischemic and bleeding events at 1 year follow-up for ACS patients undergoing PCI.

Mobilization of CD34+ KDR+ endothelial progenitor cells predicts target lesion revascularization.

BACKGROUND: Endothelial lesion and regeneration are critical events in the process leading to in-stent restenosis (ISR) after bare metal stent (BMS) percutaneous coronary intervention (PCI). OBJECTIVES: To prospectively investigate the relationship between biomarkers reflecting endothelial turnover and the occurrence of ISR. METHODS: We performed a multicenter prospective observational study that included 156 patients undergoing elective PCI with BMS. Endothelial lesion was assessed by the enumeration of circulating endothelial cells (CECs). Endothelial regeneration was evaluated by enumeration of circulating CD34+ progenitor cells (CD34+ PCs) and CD34+ KDR+ endothelial progenitor cells (EPCs). Measurements were performed before PCI, and 6 and 24 h after PCI. Dynamic changes were evaluated by calculating the delta value of each marker. The primary and secondary endpoints of the study were clinical target lesion revascularizations (TLRs) and major adverse cardiovascular events (MACEs) after 6 months of follow-up. RESULTS: During follow-up, 28 MACEs were recorded, including 27 TLRs. PCI induced a significant rise in the numbers of CECs, CD34+ PCs, and CD34+ KDR+ EPCs. Baseline, 6-h and 24-h levels of these markers did not differ between patients with and without TLR. The delta percentage of CD34+ KDR+ EPCs was significantly reduced in patients with TLR as compared with patients without TLR (- 0.56 ± 8.1 vs. 2.91 ± 6.2; P = 0.015). In multivariate analysis, the delta percentage of CD34+ KDR+ EPCs independently predicted the occurrence of TLR and MACEs (P = 0.02 and P = 0.014, respectively). CONCLUSION: The endothelial regenerative response to injury induced by PCI, assessed by CD34+ KDR+ EPCs mobilized among progenitor cells, determines the risk of TLR and MACEs in stable coronary artery disease patients.

Premature birth is associated with not fully differentiated contractile smooth muscle cells in human umbilical artery.

Smooth muscle cells (SMCs) participate to the regulation of peripheral arterial resistance and blood pressure. To assume their function, SMCs differentiate throughout the normal vascular development from a synthetic phenotype towards a fully differentiated contractile phenotype by acquiring a repertoire of proteins involved in contraction. In human fetal muscular arteries and umbilical arteries (UAs), no data are available regarding the differentiation of SMCs during the last trimester of gestation. The objective of this study was to characterize the phenotype of SMCs during this gestation period in human UAs. We investigated the phenotype of SMCs in human UAs from very preterm (28-31 weeks of gestation), late preterm (32-35 weeks) and term (37-41 weeks) newborns using biochemical and immunohistochemical detection of α-actin, smooth muscle myosin heavy chain, smoothelin, and non-muscle myosin heavy chain. We found that the number of SMCs positive for smoothelin in UAs increased with gestational age. Western blot analysis revealed a higher content of smoothelin in term compared to very preterm UAs. These results show that SMCs in human UAs gradually acquire a fully differentiated contractile phenotype during the last trimester of gestation and thus that premature birth is associated with not fully differentiated contractile SMCs in human UAs.

Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol.

BACKGROUND: Microparticles (MP) are small vesicles of 0.1-1 µm, released in response to activation or apoptosis. Over the past decade, they received an increasing interest both as biomarkers and biovectors in coagulation, inflammation and cancer. Clinical studies were conducted to assess their contribution to the identification of patients at cardiovascular risk. However, among the limitation of such studies, pre-analytical steps remains an important source of variability and artifacts in MP analysis. OBJECTIVES: Because data from the literature are insufficient to establish recommendations, the objective of the present study was to assess the impact of various pre-analytical parameters on MP measurement. These parameters included the type of collection tube, phlebotomy conditions, transportation practices, centrifugation steps and freezing. METHODS: MP were assessed by three methods: flow cytometry using a standardized approach, a thrombin generation test (Calibrated Automated Thrombogram(®)) and a procoagulant phospholipid-dependent clotting time assay (STA(®) -Procoag-PPL). RESULTS: The main results show that the three major pre-analytical parameters which impact on MP-related data are the delay before the first centrifugation, agitation of the tubes during transportation and the centrifugation protocol. CONCLUSIONS: Based on both this work and literature data, we propose a new protocol that needs to be validated on a larger scale before being applied for multicenter studies.

Circulating lipoprotein-associated phospholipase A2 in high-grade carotid stenosis: a new biomarker for predicting unstable plaque.

OBJECTIVE: To test plasma levels of lipoprotein-associated phospholipase A2 (Lp-PLA2) in patients with high-grade carotid stenosis according to plaque histology. METHODS: This cross-sectional single-centre study included patients with ≥70% North American Symptomatic Carotid Endarterectomy Trial (NASCET) carotid stenosis, who were treated surgically. Serum Lp-PLA2 and high-sensitivity C-reactive protein (hs-CRP) were determined on the day of surgery. Histopathological analysis classified carotid plaque as stable or unstable, according to AHA classification. RESULTS: Of the 42 patients (mean age 70.4 ± 10.5 years; 67% men), neurological symptoms were present in 16 (38%). Unstable plaques were found in 23 (55%). Median plasma level of Lp-PLA2 was significantly higher in patients with unstable plaque compared to those with stable plaque (222.4 (174.9-437.5) interquartile range (IQR) 63.5 vs. 211.1 (174.9-270.6) IQR 37.2 ng ml(-1); p = 0.02). Moreover, median Lp-PLA2 level were higher in asymptomatic patients with unstable plaque (226.8 ng ml(-1) (174.9-437.5) IQR 76.8) vs. stable plaque (206.9 ng ml(-1) (174.9-270.6) IQR 33.7; p = 0.16). Logistic regression showed that only the neurological symptoms (OR = 30.9 (3.7-244.6); p < 0.001) and the plasma Lp-PLA2 level (OR = 1.7 (1.1-12.3); p = 0.03) were independently associated with unstable carotid plaque as defined by histology. CONCLUSIONS: This study showed that circulating Lp-PLA2 was increased in patients with high-grade carotid stenosis and unstable plaque. Lp-PLA2 may be a relevant biomarker to guide for invasive therapy in asymptomatic patients with carotid artery disease.