Martius Scarlet Blue Research Articles

Eosinophil Depletion as a Potential Therapeutic Strategy in Acute and Chronic Intestinal Inflammation Based on a Dextran Sulfate Sodium Colitis Model.

A role for eosinophils in intestinal inflammation and fibrosis in the context of inflammatory bowel disease has been suggested, yet the precise nature, whether causal or secondary remains debated. Hence, it remains unclear whether targeting eosinophils should be further explored as a treatment option in inflammatory bowel disease. Acute and chronic dextran sulfate sodium colitis was induced in wild-type C57BL/6 mice. Eosinophils were depleted by anti-CCR3 injections before colitis induction in a chronic model and after colitis onset in an acute model in order to investigate the impact of eosinophil depletion on pre-existing colitis. Inflammation was assessed using the disease activity index, macroscopic damage, and histological disease activity score. In the chronic model, fibrosis was assessed by examining colon weight/length ratio, collagen deposition through Martius Scarlet Blue staining, hydroxyproline assay, and COL1A1 expression. Protein and gene expression were assessed using the Meso Scale Discovery platform and real-time quantitative polymerase chain reaction. In the acute and chronic colitis model, eosinophil depletion resulted in reduced disease activity and faster recovery, as observed via the total area under the curve of the disease activity index (P = .004 and P = .02, respectively), macroscopic damage score (P = .009 and P = .08, respectively), and histological disease activity score (P = .09 and P = .002, respectively). In the acute model, the accelerated recovery was accompanied by an increase in interleukin (IL)-10 (P = .03) and a decrease in IL-4 (P = .03) and IL-6 (P = .009). Colon weight/length ratio and collagen deposition were not affected by eosinophil depletion. Eosinophil depletion prevents and decreases intestinal inflammation in a preclinical dextran sulfate sodium model without affecting fibrosis. These results pave the way for exploring eosinophil depletion as a novel treatment modality in addressing intestinal inflammation.

Contribution of Red Blood Cells and Platelets to Blood Clot Computed Tomography Imaging and Compressive Mechanical Characteristics

Thrombus computed tomography (CT) imaging characteristics may correspond with thrombus mechanical properties and thus predict thrombectomy success. The impact of red blood cell (RBC) content on these properties (imaging and mechanics) has been widely studied. However, the additional effect of platelets has not been considered. The objective of the current study was to examine the individual and combined effects of blood clot RBC and platelet content on resultant CT imaging and mechanical characteristics. Human blood clot analogues were prepared from a combination of preselected RBC volumes and platelet concentrations to decouple their contributions. The resulting clot RBC content (%) and platelet content (%) were determined using Martius Scarlet Blue and CD42b staining, respectively. Non-contrast and contrast-enhanced CT (NCCT and CECT) scans were performed to measure the clot densities. CECT density increase was taken as a proxy for clinical perviousness. Unconfined compressive mechanics were analysed by performing 10 cycles of 80% strain. RBC content is the major determinant of clot NCCT density. However, additional consideration of the platelet content improves the association. CECT density increase is influenced by clot platelet and not RBC content. Platelet content is the dominant component driving clot stiffness, especially at high strains. Both RBC and platelet content contribute to the clot’s viscoelastic and plastic compressive properties. The current in vitro results suggest that CT density is reflective of RBC content and subsequent clot viscoelasticity and plasticity, and that perviousness reflects the clot’s platelet content and subsequent stiffness. However, these indications should be confirmed in a clinical stroke cohort.

Clot composition and recanalization outcomes in mechanical thrombectomy

BackgroundMechanical thrombectomy (MT) has become standard for large vessel occlusions, but rates of complete recanalization are suboptimal. Previous reports correlated radiographic signs with clot composition and a better response to...

Morphofunctional characteristics of the vessels of the small circle of blood circulation in those who died from severe and extremely severe forms of new coronavirus infection

BACKGROUND: An important feature of COVID-19 is the development of pronounced hypercoagulation with an increased risk of thrombotic damage to the pulmonary vascular bed, mainly the pulmonary arteries. Thrombosis of the pulmonary blood vessels causes a local violation of hemodynamics with the development of hyperemia, edema, which leads to a decrease in ventilation of the lung tissue area and serves as one of the causes of respiratory failure.
 AIM: This study aimed to conduct a morphological and morphometric analysis of the vascular bed of lung tissues in deceased with severe and extremely severe forms of new coronavirus infection who were on inpatient treatment in the period 2020-2022.
 METHODS: A pathomorphologic study of 129 autopsy cases with a confirmed diagnosis of a new coronavirus infection COVID-19 was performed. Morphometric analysis and statistical data processing of the pulmonary vascular system in histologic preparations stained with hematoxylin and eosin stain, orcein stain and Martius Scarlet Blue (MSB) stain was performed. The control group consisted of 14 patients who died of cardiovascular disease with bilateral focal confluent pneumonia.
 RESULTS: It was found that the proportion of thrombosed vessels in the lung tissues of the deceased was 27.6%. In 87.2% of cases, thrombosis develops in small arteries (lumen diameter 30-500 microns) and small veins (lumen diameter 40-500 microns). The vascular-functional indices of Kernogan and Vogenworth were statistically significantly increased in small arteries and small veins of the 4th order (p=0.001), small arteries (p=0.001) and small veins of the 5th order (p=0.014) compared with the control group.
 CONCLUSION: Diffuse involvement of small caliber blood vessels in the pathological process reflects the severity of specific hemocoagulopathic disorders in the lung tissue. Such disorders lead to the development of ventilation-perfusion disorders and entail an increase in right ventricular failure.

Electrical impedance measurements can identify red blood cell–rich content in acute ischemic stroke clots ex vivo associated with first-pass successful recanalization

BackgroundElectrochemical impedance spectroscopy can determine characteristics such as cell density, size, and shape. The development of an electrical impedance–based medical device to estimate acute ischemic stroke (AIS) clot characteristics could improve stroke patient outcomes by informing clinical decision making. ObjectivesTo assess how well electrical impedance combined with machine learning identified red blood cell (RBC)–rich composition of AIS clots ex vivo, which is associated with a successfully modified first-pass effect. MethodsA total of 253 clots from 231 patients who underwent thrombectomy in 5 hospitals in France, Japan, Serbia, and Spain between February 2021 and October 2023 were analyzed in the Clotbase International Registry. Electrical impedance measurements were taken following clot retrieval by thrombectomy, followed by Martius Scarlet Blue staining. The clot components were quantified via Orbit Image Analysis, and RBC percentages were correlated with the RBC estimations made by the electrical impedance machine learning model. ResultsQuantification by Martius Scarlet Blue staining identified RBCs as the major component in clots (RBCs, 37.6%; white blood cells, 5.7%; fibrin, 25.5%; platelets/other, 30.3%; and collagen, 1%). The impedance-based RBC estimation correlated well with the RBC content determined by histology, with a slope of 0.9 and Spearman’s correlation of r = 0.7. Clots removed in 1 pass were significantly richer in RBCs and clots with successful recanalization in 1 pass (modified first-pass effect) were richer in RBCs as assessed using histology and impedance signature. ConclusionElectrical impedance estimations of RBC content in AIS clots are consistent with histologic findings and may have potential for clinically relevant parameters.

DOP85 An anti-fibrotic role for eosinophils in a DSS colitis RAG-/- and an in vitro co-culture model

Abstract Background In several disease areas, such as in the liver and lungs, a pro-fibrotic role for eosinophils has been suggested. However, information about the role of eosinophils in the development of intestinal fibrosis is scarce. We therefore aimed to assess the functional and mechanistic role of eosinophils in intestinal fibrosis and fibroblast activation. Methods Chronic DSS colitis (3 DSS cycles of 1 week DSS administration followed by 2 weeks of recovery (1.75% - 2.25% - 2.25%)) was introduced in 6-8-week-old C57BL/6 RAG-/- mice to induce intestinal fibrosis (Figure 1A). Mice were injected intraperitoneally with anti-CCR3 to deplete eosinophils (n=8) or with an isotype control (n=8). Intestinal fibrosis was assessed based on collagen deposition (hydroxyproline assay, Martius Scarlet Blue staining (MSB) and second harmonics generation (SHG)), while COL1A1 expression was assessed via RT-qPCR. Gene expression was performed using RT-qPCR and protein expression via the MSD platform. Secondly, circulating eosinophils were isolated from non-IBD controls (n=3) and co-cultured with fibroblasts, isolated from resection specimens obtained from the terminal ileum of Crohn’s disease patients undergoing right hemicolectomy (n=3). The effect of eosinophils on fibroblast activation was assessed through the expression of α-SMA via flow cytometry and immunocytochemistry. Lastly, fibroblasts were stimulated with 10ng/mL eosinophil cationic protein (ECP), a protein exclusively produced by eosinophils (n=3), in order to study whether this mediator could affect fibroblast activation. Results Anti-CCR3 mediated eosinophil depletion in the chronic DSS model resulted in increased fibrosis based on the hydroxyproline assay (p=0.06) (Figure 1B), MSB (p=0.0009) (Figure 1C) and COL1A1 expression (p=0.03) (Figure 1D). Similarly, we identified an increased collagen deposition in the eosinophil-depleted group based on the SHG (Figure 1E). Furthermore, an increase in the pro-fibrotic IL-1β and TGF-β3 on protein (p=0.03 and p=0.06) and gene level (p=0.04 and p=0.005) could be found in the eosinophil depleted mice. Co-culturing eosinophils with fibroblasts resulted in decreased α-SMA expression, indicative of a decrease in active fibroblasts. This could be observed via both flow cytometry (p=0.001) (Figure 1F) and immunocytochemistry (Figure 1G). Similarly, when fibroblasts were stimulated with ECP, a decrease in α-SMA was observed (p=0.04) (Figure 1H). Conclusion Depletion of eosinophils in a chronic DSS RAG-/- model resulted in more intestinal fibrosis. Similarly, in an in vitro setting, eosinophils, as well as ECP, decreased α-SMA expression in fibroblasts, suggesting that eosinophils may inactivate fibroblasts.

Comparative evaluation of thermal impact of 980nm diode laser on soft tissue using irrigation and non-irrigation systems: A histomorphometric study.

Diode laser is one of the most captivating technologies in dental practice. In periodontics, when used at appropriate settings, it possesses the best properties for selective surgical and nonsurgical procedures such as subgingival calculus removal without a thermal change of the root surface, and also provides tissue surface sterilization. However, lasers always produce a certain amount of thermal damage to the soft tissues. Therefore, this study aimed to comparatively evaluate the thermal impact of 980-nm diode laser incisions, when used with irrigation versus nonirrigation systems. The study was conducted on 24 gingival tissue samples obtained from the goat's mandible and maxilla. The samples were divided into two groups of 12 samples each, and laser incisions were given in 4 power settings, using 980-nm diode laser in super short pulse mode, with and without saline irrigation. Martius Scarlet Blue staining technique was used to prepare the histological slides. Then, slides were examined under a research microscope (Olympus CX 21), and the pictures of the slides were taken by mounting the camera onto the microscope mobile mount. The microscopic images hence obtained were analyzed for the depth and width of the incisions, area of carbonization, necrosis, and reversible damage, using the Digimizer image analysis software. The results of this study demonstrated that the mean incision depth was higher (592.49 ± 180.97, P < 0.05), with less carbonization (25.52 ± 29.21, P = 0.00) and less necrosis (311.63 ± 156.441, P < 0.05) in the laser incisions with irrigation, as compared to the laser incisions given without irrigation system. Within the limitations of this study, it can be concluded that using an irrigation system causes less collateral damage while maintaining the incising efficiency of the diode laser. Further studies with a higher sample size, controlled irrigation systems, and incision techniques are needed to evaluate the efficiency of diode lasers for the clinical explanation of the results.

White blood cell subtypes and neutrophil extracellular traps content as biomarkers for stroke etiology in acute ischemic stroke clots retrieved by mechanical thrombectomy

BackgroundLymphocytes, macrophages, neutrophils, and neutrophil extracellular traps (NETs) associate with stroke risk factors and form a thrombus through different mechanisms. We investigated the total WBCs, WBC subtypes and NETs composition in acute ischemic stroke (AIS) clots to identify possible etiological differences that could help us further understand the process of thrombosis that leads to AIS. MethodsAIS clots from 100 cases each of atherothrombotic (AT), cardioembolic (CE) and cryptogenic stroke etiology were collected per-pass as part of the CÚRAM RESTORE registry of AIS clots. Martius Scarlet Blue stain was used to identify the main histological components of the clots. Immunohistochemical staining was used to identify neutrophils, lymphocytes, macrophages, and NETs patterns. The cellular and histological components were quantified using Orbit Image Analysis software. ResultsAT clots were larger, with more red blood cells and fewer WBCs than CE clots. AT clots had more lymphocytes and cryptogenic clots had fewer macrophages than other etiologies. Most significantly, CE clots showed higher expression of neutrophils and extracellular web-like NETs compared to AT and cryptogenic clots. There was also a significantly higher distribution of web-like NETs around the periphery of the CE clots while a mixed distribution was observed in AT clots. ConclusionThe difference in neutrophil and NETs expression in clots from different etiologies may provide insight into the mechanism of clot formation.

Quality assessment of histopathological stainings on prolonged formalin fixed thrombus tissues retrieved by mechanical thrombectomy.

Formalin-fixed retrieved clots from mechanical thrombectomy (MT) are now routinely studied using both conventional histopathologic techniques and immunohistochemistry (IHC). However, the effects of prolonged formalin fixation on the histological results of clot analysis remain unknown. The objective of this study was to investigate the effects of prolonged formalin fixation on quality of histopathologic stainings of thrombus tissues retrieved by MT. As part of the multicenter EXCELLENT registry, a total of 80 clots extracted by MT from acute ischemic stroke patients were randomly selected from the tissue database and assigned into four groups according to 10% neutral buffered formalin (NBF) fixation duration (1-30, 30-60, 60-90, and 90+ days, up to 2 years). Samples underwent processing and sectioning. Two serial sections for each case were stained with hematoxylin and eosin (H&E), Martius Scarlet Blue (MSB), and IHC for CD42b (platelet marker). An expert pathologist, who was blinded to tissue fixation duration and patient clinical data, assessed the quality of each stain including stainability, sensitivity, specificity, and consistency of stainings. No significant issues were encountered during tissue processing and sectioning. On H&E stain, 97.5% (78/80) of slides showed good-quality staining, demonstrating clear histological properties of the thrombus tissue as red blood cells (RBC) stained in red, fibrin/platelet stained in pink, and nuclei stained in blue with intranuclear detail. The same histological features were also successfully demonstrated on MSB for all 80 samples. One of the 80 samples (1.2%) showed that RBC lost stainability on H&E due to tissue autolysis. Clear positive signal of platelet staining was expressed in 98.8% of the samples (79/80) with minimal background staining on IHC. There was no significant difference in staining quality across different formalin fixation groups. A good quality of histopathological staining is achievable for the thrombus tissue fixed in 10% neutral buffered formalin for up to 2 years. The findings are limited to the thrombus tissue retrieved by MT and specific fixation and staining protocols used in the study. To apply these results to other tissue or experimental setups, further studies and validations would be necessary. This study was conducted as part of the EXCELLENT study: www.clinicaltrials.gov, unique identifier: NCT03685578.

Endotheliitis and cytokine storm as a mechanism of clot formation in COVID-19 ischemic stroke patients: A histopathologic study of retrieved clots.

Studies during the COVID-19 pandemic have demonstrated an association between COVID-19 virus infection and the development of acute ischemic stroke, particularly large vessel occlusion (LVO). Studying the characteristics and immunohistochemistry of retrieved stroke emboli during mechanical thrombectomy for LVO may offer insights into the pathogenesis of LVO in COVID-19 patients. We examined retrieved COVID-19 emboli from the STRIP, EXCELLENT, and RESTORE registries and compared their characteristics to a control group. We identified COVID-positive LVO patients from the STRIP, RESTORE, and EXCELLENT studies who underwent mechanical thrombectomy. These patients were matched to a control group controlling for stroke etiology based on Trial of Org 10172 in Acute Stroke Treatment criteria. All clots were stained with Martius Scarlet Blue (MSB) along with immunohistochemistry for interleukin-6 (IL-6), C-reactive protein (CRP), von Willebrand factor (vWF), CD66b, fibrinogen, and citrullinated Histone H3. Clot composition was compared between groups. Nineteen COVID-19-positive patients and 38 controls were included. COVID-19-positive patients had a significantly higher percentage of CRP and vWF. There was no difference in IL-6, fibrin, CD66b, or citrullinated Histone H3 between groups. Based on MSB staining, there was no statistically significant difference regarding the percentage of red blood cells, white blood cells, fibrin, and platelets. Our study found higher concentrations of CRP and vWF in retrieved clots of COVID-19-positive stroke patients compared to COVID-19-negative controls. These findings support the potential role of systemic inflammation as indicated by elevated CRP and endothelial injury as indicated by elevated vWF as precipitating factors in thrombus development in these patients.

Abstract 99: Clot Composition And Reperfusion Outcomes In 1430 Mechanical Thrombectomy Patients: Analysis Of The Stroke Thromboembolism Registry Of Imaging And Pathology

Background: Understanding clot composition may allow for better technical planning and improved outcomes in mechanical thrombectomy (MT). We sought to correlate clot composition with reperfusion outcomes in MT. Methods: Clinical, imaging, and clot data from patients enrolled in the STRIP Registry from September 2016 to September 2020 were analyzed. Samples were fixed in 10% phosphate-buffered formalin and stained with hematoxylin and eosin and Martius Scarlett Blue. Percent composition, richness, and gross appearance were evaluated. Outcome measures included the rate of First-Pass Effect (mTICI2c/3, FPE) and the number of device passes. Results: A total of 1430 patients (mean age, 68.4±13.5years; median [IQR] baseline NIHSS,17.2 [10.5-23]; IV-tPA use, 36%; Stent-Retrievers [SR], 27%; Contact Aspiration [CA], 27%; Combined SR + CA, 43%) were included. The median [IQR] number of passes was 1 [1-2]. FPE was achieved in 39.3% of the cases. There was no association between percent histological composition or clot richness and FPE in the overall population. However, the combined technique resulted in lower FPE rates for RBC-rich (P&lt;0.0001), platelet-rich (P=0.003), and mixed (P&lt;0.0001) clots. Fibrin-rich and platelet-rich clots required a higher number of passes compared to RBC-rich and mixed clots (median, 2 and 1.5 vs.1, respectively, P=0.02). CA displayed a trend towards a higher number of passes with fibrin-rich clots (2 vs.1, P=0.12). By gross appearance, mixed/heterogeneous clots had lower FPE rates than red and white clots. Conclusion: Despite the lack of correlation between clot histology and FPE in the overall population, our study adds to the growing body of evidence supporting the notion that clot composition influences reperfusion treatment strategy outcomes. Additional studies are needed.

DOP09 Eosinophils exert a pro-inflammatory role in a chronic DSS colitis model without an impact on fibrosis

Abstract Background Eosinophils might play a pro-inflammatory role in inflammatory bowel disease (IBD). However, the role of eosinophils in intestinal fibrosis remains poorly understood, although a pro-fibrotic function has been hypothesized based on data outside the gastrointestinal tract. Therefore, we aimed to unravel the role of eosinophils in chronic intestinal inflammation and fibrosis and to explore eosinophil depletion as a potential therapeutic target. Methods A 3-cycles chronic dextran sodium sulphate (DSS) model was induced in 6-8-week-old C57BL/6 wild type mice. Mice received 3 DSS cycles (1.5% - 2.00% - 2.00%) in which 1 DSS cycle consisted of 1 week of DSS administration followed by 2 weeks of recovery. During the 3 cycles, and starting 3 days prior to colitis induction, anti-CCR3 antibody or isotype injections were given twice weekly to study the effect of the eosinophil depletion (total of 18 injections). The disease activity index (DAI; weight loss, stool consistency and presence of blood) was determined twice weekly. At sacrifice, colonic damage was scored macroscopically (presence of hyperaemia, adhesions and length and degree of colon affected by inflammation) and colonic single cells were isolated and fluorescently stained for flow cytometry. Eosinophils were thereby identified as CD45+ CD11b+ Siglec-F+ CD117- cells. Lastly, a histological active disease score was determined comprising of the sum of neutrophil infiltration, mononuclear cell infiltration, changes in mucosal architecture, goblet cell loss and epithelial defects (Creyns et al., 2019). Intestinal fibrosis was assessed via colon weight/length ratio, COL1A1 gene expression and collagen deposition with a Martius Scarlet Blue (MSB) staining and a colorimetric hydroxyproline assay. Results Blood and colonic eosinophil depletion via anti-CCR3 injections was confirmed via flow cytometry (figure 1). The DAI in the eosinophil depleted group was decreased compared to the isotype injected group (area under the curve 115.4 ± 29 vs 160.6 ± 28; p=0.02). A similar trend was seen in the macroscopic damage score (2.0 ± 1.3 vs 3.9 ± 2.1; p=0.08). Furthermore, a lower histological active disease score was found in the mice in which the eosinophils were depleted compared to the isotype injected mice (8.8 ± 0.8 vs 12.8 ± 1.5; p=0.002) (figure 2). Lastly, no differences in the parameters for fibrosis between the anti-CCR3 injected and isotype injected groups were observed (figure 3). Conclusion Eosinophil depletion via intraperitoneal anti-CCR3 injections resulted in partial protection from intestinal inflammation in chronic DSS, and could therefore be further explored as a potential therapeutic agent. In contrast, eosinophil depletion does not seem to have any anti-fibrotic effect

Pulmonary Vascular Thrombosis in COVID-19: Clinical and Morphological Parallels

Aim. We aimed to study the histological and thrombotic changes in lung vessels in patients who died with COVID-19, to access the correlation between anticoagulation therapy (ACT) and thrombotic events (TE), treatment results, clinical and laboratory patients' characteristics.Material and Methods. We retrospectively analyzed treatment results of patients hospitalized with COVID-19 and lung vessel samples of the deceased patients. Dynamic changes and highest levels of D-dimer and fibrinogen were studied in its correlation with the disease severity according to SOFA score, computer tomographic (CT) results, lung, renal and hepatic dysfunction. The association between different doses of ACT and treatment results, laboratory indicators and thrombotic events was accessed. The histological lung vessels examination was performed using Martius Scarlet Blue (MSB)staining.Results. 313 patients were included in the study (61 patients died). The median age of hospitalized patients was 60 years (IQR 51-66 years). The frequency of the intravitallyconfirmed TE was 4,8%. The strong statistical association was revealed between D-dimer level and 3-4 points SOFA score, patients' mortality, oxygen support requirement, CT3-CT4 pneumonia, glomerular filtration rate and TE. There was no mortality in patients with D-dimer normal references, but in cases with three times elevation reached 13%, 48,5% - in cases with 3-6 times elevation and 64,6% - in cases with more than 6 times elevation. The strong statistical association was registered between fibrinogen and SOFA score, CT 3-4 pneumonia, patients' mortality. D-dimer and fibrinogen levels demonstrated weak correlation. There was no statistical correlation between prophylactic, intermediate and therapeutic ACT and D-dimer and fibrinogen levels, CT results, patients' mortality. MSBstaining was used in 36 deceased patients tissue samples. 1394 lung vessels were analyzed. Lung vessels thrombi persisted in samples of all 36 patients (100%). Vessels with the diameter 3,5-30 mm were thrombosed in 7%, with the diameter 0,034-0,84 mm - in 48%, with the diameter 0,85-3,4 mm - in 45%. The frequency of thrombi persisted 06 hours, 6-12 hours, 12-18hours, 18-24 hours and more than 24 hours was12%, 14%, 62%, 5% and 7% respectively.Conclusion. Thrombi of different ages from fresh to organized were observed in one third of lung vessels in all deceased patients. Lung vessels thrombosis plays an important role in pathogenesis and thanatogenesis of COVID-19. The D-dimer level correlates with lung, renal dysfunction, patients' mortality and doesn't show any correlation with ACT and can be accepted as a criterion of lung vessel thrombotic progression.

A nomogram for predicting thrombus composition in stroke patients with large vessel occlusion: combination of thrombus density and perviousness with clinical features.

To establish a nomogram incorporating pretreatment imaging parameters and clinical characteristics for predicting the thrombus composition of acute ischemic stroke (AIS) with large vessel occlusion (LVO). We retrospectively enrolled patients with occlusion of the Middle Cerebral Artery (MCA) who underwent Mechanical Thrombectomy (MT). Retrieved thrombi were stained with Hematoxylin and Eosin (H&E) and Martius Scarlet Blue (MSB). Thrombi are assigned to the Fibrin-rich or RBC-rich group based on the relative fractions of Red Blood Cells (RBC), fibrin, and platelet. The independent risk factors for Fibrin-rich clots were determined via univariate and multivariate logistic regression analysis and were then integrated to establish a nomogram. In total, 98 patients were included in this study. Patients with fibrin-rich clots had worse functional outcome [modified Rankin scale (mRS) 0-2, 34.7% vs 63.2%, p = 0.005], longer procedure time (76.8min vs 50.8min, p = 0.001), and increased maneuvers of MT (1.84 vs 1.46, p = 0.703) than those with RBC-rich clots. The independent risk factors for Fibrin-rich clots were lower perviousness measured by Non-Contrast Computer Tomography (NCCT) and CT Angiography (CTA), lower thrombus relative attenuation on NCCT, elevated Platelet-WBC ratio (PWR) of admission peripheral blood, and previous antithrombotic medication. The nomogram showed good discrimination with an area under the Receiver Operating Characteristic (ROC) curve (AUC) of 0.852 (95% CI: 0.778-0.926). The calibration curve and decision curve analysis also displayed satisfactory accuracy and clinical utility. This study has developed and internally validated an easy-to-use nomogram which can help predict clot composition and optimize therapeutic strategies for thrombectomy.

Histological composition of retrieved emboli in acute ischemic stroke is independent of pre-thrombectomy alteplase use

Background and purposeGiven recent evidence suggesting the clot composition may be associated with revascularization outcomes and stroke etiology, clot composition research has been a topic of growing interest. It is currently unclear what effect, if any, pre-thrombectomy thrombolysis has on clot composition. Understanding this association is important as it is a potential confounding variable in clot composition research. We retrospectively evaluated the composition of retrieved clots from ischemic stroke patients who did and did not receive pre-treatment tPA to study the effect of tPA on clot composition. Materials and methodsConsecutive patients enrolled in the Stroke Thromboembolism Registry of Imaging and Pathology (STRIP) were included in this study. All patients underwent mechanical thrombectomy and retrieved clots were sent to a central core lab for processing. Histological analysis was performed using Martius Scarlett Blue (MSB) staining and area of the clot was also measured on the gross photos. Student's t test was used for continuous variables and chi-squared test for categorical variables. ResultsA total of 1430 patients were included in this study. Mean age was 68.4±13.5 years. Overall rate of TICI 2c/3 was 67%. A total of 517 patients received tPA (36%) and 913 patients did not (64%). Mean RBC density for the tPA group was 42.97±22.62% compared to 42.80±23.18% for the non-tPA group (P=0.89). Mean WBC density for the tPA group was 3.74±2.60% compared to 3.42±2.21% for the non-tPA group (P=0.012). Mean fibrin density for the tPA group was 26.52±15.81% compared to 26.53±15.34% for the non-tPA group (P=0.98). Mean platelet density for the tPA group was 26.22±18.60% compared to 26.55±19.47% for the non-tPA group (P=0.75). tPA group also had significantly smaller clot area compared to non-tPA group. ConclusionsOur study 1430 retrieved emboli and ischemic stroke patients shows no interaction between tPA administration and clot composition. These findings suggest that tPA does not result in any histological changes in clot composition.

Light microscopic observations of the ruminal papillae of cattle on diets with divergent forage to cereal ratios

High levels of supplementation with cereal increases production rates in cattle but can increase incidence of disease, ranging from mild indigestion to acute ruminal acidosis and death. Therefore, there is motivation to determine biological markers which can be used to identify whether animals have been, or are being fed, sufficient or excessive cereals. This study aimed to describe light microscopic findings from animals being fed diverse dietary cereal proportions and to test the performance of a novel rumen epithelial scoring system. Rumen wall tissue samples were obtained from the abattoir from 195 cattle from 11 Scottish farms and processed for histological examination. Light microscopic examination was used to characterise ruminal epithelial response to dietary challenge. Secondary objectives included describing the distribution of immune-related cells in bovine ruminal epithelium and assessing the use of a modified Elastin Martius Scarlet Blue stain (EMSB) for histological examination of the rumen epithelium. Cells staining positive for cluster of differentiation 3 were distributed mainly in the lower layers of the stratum basale and were found in higher densities in animals offered lower cereal proportion diets. Cells staining positive for major histocompatibility complex class 2 (MHCII) were most common in perivascular locations and in the junction between the lower stratum basale and the propria-submucosa. The density of MHCII positive staining cells was higher in animals on lower cereal diets. The level of supplementation with cereal was also associated with the thickness of the stratum corneum (SCT) and stratum granulosum (SGT), the integrity of the stratum corneum and sloughing of cornified cells. There were no advantages in using EMSB stain over haematoxylin and eosin (H&E) in this scoring system. We concluded that a scoring system that included only SCT, SGT and a measure of the loss of appearance of intercellular space allowed differentiation of groups of animals according to the level of cereal supplementation.

Association Between Prior Anticoagulation and Thrombus Composition in Mechanical Thrombectomy Patients with Atrial Fibrillation

PurposeAnticoagulation (AC) is the main preventive strategy for ischemic stroke in atrial fibrillation (AF) patients. We aim to investigate the association of prior AC with thrombus composition and clinical outcome in AF patients with acute ischemic stroke (AIS). Materials and MethodsFrom January 2019 to December 2020, consecutive AIS patients with AF treated with mechanical thrombectomy (MT) in our center were included in this analysis. Retrieved thrombi were stained with hematoxylin and eosin (H&E) and Martius Scarlet blue (MSB). The relative fractions of red blood cell (RBC), white blood cell (WBC), fibrin, and platelet were quantitatively analyzed. Procedural and clinical outcomes were compared between patients with and without prior AC. ResultsA total of 133 patients were enrolled in this study, with 39 in AC group and 94 in non-AC (NAC) group. Thrombi in AC group contained more fibrins (36% vs 20%, p<0.001), more platelets (36% vs 24%, p<0.001) and fewer RBCs (25% vs 54%, p<0.001). No difference was detected in terms of successful recanalization evaluated with modified Thrombolysis in Cerebral Infarction scale (mTICI 2b-3, 97% vs 86%, p=0.065), functional independence at 90 days with modified Rankin Score (mRS 0-2, 44% vs 33%, p=0.246). ConclusionThrombi retrieved from AF patients with prior AC contained more fibrins, more platelets and fewer RBCs compared with those of NAC patients. A trend of higher successful reperfusion rate was observed in AC patients but failed to reach statistical significance.

Abstract TP159: The Effect Of Diabetes Mellitus On Thrombus Composition In Patients With Stroke

Introduction: Outcomes of mechanical thrombectomy (MT) are influenced by the composition of thrombi. Amongst the risk factors affecting clot composition, diabetes mellitus (DM) may be associated with more thrombus structural and mechanical alterations resulting in fibrinolysis-resistant thrombi. However, extensive analysis of the composition of thrombi obtained from patients is needed to elucidate this relationship. Hypothesis: Thrombi retrieved from patients with DM may have different compositions compared to that of patients with no history of DM. Methods: MT retrieved-thrombi from patients suffering from large vessel occlusion (LVO) stroke were included. Thrombi sections were stained with Martius Scarlet Blue (MSB) for main thrombus components including red blood cells (RBCs), white blood cells (WBCs), fibrin and platelet. Additionally, immunohistochemistry (IHC) was performed to identify neutrophil extracellular traps (NETs) (anti-citrulinated H3; CitH3; NETs-specific marker) and von Willebrand Factor (vWF) in thrombi. Slides were then scanned and analyzed and the thrombus composition was compared between patients with or without a DM diagnosis at the time of stroke presentation. Results: A total of 140 patients were included of which 35 had a diagnosis of DM at stroke presentation. Baseline characteristics (excluding age), IV-tPA use prior to MT, and MT outcomes (number of device passes and final mTICI score) were similar between DM and non DM patients. The overall average percentage of RBCs, WBCs, fibrin, platelet, NETs (CitH3), and vWF components in thrombi were 47.4%, 3.2%, 27.4%, 21.8%, 25.9% and 17.6%, respectively. Thrombi from patients with DM were not statistically significant different from those of patients with no DM in terms of RBCs (44.3 vs 48.4 p=0.37), WBCs (2.7 vs 3.3 p=0.05), fibrin (29.5 vs 26.7 p=0.37), platelet (23.3 vs 21.3 p=0.57), NETs (25.6 vs 22.3 p=0.54), and vWF (17.5 vs 17.7 p=0.96). Conclusion: DM has no effect on the composition of thrombi as no significant difference was found in thrombi obtained from patients with and without DM.

Abstract WP156: Association Of Thrombus Histopathology And Prognosis Of Successful Recanalization

Introduction: Mechanical thrombectomy (MT) has become the standard of care for acute ischemic stroke (AIS) patients due to large vessel occlusion (LVO). Clinical outcome of successful recanalization (mTICI 2b or higher) varies greatly and is associated with some baseline characteristics. Many studies have reported the relation of thrombus composition with MT outcomes however the association of thrombus composition with the prognosis of successful recanalization remains to be elucidated. Hypothesis: thrombi retrieved from patients with good clinical outcome may have different compositions compared to that of patients with poor clinical outcome. Methods: Thrombi retrieved from AIS-LVO patients with successful recanalization were included. Patients were divided according to modified Rankin Scale at 90 days (mRS-90days) into good clinical outcome (mRS-90days 0-2) and poor clinical outcome (mRS-90 days 3-6). Representative slide for each thrombus was stained with Martius Scarlet Blue (MSB) for red blood cells (RBCs), white blood cells (WBCs), fibrin (FIB), and platelet (PLT). Immunohistochemistry (IHC) was also performed for NETs (anti-citrullinated H3; CitH3) and vWF in thrombi. The overall mean value of each thrombus composition was calculated. Thrombus was considered rich in given composition if that composition was higher than its overall mean. We compared the thrombus composition and type between patients with good and poor clinical outcomes. Results: A total of 94 patients were included. Fifty-six patients had good clinical outcome. The overall average percentage of RBCs, WBCs, fibrin, platelet, NETs (CitH3), and vWF components in thrombi were 44.3%, 3.6%, 29.3%, 22.8%, 20.3% and 12.5% respectively. There was no association between thrombus type (including RBC-rich, FIB-rich, PLT-rich, vWF-rich, and NETs-rich) and clinical outcomes (P values&gt;0.05). Multivariate logistic regression analysis showed no association between thrombus composition and clinical outcome (good vs poor) while adjusting for age, initial NIHSS score, ASPECT score, pre-MT IV-tPA, and MT technique and number of the device passes. Conclusion: we found no association between thrombus composition and clinical outcome after successful recanalization.

Abstract TP146: The Effect Of Statins On Thrombus Composition Retrieved By Mechanical Thrombectomy In Acute Ischemic Stroke

Introduction: Ample evidence exists in support of the anti-inflammatory properties of statins which in turn could affect arterial thrombosis. Compositional analysis of thrombi retrieved by MT may provide us with knowledge useful for evaluating the effect of current Statin therapy on thrombus composition. Methods: Consecutive AIS-LVO patients were included. Histopathological evaluation of thrombi retrieved during MT was performed using Martius Scarlet Blue staining (MSB) and immunohistochemistry for von Willebrand Factor (vWF) and anti-citrulinated H3 (CitH3; NETs marker) and the composition of thrombi was determined using image analysis software. Thrombus was considered rich in a given composition if that composition was higher than its overall mean. We reviewed patient’s medication records and cataloged any statin therapy prior to MT. Results: 117 patients were included in this study. The mean density of red blood cells (RBCs), white blood cells (WBCs), platelets (PLT), fibrin (FIB), vWF and NETs (CitH3) in thrombi was 43.4%, 3.7%, 23.6%, 29.3%, 14.3% and 21.1% respectively. Twenty-seven patients were taking Statins at the time of MT. Statins were not associated with pre-thrombectomy IV-tPA, and stroke etiology (p values: &gt;0.05). There was no association between Statins and thrombi composition and type (p values: &gt;0.05 for all thrombus components and type). Additionally, Statins were not associated with MT outcomes including number of device passes and final mTICI score. Conclusion: Pre-stroke Statin therapy was not associated with differences in thrombus composition.