Thrombus Specimens Research Articles

The clinical characteristics of neutrophil extracellular trap in patients with severe cerebral venous sinus thrombosis and its prognostic value in acute and subacute patients

Objective: To explore the clinical characteristics of neutrophil extracellular trap (NET) in patients with severe cerebral venous sinus thrombosis (CVST) and to study their prognostic value in the acute and subacute phases. Methods: This study is a retrospective case series analysis. Clinical and pathological data of 52 patients with severe cerebral venous sinus thrombosis who underwent endovascular treatment in the Department of Neurosurgery, Tianjin Huanhu Hospital from June 2019 to June 2022 were retrospectively analyzed. There were 20 males and 32 females, with an age of (40.1±13.6) years(range:18 to 66 years). Forty-five healthy physical examinees were included in the control group. High-resolution MRI was used to stage the thrombus, with 11 cases in the acute group, 28 cases in the subacute group, and 13 cases in the chronic group. Thrombus specimens were obtained through endovascular treatment, and the fluorescence intensity of NET in peripheral blood at different time points was analyzed by immunofluorescence contrast,including the double-stranded DNA structure and adhesion protein components (citrolinated histone H3 (CitH3), myeloperoxidase-DNA complex(MPO-DNA), neutrophil elastase (NE)). The NET markers were determined by ELISA. Spearman rank correlation analysis was used to analyze the correlation between the NET markers in peripheral blood of patients with severe cerebral venous sinus thrombosis in the acute and subacute phases and the volume of venous sinus thrombus, the degree of venous sinus recanalization after treatment, and the discharge modified Rankin scale(mRS)score. The accuracy of NET markers in predicting the prognosis of patients with severe cerebral venous sinus thrombosis was analyzed by drawing receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC). Results: The results of immunofluorescence staining and ELISA showed that no NET structure was formed in the peripheral blood of the control group, while CitH3, MPO-DNA and NE levels in the peripheral blood of CVST patients were increased, among which the acute stage group was the highest, followed by the subacute group, and the chronic group was the lowest. Spearman correlation analysis showed that CitH3, MPO-DNA and NE levels in peripheral blood of patients in acute group and subacute group were positively correlated with thrombus volume and mRS score at discharge (P<0.05). The levels of CitH3 and MPO-DNA in peripheral blood of patients with complete venous sinus recanalization were lower than those of patients with partial venous sinus recanalization (P<0.01). ROC curve analysis results showed that MPO-DNA and NE had no predictive ability for the prognosis of CVST patients (P values were 0.614 and 0.324, respectively), and the AUC of CitH3 was 0.800 (95%CI: 0.638~0.962, P=0.032), the best cut-off value was 13.5 μg/L, the sensitivity was 100%, and the specificity was 58.8%. Conclusions: A large number of NET are formed in patients with severe cerebral venous sinus thrombosis in acute stage. Patients with severe cerebral venous sinus thrombosis in acute stage and subacute stage with high peripheral blood NET content has a low rate of complete sinus revascularization and poor neurological function recovery after treatment.

Correlation between thrombus composition and regulatory T cell counts with clinical outcomes of acute ischemic stroke patients with thrombectomy.

To analyze the relationship of thrombus composition and regulatory T cell expression with clinical outcome in acute ischemic stroke (AIS) patients with thrombectomy. A total of 44 AIS patients who underwent thrombectomy in the Department of Neurology of Shaoxing Hospital from June 2021 to October 2022 were enrolled. All thrombus specimens were subjected to hematoxylin-eosin staining and immunohistochemistry. Semi-quantitative analysis was performed to determine the content of red blood cells, fibrinogen/platelets, and regulatory T (CD4+CD25+) cells. Clinical data, vascular recanalization status, and neurologic outcomes at 3 months after thrombectomy were collected. A modified Rankin Scale score of 0-2 was defined as a favorable outcome. Among 44 patients with complete thrombus data there were 15 cases of red cell type, 11 cases of mixed type and 18 cases of fibrin/platelet type. There was a significant difference in trial of ORG 10172 in acute stroke treatment (TOAST) etiological classification among the three groups (P<0.01), while no significant differences were found in other general clinical and surgical data (all P>0.05). According to the TOAST etiology, 28 cases were classified as large atherosclerosis type and 16 cases as cardioembolic type. The proportion of red blood cells in thrombus was significantly higher in patients with large atherosclerosis type than that in those with cardioembolic type [58.0% (44.2%, 72.5%) vs. 24.5% (12.7%, 48.0%), P<0.01]. The ratio of fibrin to platelet in patients with cardiogenic embolism was significantly higher than that in patients with large atherosclerosis [73.0% (49.2%, 84.5%) vs. 40.0% (25.2%, 54.5%), P<0.01). Among the 44 patients, 19 had good while 25 had poor neurological outcomes. Univariate binary logistic regression analysis showed that age, operation time, CD4+CD25+T cell number were correlated with the functional outcomes of the patients (all P<0.05). Multivariate binary logistic regression analysis showed that thrombus CD4+CD25+T cell count was an independent factor affecting the functional outcome of patients (OR=1.369, 95%CI: 1.101-1.701, P<0.01). There is no significant correlation of erythrocyte and fibrin/platelet components in thrombus with functional outcome in AIS patients, but an increased count of regulatory T cells associates with good functional outcome.

Abstract WP304: Investigating Thrombus Architecture Using Spatial Transcriptomics

Introduction: Spatial transcriptomic (ST) platforms provide high-resolution localization of gene expression profiles within intact tissue, offering insight to cellular phenotype and ultimately tissue function. While cerebral thrombus morphology has been well-characterized, their functional architecture has not. Although this study is insufficiently powered to infer specific pathophysiologic conclusions, our findings offer preliminary insight into the functional segmentation of cerebral thrombi using ST. Methods: Four thrombi retrieved from cerebral arteries of ischemic stroke patients during standard of care thrombectomy were formalin-fixed and paraffin-embedded. Morphology marker antibodies (vimentin, CD45) and RNA probes coupled to photocleavable oligonucleotide tags were applied to thrombus specimens. Regions of interest (ROI) were randomly selected based on visualized cellular clustering within each specimen. ST analysis was performed using NanoString's GeoMx Digital Spatial Profiling platform. Results: Of the 24 ROIs, 0 samples were below the 50% warning, and 18,676 targets were analyzed. 1,127 genes were expressed in 50% of ROIs. 8,793 genes normalized by 3rd quartile, expressed above the Limit of Quantitation in at least 1% of ROIs. Differential expression analysis using a false discovery rate of &lt; 0.05 revealed 54 differentially expressed genes (DEG), all of which were protein-coding using edgeR’s exact test. Conclusions: Although ST does not offer visibility into the presence of fully formed ribosomes, the identification of intact cytoplasmic RNA suggests there is significant transcriptional activity within thrombi. We cautiously present pilot data to support feasibility for spatially-resolved transcriptomic analysis of cerebral thrombi which, upon further technical refinement, may facilitate discovery of new targets for stroke prevention or treatment.

Ticagrelor versus clopidogrel in reducing inflammatory cell infiltration of thrombus aspirated in patients with ST-elevation myocardial infarction.

Ticagrelor provides more rapid, potent, and consistent anti-platelet efficacy than clopidogrel. This randomized trial aimed to evaluate the anti-inflammation effects of ticagrelor versus clopidogrel on thrombus aspirated from the ST-elevation myocardial infarction (STEMI) patients. A total of 98 patients with STEMI and intended percutaneous coronary intervention (PCI) were randomly assigned to receive clopidogrel (600-mg loading dose) or ticagrelor (180-mg loading dose), of whom 55 with large thrombus burden underwent thrombus aspiration during PCI. Thrombus specimens were successfully aspirated from 49 patients. Finally, 24 patients in the clopidogrel group and 23 in the ticagrelor group completed the study. Inflammatory cells within thrombi were assessed by hematoxylin-eosin and immunohistochemistry stainings. Compared with the clopidogrel group, the number of total inflammatory cells per mm2 thrombus area in the ticagrelor group was decreased by 28% (P = 0.009). The numbers of neutrophils and myeloperoxidase-positive cells per mm2 thrombus area in the ticagrelor group were respectively decreased by 35% (P = 0.016) and 28% (P = 0.047), as compared with those in the clopidogrel group. Moreover, ticagrelor treatment reduced the ratio of monocytes number higher than 250 per mm2 thrombus area compared with clopidogrel treatment (4% versus 29%, P = 0.048). In patients with undergoing PCI for STEMI, the loading dose ticagrelor regimen was associated with a reduction in inflammatory cell infiltration within thrombus compared with the loading dose clopidogrel regimen.

Insights Into Immunothrombotic Mechanisms in Acute Stroke due to Vaccine-Induced Immune Thrombotic Thrombocytopenia.

During the COVID-19 pandemic, vaccination is the most important countermeasure. Pharmacovigilance concerns however emerged with very rare, but potentially disastrous thrombotic complications following vaccination with ChAdOx1. Platelet factor-4 antibody mediated vaccine-induced immune thrombotic thrombocytopenia (VITT) was described as an underlying mechanism of these thrombotic events. Recent work moreover suggests that mechanisms of immunothrombosis including neutrophil extracellular trap (NET) formation might be critical for thrombogenesis during VITT. In this study, we investigated blood and thrombus specimens of a female patient who suffered severe stroke due to VITT after vaccination with ChAdOx1 in comparison to 13 control stroke patients with similar clinical characteristics. We analyzed cerebral thrombi using histological examination, staining of complement factors, NET-markers, DNase and LL-37. In blood samples at the hyper-acute phase of stroke and 7 days later, we determined cell-free DNA, myeloperoxidase-histone complexes, DNase activity, myeloperoxidase activity, LL-37 and inflammatory cytokines. NET markers were identified in thrombi of all patients. Interestingly, the thrombus of the VITT-patient exclusively revealed complement factors and high amounts of DNase and LL-37. High DNase activity was also measured in blood, implying a disturbed NET-regulation. Furthermore, serum of the VITT-patient inhibited reactive oxygen species-dependent NET-release by phorbol-myristate-acetate to a lesser degree compared to controls, indicating either less efficient NET-inhibition or enhanced NET-induction in the blood of the VITT-patient. Additionally, the changes in specific cytokines over time were emphasized in the VITT-patient as well. In conclusion, insufficient resolution of NETs, e.g. by endogenous DNases or protection of NETs against degradation by embedded factors like the antimicrobial peptide LL-37 might thus be an important factor in the pathology of VITT besides increased NET-formation. On the basis of these findings, we discuss the potential implications of the mechanisms of disturbed NETs-degradation for diagnostic and therapeutic approaches in VITT-related thrombogenesis, other auto-immune disorders and beyond.

Impacts of eosinophil percentage on prognosis acute type A aortic dissection patients

BackgroundEosinophils are pro-inflammatory cells involved in thrombosis and have been proposed as a prognosis marker in acute ischemic stroke and ST-elevation myocardial Infarction. Here, we sought to clarify the prognostic value of eosinophil percentage (EOS%) in patients with acute type A aortic dissection (AAAD).MethodsWe examined 183 consecutive AAAD patients. Based on the optimum cut-off value of EOS% determined by X-tile software, patients were classified into the low EOS% (EOS% ≤ 0.1) and high EOS% groups (EOS% > 0.1). We performed multivariate regression analysis and Kaplan–Meier (KM) survival curves to assess the association between EOS% and mortality. Eosinophil accumulation in aortic dissection intraluminal thrombus was confirmed using hematoxylin–eosin (H&E) staining. An external cohort from Medical Information Mart for Intensive Care IV was performed to validate the results.ResultsRelative to surviving patients, those who died during hospitalization had significantly lower EOS% (p = 0.001) but significantly higher WBC (p = 0.002) and neutrophil (p = 0.001) counts. Multivariate regression analysis identified EOS% as an independent predictor of in-hospital and 1-year mortality. KM curves revealed that 1-year cumulative mortality was significantly higher in the low EOS% group, although it was mainly attributed to the higher 30-day mortality. H&E staining revealed massive infiltration of eosinophils in all 20 thrombus specimens. The external validation confirmed that relative to survivors, patients with in-hospital mortality (p = 0.010) had significantly lower EOS%. Moreover, multivariate regression analyses identified that decreased EOS% was independently significantly associated with in-hospital mortality.ConclusionsLow EOS% is significantly related to increased mortality rates in AAAD patients.

No Histological Difference between Large Atherosclerotic and Cardiogenic Embolic Thrombus.

Background The continuous development of endovascular treatment technology provides more opportunities for the histological study of thrombus. According to Trial of Org 10 172 in Acute Stroke Treatment (TOAST), clinicians take different strategies in anticoagulant or antiplatelet therapy. There are some patients still suffering from recurrent stroke while they took anticoagulant or antiplatelet drugs regularly for secondary prevention. In view of that, we found that histological analysis of thrombus can provide guidance for secondary prevention. Aim Exploring the histological characteristics differences between large atherosclerotic and cardiogenic embolic thrombosis in order to guide clinical secondary prevention of the two stroke subtypes. Methods A total of 54 patients with acute ischemic stroke were collected from December 2019 to April 2021. Identify stroke subtypes according to TOAST classification. Stain thrombus specimens with hematoxylin-eosin staining, and perform statistical analysis on the components (red blood cells and fibrin/platelets) of thrombus. Results In cardiogenic thrombi, the composition of RBCs was dominant (51.38 ± 18.463%) compared to that of fibrin/platelets (48.62 ± 18.463%). Similarly, among the thrombi of large artery atherosclerotic, RBCs (50.40 ± 20.100%) compared to fibrin/platelets (49.60 ± 20.100%). There was no statistical difference in RBCs or fibrin/platelet composition of both cardiogenic and atherosclerotic thrombi (P = 0.89). Conclusions The histologic composition of thrombi in cardiogenic and atherosclerotic had no statistical difference. These thrombi are all mixed thrombus, which are rich in RBCs, fibrinogen, and platelets. Anticoagulation combined with antiplatelet may be a more effective secondary prevention strategy.

SARS-COV-2 colonizes coronary thrombus and impairs heart microcirculation bed in asymptomatic SARS-CoV-2 positive subjects with acute myocardial infarction

BackgroundThe viral load of asymptomatic SAR-COV-2 positive (ASAP) persons has been equal to that of symptomatic patients. On the other hand, there are no reports of ST-elevation myocardial infarction (STEMI) outcomes in ASAP patients. Therefore, we evaluated thrombus burden and thrombus viral load and their impact on microvascular bed perfusion in the infarct area (myocardial blush grade, MBG) in ASAP compared to SARS-COV-2 negative (SANE) STEMI patients.MethodsThis was an observational study of 46 ASAP, and 130 SANE patients admitted with confirmed STEMI treated with primary percutaneous coronary intervention and thrombus aspiration. The primary endpoints were thrombus dimension + thrombus viral load effects on MBG after PPCI. The secondary endpoints during hospitalization were major adverse cardiovascular events (MACEs). MACEs are defined as a composite of cardiovascular death, nonfatal acute AMI, and heart failure during hospitalization.ResultsIn the study population, ASAP vs. SANE showed a significant greater use of GP IIb/IIIa inhibitors and of heparin (p < 0.05), and a higher thrombus grade 5 and thrombus dimensions (p < 0.05). Interestingly, ASAP vs. SANE patients had lower MBG and left ventricular function (p < 0.001), and 39 (84.9%) of ASAP patients had thrombus specimens positive for SARS-COV-2. After PPCI, a MBG 2–3 was present in only 26.1% of ASAP vs. 97.7% of SANE STEMI patients (p < 0.001). Notably, death and nonfatal AMI were higher in ASAP vs. SANE patients (p < 0.05). Finally, in ASAP STEMI patients the thrombus viral load was a significant determinant of thrombus dimension independently of risk factors (p < 0.005). Thus, multiple logistic regression analyses evidenced that thrombus SARS-CoV-2 infection and dimension were significant predictors of poorer MBG in STEMI patients. Intriguingly, in ASAP patients the female vs. male had higher thrombus viral load (15.53 ± 4.5 vs. 30.25 ± 5.51 CT; p < 0.001), and thrombus dimension (4.62 ± 0.44 vs 4.00 ± 1.28 mm2; p < 0.001). ASAP vs. SANE patients had a significantly lower in-hospital survival for MACE following PPCI (p < 0.001).ConclusionsIn ASAP patients presenting with STEMI, there is strong evidence towards higher thrombus viral load, dimension, and poorer MBG. These data support the need to reconsider ASAP status as a risk factor that may worsen STEMI outcomes.

Iatrogenic Foreign Materials Associated with Retrieved Clot Tissue via Mechanical Thrombectomy.

Hydrophilic polymers and polytetrafluoroethylene liners, commonly used in the construction of endovascular devices, occasionally separate from devices with subsequent embolization. We determined the frequency of such materials in thrombus specimens retrieved by mechanical thrombectomy in patients with stroke. We retrospectively reviewed H&E-stained thrombus sections for presence and types of foreign materials. We identified 4 types of foreign materials-Type I: material was light green with refraction and had a homogeneous texture; type II: material was light gray and/or dark gray, thin, and loose or attenuated in texture; type III: material was light green with refraction, solitary in texture, irregular in shape, and was often associated with round or oval bubblelike particles and/or diffuse black particles; and type IV: material had homogeneous texture and was light pink or red. In addition, polymer materials from different layers of used mechanical thrombectomy catheters were compared with the foreign materials found in thrombus specimens. A total of 101 thrombi were evaluated. Foreign materials were found in 53 (52.5%) thrombus samples. The most common type was type I (92%), followed by type II (30%). The histopathologic features of the polymer materials from mechanical thrombectomy catheters were similar to the foreign materials found in thrombus specimens. The inner polytetrafluoroethylene liner and coating layer of catheters resembled type I and type II of the foreign materials, respectively. Foreign polymer materials are present in approximately half of retrieved thrombi, most commonly polytetrafluoroethylene from catheter liners and less from hydrophilic coatings.

Proteomic Analysis of Cardioembolic and Large Artery Atherosclerotic Clots Using Reverse Phase Protein Array Technology Reveals Key Cellular Interactions Within Clot Microenvironments.

Thrombus characteristics are dependent on clot composition, but identification of the etiology based on histological analysis has proved inconclusive. Identification of proteomic signatures may help to differentiate between clots of different etiologies such as cardioembolic, large artery atherosclerotic, and other known etiologies, information that could enhance an individualized medicine approach to secondary stroke prevention. In this study, total protein extracts from cardioembolic (n=25) and large artery atherosclerotic (n=23) thrombus specimens were arrayed in quadruplicate on nitrocellulose slides and immunostained for 31 proteins using a Dako Autostainer (Agilent Technologies, Inc., Santa Clara, USA). We quantified 31 proteins involved in platelet and/or endothelial function, inflammation, oxidative stress, and metabolism. Pathway analysis showed more heterogeneity and protein network interactions in the cardioembolic clots but no specific correlations with clot etiology. Reverse-phase protein arrays are a powerful tool for assessing cellular interactions within the clot microenvironment and may enhance understanding of clot formation and origination. This tool could be further explored to help in identifying stroke etiology in large vessel occlusion patients with embolic stroke of an undetermined source.

Interventional stroke treatment - Is it also safe for arteries? Looking at thrombectomy wall damage through clot histology.

The aim of this study was to investigate whether morphological evidence of intracranial vascular injury can be found in the occluding thrombi of patients with ischemic stroke. From 2015 until 2018 specimens of thrombi from patients with large vessel occlusion treated either by stent-assisted aspiration thrombectomy (TE) or by aspiration thrombectomy were prospectively collected. Thrombus specimens were formalin-fixed, paraffin-embedded and stained. Architectural features, presence of parts of vessel wall or atheromatous material, organisation of the thrombi, technique and number of thrombectomy manoeuvers were evaluated. Thrombus specimens from 302 patients were analyzed. 238 (80%) patients were treated with stent-assisted aspiration TE and 64 (20%) patients with aspiration TE only. 286 (95%) had fresh thrombi, 14 (4.6%) showed initial/complete thrombus organisation and multi-staged thrombi were found in 2 (<1%) patients. In 8 patients (2.6%) we found atypical thrombus content after a time interval longer 6 hours after onset and final thrombectomy manoeuvre: 4 with atheromatous material and 4 with parts of a vessel wall. In 1 patient with parts of vessel wall angiographically a dissection was detected. No parts of the vessel wall were found after sole aspiration thrombectomy. The overall risk of arterial vessel wall injury aspiration thrombectomy was low in our study. Aspiration thrombectomy and the procedure related with a retrieving device together with an ischemia time longer than 6 hours may increase the risk of vessel injury through the thrombectomy procedure. Further investigations are necessary to elucidate the influence of the mentioned aspects.

Roles of microRNA‑186 and vascular endothelial growth factor in hepatocellular carcinoma complicated with portal vein tumor thrombus

The present study aims to investigate the role and underlying mechanism of microRNA (miR)-186 in patients with hepatocellular carcinoma (HCC) complicated with portal vein tumor thrombus. Blood samples from 29 HCC patients with portal vein tumor thrombus were collected between January 2014 and September 2015 in Huai'an First People's Hospital, while blood from 36 HCC patients without vein tumor thrombus was also collected in the same period. In addition, tumor thrombus specimens were collected from the HCC patients with portal vein tumor thrombus, and peritumoral tissues of the tumor thrombus were used as the control. Reverse transcription-quantitative polymerase chain reaction, ELISA and western blot analyses were applied to detect vascular endothelial growth factor (VEGF) expression at the mRNA and protein levels. Bioinformatics prediction was used to predict the target of miR-186, and then miR-186 expression was detected. Furthermore, dual-luciferase reporter assay was used to validate whether miR-186 directly targeted VEGF. Following transfection with agomiR-186, the expression levels of miR-186 and VEGF were detected, while MTT assay was used to detect EA.hy926 cell proliferation subsequent to small interfering RNA (siRNA) silencing. The results identified that VEGF was significantly increased in the tumor thrombus and blood samples of HCC patients with vein tumor thrombus at the mRNA and protein levels, while miR-186 expression was significantly decreased (P<0.05). Following silencing VEGF by siRNA transfection, the proliferation of EA.hy926 cells was inhibited. In addition, VEGF expression was significantly decreased and cell proliferation was reduced when upregulating miR-186. Dual-luciferase reporter assay demonstrated that miR-186 regulated VEGF expression through complementary binding to 3'-untranslated region. In conclusion, VEGF was significantly increased in tumor thrombus and blood samples from HCC patients with vein tumor thrombus, which may be associated with the downregulation of miR-186. Thus, miR-186 may promote the development and progression of vein tumor thrombus in HCC.

Hydrophilic Polymer in Thrombus Specimens Obtained During Thrombus Aspiration in the Setting of Percutaneous Interventions in Japan.

Although hydrophilic polymer (HP) is used on vascular catheters to prevent medical device-related complications, HP emboli are well established as a potentially fatal iatrogenic phenomenon. HP embolus is thromboembolism by HP, which is mechanically disrupted from catheter during procedure. We reviewed 119 thrombus specimens obtained by percutaneous interventions from the coronary artery, vessels of inferior and superior limbs, cerebral artery, carotid artery, and renal vein. The frequency of HP was 28.6% (34/119 cases); 26.4% (24/91 cases), coronary artery; 50% (6/12 cases), artery of the lower limb; 28.6% (2/7 cases), vein of the lower limb; 0% (0/2), artery of the upper limb; 33.3% (1/3 cases), cerebral artery; 50% (1/2 cases), pulmonary artery; 0% (0/1 case), carotid artery; and 0% (0/1 case), renal vein. The range of numbers and diameter of HP was 1 to 127 per case and 10 to 934 µm, respectively. This is the first study concerning HP in thrombus specimens in Japan. We think that this study is significant because HP in thrombus specimens obtained from coronary artery in Japan seems to be of lower frequency than that of Europe. Although we suspect that the difference was caused by types of catheter, protocol, and procedure time of percutaneous interventions, we could not investigate correlation of HP in thrombus specimens with these factors. For future investigation, we should accumulate thrombus specimens obtained by routine procedure and device to clarify specific device-associated risk of disruption of HP.

Development of immunohistochemical triple staining method for the evaluation of different types of cell death in coronary thrombus

Immunohistochemistry is a powerful technique to identify the presence, distribution and extent of certain antigens at protein level in tissue specimen. Multiple immunostainings enable the detection, colocalization and comparison of several markers within one specimen. However, an optimization study is firstly needed to ensure the expression of multiple markers is clear and distinguishable. We formulated an optimized sequential triple immunostaining protocol which could identify the simultaneous presence of three types of cell death namely necrosis, apoptosis and etosis. These following antibodies were used: C-reactive protein (CRP, necrosis), (cleaved) Caspase-3 (Casp3, apoptosis) and citrullinated-Histone3 (CitH3, etosis). Several antigen retrieval methods, various concentration and order combinations of those antibodies as well as different combination color of chromogens were tested on coronary thrombus materials obtained from patients with acute myocardial infarction (AMI). The results showed that CRP (1:4000) visualized with 3,3’-diaminobenzidine (DAB) in brown, is better performed as the first staining, followed by CitH3 (1:8000) visualized with Perma Blue as the second staining and Casp3 (1:500) visualized with Perma Red as the last staining. In conclusion, we presented an immunohistochemical triple staining protocol to identify the comparative presence of different types of cell deaths: necrosis, apoptosis and etosis in coronary thrombus specimens.

Neutrophils in Acute Coronary Syndrome

Acute coronary syndrome (ACS) encompasses a spectrum of clinical disorders of myocardial ischaemia or infarction, with atherosclerosis leading to coronary plaque formation the predominant disease process. Alterations of endothelial cell integrity involving atherosclerotic plaque surfaces, such as plaque rupture or erosion, can lead to atherothrombosis with subsequent interruption to myocardial blood supply. Over the past two decades, it has become increasingly apparent that inflammation plays a pivotal role in the initiation and progression of atherosclerosis. Inflammatory cytokines have been shown to correlate with the risk and burden of coronary artery disease and there is a growing body of evidence demonstrating the presence of various immune cells in atherosclerotic plaques and coronary thrombus specimens. Due to improved cellular detection methods compared to earlier studies, neutrophils are being increasingly recognised as a key player in the process of athero-inflammation. The aim of this review is to: i) outline the role of neutrophils in ACS and atherothrombosis, ii) describe the process of inflammasome-mediated release of inflammatory cytokines from neutrophils, and iii) discuss multiple parameters of neutrophil activity in ACS, including peripheral neutrophil/lymphocyte ratio; neutrophil microparticle release; expression of neutrophilic granular proteins, including myeloperoxidase, neutrophil elastase, and metalloproteinases; neutrophil extracellular traps release; tissue factor; and neutrophil-macrophage interactions.

Kounis Syndrome (Type I) Following Bee Sting

Bee stings are commonly encountered worldwide. Various manifestations after bee sting have been described 1 . Local reactions are common. Unusually, manifestations like vomiting, diarrhoea, dyspnoea, generalised oedema, acute renal failure, hypotension and collapse may occur. Rarely vasculitis, serum sickness, neuritis and encephalitis have been described which generally develop days to weeks after a sting. Acute coronary syndromes after hymenoptera stings and other environmental exposures are referred to as the Kounis syndrome or allergic myocardial ischaemia and infarction. The three variants of Kounis syndrome are Type I variant: includes patients with normal coronary arteries without predisposing factors for coronary artery disease in whom the acute release of inflammatory mediators can induce either coronary artery spasm without increase of cardiac enzymes and troponins or coronary artery spasm progressing to acute myocardial infarction with raised cardiac enzymes and troponins. Type II variant: includes patients with culprit but quiescent pre-existing atheromatous disease in whom the acute release of inflammatory mediators can induce either coronary artery spasm with normal cardiac enzymes and troponins or plaque erosion or rupture manifesting as acute myocardial infarction. Type III variant: includes patients with coronary thrombosis (including stent thrombosis) in whom aspirated thrombus specimens stained with hematoxylin-eosin and Giemsa demonstrate the presence of eosinophils and mast cells respectively. We report a 23 year old male who developed myocardial infarction after multiple bee stings over his body.

A splicing variant of Merlin promotes metastasis in hepatocellular carcinoma.

Merlin, which is encoded by the tumour suppressor gene Nf2, plays a crucial role in tumorigenesis and metastasis. However, little is known about the functional importance of Merlin splicing forms. In this study, we show that Merlin is present at low levels in human hepatocellular carcinoma (HCC), particularly in metastatic tumours, where it is associated with a poor prognosis. Surprisingly, a splicing variant of Merlin that lacks exons 2, 3 and 4 (Δ2–4Merlin) is amplified in HCC and portal vein tumour thrombus (PVTT) specimens and in the CSQT2 cell line derived from PVTT. Our studies show that Δ2–4Merlin interferes with the capacity of wild-type Merlin to bind β-catenin and ERM, and it is expressed in the cytoplasm rather than at the cell surface. Furthermore, Δ2–4Merlin overexpression increases the expression levels of β-catenin and stemness-related genes, induces the epithelium–mesenchymal-transition phenotype promoting cell migration in vitro and the formation of lung metastasis in vivo. Our results indicate that the Δ2–4Merlin variant disrupts the normal function of Merlin and promotes tumour metastasis.

Histopathological evaluation of thrombus in patients presenting with stent thrombosis. A multicenter European study: a report of the prevention of late stent thrombosis by an interdisciplinary global European effort consortium.

BackgroundStent thrombosis (ST) is a rare but serious complication following percutaneous coronary intervention. Analysis of thrombus composition from patients undergoing catheter thrombectomy may provide important insights into the pathological processes leading to thrombus formation. We performed a large-scale multicentre study to evaluate thrombus specimens in patients with ST across Europe.MethodsPatients presenting with ST and undergoing thrombus aspiration were eligible for inclusion. Thrombus collection was performed according to a standardized protocol and specimens were analysed histologically at a core laboratory. Serial tissue cross sections were stained with haematoxylin–eosin (H&E), Carstairs and Luna. Immunohistochemistry was performed to identify leukocyte subsets, prothrombotic neutrophil extracellular traps (NETs), erythrocytes, platelets, and fibrinogen.ResultsOverall 253 thrombus specimens were analysed; 79 (31.2%) from patients presenting with early ST, 174 (68.8%) from late ST; 79 (31.2%) were from bare metal stents, 166 (65.6%) from drug-eluting stents, 8 (3.2%) were from stents of unknown type. Thrombus specimens displayed heterogeneous morphology with platelet-rich thrombus and fibrin/fibrinogen fragments most abundant; mean platelet coverage was 57% of thrombus area. Leukocyte infiltrations were hallmarks of both early and late ST (early: 2260 ± 1550 per mm2 vs. late: 2485 ± 1778 per mm2; P = 0.44); neutrophils represented the most prominent subset (early: 1364 ± 923 per mm2 vs. late: 1428 ± 1023 per mm2; P = 0.81). Leukocyte counts were significantly higher compared with a control group of patients with thrombus aspiration in spontaneous myocardial infarction. Neutrophil extracellular traps were observed in 23% of samples. Eosinophils were present in all stent types, with higher numbers in patients with late ST in sirolimus-and everolimus-eluting stents.ConclusionIn a large-scale study of histological thrombus analysis from patients presenting with ST, thrombus specimens displayed heterogeneous morphology. Recruitment of leukocytes, particularly neutrophils, appears to be a hallmark of ST. The presence of NETs supports their pathophysiological relevance. Eosinophil recruitment suggests an allergic component to the process of ST.

Platelet endothelial cell adhesion molecule 1 deficiency misguides venous thrombus resolution

AbstractPlatelet endothelial cell adhesion molecule 1 (PECAM-1) is involved in leukocyte migration and angiogenesis, which are key components of venous thrombus resolution. This study investigated the effect of PECAM-1 deficiency on thrombus resolution in FVB/n mice and the extent to which levels of soluble PECAM-1 (sPECAM-1) correlate with delayed thrombus resolution in humans after acute symptomatic deep vein thrombosis (DVT). In a mouse stagnant flow venous thrombosis model Pecam-1−/− thrombi were larger, persisted for longer periods of time, and displayed attenuated macrophage invasion and decreased vessel formation in the presence of increased fibrosis. In humans, higher levels of truncated plasma sPECAM-1 possibly cleaved from cell surfaces, were found in patients with delayed thrombus resolution (assessed via duplex-based thrombus scoring) relative to those whose thrombi resolved (median, 25th/75th percentile): 92.5 (87.7/103.4) ng/mL vs 71.5 (51.1/81.0) ng/mL; P < .001. Furthermore, unresolved human deep vein thrombus specimens stained positively with antibodies specific for the extracellular, but not the cytoplasmic domain of PECAM-1, consistent with accumulation of cleaved PECAM-1. Our data suggest a regulatory role of PECAM-1 in venous thrombus resolution and suggest a predictive value of sPECAM-1 for postthrombotic syndrome (PTS) after acute DVT.

Isolation and characterisation of endothelial outgrowth from coronary arteries in patients with acute myocardial infarction

Purpose: Our understanding of endothelial cell biology is primarily derived from studies of human umbilical vein endothelial cells (HUVECs). However, HUVECs provide limited insight into disease pathogenesis. We describe a novel method for isolation of coronary artery endothelial cells from thrombectomy specimens obtained during the treatment of patients with acute myocardial infarction. We have expanded and characterised human coronary endothelial outgrowth (CEO) cells compared to HUVECs, to investigate their potential as a model of endothelial dysfunction. Methods: Patients presenting to the Edinburgh Royal Infirmary with ST-segment elevation myocardial infarction (n=15) underwent emergency percutaneous coronary intervention and thrombus aspiration. Thrombus specimens were dissected, plated onto collagen-I coated plates and maintained in vitro to encourage cellular outgrowth. Population doubling time was evaluated and angiogenic potential assessed by tubule formation. Multiparameter flow cytometry using antibodies to endothelial cell markers was performed (CD31, KDR, CD146 and CD34) and cells were immunostained for vonWillenbrand factor (vWF). Results: CEO cell outgrowth was observed in 9/15 samples. Population doubling times of CEO cells were comparable to HUVECs (mean±SD: CEO 2.6±0.5, HUVEC 2.3±0.1 days; p=0.50 student's t-test). CEO cells had typical "cobblestone" morphology and were immunoreactive for vWF. Surface expression of CD31 and KDR was comparable in both cell types (CD31 mean±SD: CEO 74.9±21.3 vs. HUVEC 89.3±11.4, p=0.13; KDR mean±SD: CEO 46.7±31.7 vs. HUVEC 22.2±15.9, p=0.11), however CD146 and CD34 expression was increased in CEO cells (CD146 mean±SD: CEO 96±5.3 vs. HUVEC 73.5±30.7, p<0.05; CD34 mean±SD: CEO 80.9±16.92 vs. HUVEC 40.63±18.06, p<0.001). Only 44% of CEO cells were capable of tubule formation compared to 100% of HUVECs (p=0.01, contingency tables, fishers exact test). CEO cells that were capable of tubule formation had reduced capacity to form connections compared to HUVECs (mean±SD: CEO 12±8, versus HUVEC 63±16 connections; p<0.0001). Conclusions: Viable coronary arterial endothelial cells can be isolated from thrombus extracted during emergency percutaneous coronary intervention. These cells have a mature endothelial phenotype comparable to HUVECs, but have reduced angiogenic potential suggesting they retain the functional characteristics of in situ endothelium. This novel approach to isolate dysfunctional endothelial cells may have applications in studies of the cellular and molecular basis of endothelial dysfunction in patients with coronary artery disease.