Investigating the Role of Brain Natriuretic Peptide (BNP) and N-Terminal-proBNP in Thrombosis and Acute Ischemic Stroke Etiology.

The need for biomarkers for acute ischemic stroke (AIS) to understand the mechanisms implicated in pathological clot formation is critical. The levels of the brain natriuretic peptides known as brain natriuretic peptide (BNP) and NT-proBNP have been shown to be increased in patients suffering from heart failure and other heart conditions. We measured their expression in AIS clots of cardioembolic (CE) and large artery atherosclerosis (LAA) etiology, evaluating their location inside the clots, aiming to uncover their possible role in thrombosis. We analyzed 80 thrombi from 80 AIS patients in the RESTORE registry of AIS clots, 40 of which were of CE and 40 of LAA etiology. The localization of BNP and NT-BNP, quantified using immunohistochemistry and immunofluorescence, in AIS-associated white blood cell subtypes was also investigated. We found a statistically significant positive correlation between BNP and NT-proBNP expression levels (Spearman's rho = 0.668 p < 0.0001 *). We did not observe any statistically significant difference between LAA and CE clots in BNP expression (0.66 [0.13-3.54]% vs. 0.53 [0.14-3.07]%, p = 0.923) or in NT-proBNP expression (0.29 [0.11-0.58]% vs. 0.18 [0.05-0.51]%, p = 0.119), although there was a trend of higher NT-proBNP expression in the LAA clots. It was noticeable that BNP was distributed throughout the thrombus and especially within platelet-rich regions. However, NT-proBNP colocalized with neutrophils, macrophages, and T-lymphocytes, suggesting its association with the thrombo-inflammatory process.

MRI characterization of in vitro clots at 3T and 7T: A technical note.

In acute ischemic stroke, the composition of the occlusive clot can be associated with the underlying pathophysiology and the response to treatment. For these reasons, it is important to characterize the clot composition from clinical scans. We examine the ability of 3T and 7T MRI to distinguish the composition of in vitro clots, using quantitative T1 and T2*, alternatively R2*, mapping. When comparing the two field strengths, we found a tradeoff between sensitivity for clot composition and confidence in the clot depiction associated with spatial resolution. The loss of sensitivity at 7T can be mitigated by combining the T1 and T2* signals.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Implications of COVID-19 to Stroke Medicine: An Epidemiological and Pathophysiological Perspective.

The neurological complications of Coronavirus 2019 (COVID-19) including stroke have been documented in the recent literature. COVID-19-related inflammation is suggested to contribute to both a hypercoagulable state and haemorrhagic transformation, including in younger individuals. COVID-19 is associated with a heightened risk of ischaemic stroke. Haemorrhagic stroke in COVID-19 patients is associated with increased morbidity and mortality. Cerebral venous sinus thrombosis (CVST) accounts for <1% of stroke cases in the general population but has come to heightened public attention due to the increased risk associated with adenoviral COVID-19 vaccines. However, recent evidence suggests the prevalence of stroke is less in vaccinated individuals than in unvaccinated COVID-19 patients. This review evaluates the current evidence of COVID-19-related ischaemic and haemorrhagic stroke, with a focus on current epidemiology and inflammatory-linked pathophysiology in the field of vascular neurology and stroke medicine.

Potential Biomarkers of Acute Ischemic Stroke Etiology Revealed by Mass Spectrometry-Based Proteomic Characterization of Formalin-Fixed Paraffin-Embedded Blood Clots.

Background and Aims: Besides the crucial role in the treatment of acute ischemic stroke (AIS), mechanical thrombectomy represents a unique opportunity for researchers to study the retrieved clots, with the possibility of unveiling biological patterns linked to stroke pathophysiology and etiology. We aimed to develop a shotgun proteomic approach to study and compare the proteome of formalin-fixed paraffin-embedded (FFPE) cardioembolic and large artery atherosclerotic (LAA) clots. Methods: We used 16 cardioembolic and 15 LAA FFPE thrombi from 31 AIS patients. The thrombus proteome was analyzed by label-free quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant v1.5.2.8 and Perseus v.1.6.15.0 were used for bioinformatics analysis. Protein classes were identified using the PANTHER database and the STRING database was used to predict protein interactions. Results: We identified 1,581 protein groups as part of the AIS thrombus proteome. Fourteen significantly differentially abundant proteins across the two etiologies were identified. Four proteins involved in the ubiquitin-proteasome pathway, blood coagulation or plasminogen activating cascade were identified as significantly abundant in LAA clots. Ten proteins involved in the ubiquitin proteasome-pathway, cytoskeletal remodeling of platelets, platelet adhesion or blood coagulation were identified as significantly abundant in cardioembolic clots. Conclusion: Our results outlined a set of 14 proteins for a proof-of-principle characterization of cardioembolic and LAA FFPE clots, advancing the proteome profile of AIS human thrombi and understanding the pathophysiology of ischemic stroke.

MicroCT Can Characterize Clots Retrieved With Mechanical Thrombectomy From Acute Ischemic Stroke Patients-A Preliminary Report.

Background: Characterization of the clot occluding the arteries in acute ischemic stroke received ample attention, in terms of elucidating the relationship between the clot composition, its etiology and its amenability for pharmacological treatment and mechanical thrombectomy approaches. Traditional analytical techniques such as conventional 2D histopathology or electron microscopy sample only small parts of the clot. Visualization and analysis in 3D are necessary to depict and comprehend the overall organization of the clot. The aim of this study is to investigate the potential of microCT for characterizing the clot composition, structure, and organization. Methods: In a pilot study, we analyzed with microCT clots retrieved from 14 patients with acute ischemic stroke. The following parameters were analyzed: overall clot density, clot segmentation with various density thresholds, clot volume. Results: Our findings show that human clots are heterogeneous in terms of CT intra-clot density distribution. After fixation in formalin, the clots display a shift toward negative values. On average, we found the mean HU values of red clots retrieved from patients to be -153 HU, with SD = 23.8 HU, for the intermediate clots retrieved from patients -193 HU, SD = 23.7 HU, and for the white clots retrieved from patients -229 HU, SD = 64.8 HU. Conclusion: Our study shows that volumetric and density analysis of the clot opens new perspectives for clot characterization and for a better understanding of thrombus structure and composition.

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

BACKGROUND AND PURPOSE: Given 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 METHODS: Consecutive 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. RESULTS: A 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. CONCLUSIONS: Our 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.

Does prior administration of rtPA influence acute ischemic stroke clot composition? Findings from the analysis of clots retrieved with mechanical thrombectomy from the RESTORE registry.

BACKGROUND AND PURPOSE: There is still much debate whether bridging-therapy [intravenous thrombolysis (IVT) prior to mechanical thrombectomy (MT)] might be beneficial compared to MT alone. We investigated the effect of IVT on size and histological composition of the clots retrieved from patients undergoing bridging-therapy or MT alone. METHODS: We collected mechanically extracted thrombi from 1000 acute ischemic stroke (AIS) patients included in RESTORE registry. Patients were grouped according to the administration (or not) of IVT before thrombectomy. Gross photos of each clot were taken and Extracted Clot Area (ECA) was measured using ImageJ software. Martius Scarlett Blue stain was used to characterize the main histological clot components [red blood cells (RBCs), fibrin (FIB), platelets/other (PTL)] and Orbit Image Analysis was used for quantification. Additionally, we calculated the area of each main component by multiplying the component percent by ECA. Chi-squared and Kruskal-Wallis tests were used for statistical analysis. RESULTS: 451 patients (45%) were treated with bridging-therapy while 549 (55%) underwent MT alone. When considering only percent histological composition, we did not find any difference in RBC% (P = 0.895), FIB% (P = 0.458) and PTL% (P = 0.905). However, bridging-therapy clots were significantly smaller than MT-alone clots [32.7 (14.8-64.9) versus 36.8 (20.1-79.8) mm2, N = 1000, H1 = 7.679, P = 0.006*]. A further analysis expressing components per clot area showed that clots retrieved from bridging-therapy cases contained less RBCs [13.25 (4.29-32.06) versus 14.97 (4.93-39.80) mm2, H1 = 3.637, P = 0.056] and significantly less fibrin [9.10 (4.62-17.98) versus 10.54 (5.57-22.48) mm2, H1 = 7.920, P = 0.005*] and platelets/other [5.04 (2.26-11.32) versus 6.54 (2.94-13.79) mm2, H1 = 9.380, P = 0.002*] than MT-alone clots. CONCLUSIONS: Our results suggest that previous IVT administration significantly reduces thrombus size, proportionally releasing all the main histological components.

Histological evaluation of acute ischemic stroke thrombi may indicate the occurrence of vessel wall injury during mechanical thrombectomy.

BACKGROUND: Several animal studies have demonstrated that mechanical thrombectomy (MT) for acute ischemic stroke (AIS) may cause vessel wall injury (VWI). However, the histological changes in human cerebral arteries following MT are difficult to determine. OBJECTIVE: To investigate the occurrence of VWI during MT by histological and immunohistochemical evaluation of AIS clots. METHODS: As part of the multicenter STRIP registry, 277 clots from 237 patients were analyzed using Martius Scarlett Blue stain and immunohistochemistry for CD34 (endothelial cells) and smooth muscle actin (smooth muscle cells). RESULTS: MT devices used were aspiration catheters (100 cases), stentriever (101 cases), and both (36 cases). VWI was found in 33/277 clots (12%). There was no significant correlation between VWI and MT device. The degree of damage varied from grade I (mild intimal damage, 24 clots), to grade II (relevant intimal and subintimal damage, 3 clots), and III (severe injury, 6 clots). VWI clots contained significantly more erythrocytes (p=0.006*) and less platelets/other (p=0.005*) than non-VWI clots suggesting soft thrombus material.Thrombolysis correlated with a lower rate of VWI (p=0.04*). VWI cases showed a significantly higher number of passes (2 [1-4] vs 1 [1-3], p=0.028*) and poorer recanalization outcome (p=0.01*) than cases without VWI. CONCLUSIONS: Histological markers of VWI were present in 12% of AIS thrombi, suggesting that VWI might be related to MT. VWI was associated with soft thrombus consistency, higher number of passes and poorer revascularization outcome. There was no significant correlation between VWI and MT device.

S100b in acute ischemic stroke clots is a biomarker for post-thrombectomy intracranial hemorrhages.

Background and purpose: Post-thrombectomy intracranial hemorrhages (PTIH) are dangerous complications of acute ischemic stroke (AIS) following mechanical thrombectomy. We aimed to investigate if S100b levels in AIS clots removed by mechanical thrombectomy correlated to increased risk of PTIH. Methods: We analyzed 122 thrombi from 80 AIS patients in the RESTORE Registry of AIS clots, selecting an equal number of patients having been pre-treated or not with rtPA (40 each group). Within each subgroup, 20 patients had developed PTIH and 20 patients showed no signs of hemorrhage. Gross photos of each clot were taken and extracted clot area (ECA) was measured using ImageJ. Immunohistochemistry for S100b was performed and Orbit Image Analysis was used for quantification. Immunofluorescence was performed to investigate co-localization between S100b and T-lymphocytes, neutrophils and macrophages. Chi-square or Kruskal-Wallis test were used for statistical analysis. Results: PTIH was associated with higher S100b levels in clots (0.33 [0.08-0.85] vs. 0.07 [0.02-0.27] mm2, H1 = 6.021, P = 0.014*), but S100b levels were not significantly affected by acute thrombolytic treatment (P = 0.386). PTIH was also associated with patients having higher NIHSS at admission (20.0 [17.0-23.0] vs. 14.0 [10.5-19.0], H1 = 8.006, P = 0.005) and higher number of passes during thrombectomy (2 [1-4] vs. 1 [1-2.5], H1 = 5.995, P = 0.014*). S100b co-localized with neutrophils, macrophages and with T-lymphocytes in the clots. Conclusions: Higher S100b expression in AIS clots, higher NIHSS at admission and higher number of passes during thrombectomy are all associated with PTIH. Further investigation of S100b expression in AIS clots by neutrophils, macrophages and T-lymphocytes could provide insight into the role of S100b in thromboinflammation.

Correlation between acute ischaemic stroke clot length before mechanical thrombectomy and extracted clot area: Impact of thrombus size on number of passes for clot removal and final recanalization.

INTRODUCTION: We assessed the correlation between thrombus size before and after mechanical thrombectomy, measured as length by Computed Tomography Angiography/Non-Contrast Computed Tomography (CTA/NCCT) and Extracted Clot Area, ECA, respectively. We also assessed the influence of thrombus size on the number of passes required for clot removal and final recanalization outcome. MATERIALS AND METHODS: Acute ischaemic stroke (AIS) thrombi retrieved by mechanical thrombectomy from 500 patients and data of clot length by CTA/NCCT were collected from three hospitals in Europe. ECA was obtained by measuring the area of the extracted clot. Non-parametric tests were used for data analysis. RESULTS: A strong positive correlation was found between clot length on CTA/NCCT and ECA (rho = 0.619,N = 500,P < 0.0001*). Vessel size influences clot length on CTA/NCCT (H2 = 98.6, P < 0.0001*) and ECA (H2 = 105.6,P < 0.0001*), but the significant correlation between CTA/NCCT length and ECA was evident in all vessels. Poorer revascularisation outcome was associated with more passes (H5 = 73.1, P < 0.0001*). More passes were required to remove longer clots (CTA/NCCT; H4 = 31.4, P < 0.0001*; ECA; H4 = 50.2, P < 0.0001*). There was no significant main association between recanalization outcome and length on CTA/NCCT or ECA, but medium sized clots (ECA 20-40 mm2) were associated with least passes and highest revascularisation outcome (N = 500, X2 = 16.2, P < 0.0001*). CONCLUSION: Clot length on CTA/NCCT strongly correlates with ECA. Occlusion location influences clot size. More passes are associated with poorer revascularisation outcome and bigger clots. The relationship between size and revascularisation outcome is more complex. Clots of medium ECA take less passes to remove and are associated with better recanalization outcome than both smaller and larger clots.

Studying Stroke Thrombus Composition After Thrombectomy: What Can We Learn?

The composition of ischemic stroke thrombi has gained an increasing amount of interest in recent years. The implementation of endovascular procedures in standard stroke care has granted researchers the unique opportunity to examine patient thrombus material. Increasing evidence indicates that stroke thrombi are complex and heterogenous, consisting of various biochemical (eg, fibrin, von Willebrand Factor, and neutrophil extracellular traps) and cellular (eg, red blood cells, platelets, leukocytes, and bacteria) components. This complex composition may explain therapeutic limitations and also offer novel insights in several aspects of stroke management. Better understanding of thrombus characteristics could, therefore, potentially lead to improvements in the management of patients with stroke. In this review, we provide a comprehensive overview of the lessons learned by examining stroke thrombus composition after endovascular thrombectomy and its potential relevance for thrombectomy success rates, thrombolysis, clinical outcomes, stroke etiology, and radiological imaging.

Characterization of the 'White' Appearing Clots that Cause Acute Ischemic Stroke.

OBJECTIVES: Most clots retrieved from patients with acute ischemic stroke are 'red' in color. 'White' clots represent a less common entity and their histological composition is less known. Our aim was to investigate the composition, imaging and procedural characteristics of 'white' clots retrieved by mechanical thrombectomy. MATERIALS AND METHODS: Seventy five 'white' thrombi were selected by visual inspection from a cohort of 760 clots collected as part of the RESTORE registry. Clots were evaluated histopathologically. RESULTS: Quantification of Martius Scarlett Blue stain identified platelets/other as the major component in 'white' clots' (mean of 55% of clot overall composition) followed by fibrin (31%), red blood cells (6%) and white blood cells (3%). 'White' clots contained significantly more platelets/other (p<0.001*) and collagen/calcification (p<0.001*) and less red blood cells (p<0.001*) and white blood cells (p=0.018*) than 'red' clots. The mean platelet and von Willebrand Factor expression was 43% and 24%, respectively. Adipocytes were found in four cases. 'White' clots were significantly smaller (p=0.016*), less hyperdense (p=0.005*) on computed tomography angiography/non-contrast CT and were associated with a smaller extracted clot area (p<0.001*) than 'red' clots. They primarily caused the occlusion of middle cerebral artery, were less likely to be removed by aspiration and more likely to require rescue-therapy for retrieval. CONCLUSIONS: 'White' clots represented 14% of our cohort and were platelet, von Willebrand Factor and collagen/calcification-rich. 'White' clots were smaller, less hyperdense, were associated with significantly more distal occlusions and were less successfully removed by aspiration alone than 'red' clots.

Per pass analysis of thrombus composition retrieved by mechanical thrombectomy.

BACKGROUND AND AIM: Mechanical thrombectomy (MT) for large vessel occlusion often requires multiple passes to retrieve the entire thrombus load. In this multi-institutional study we sought to examine the composition of thrombus fragments retrieved with each pass during MT. METHODS: Patients who required multiple passes during thrombectomy were included. Histopathological evaluation of thrombus fragments retrieved from each pass was performed using Martius Scarlet Blue staining and the composition of each thrombus component including RBC, fibrin and platelet was determined using image analysis software. RESULTS: 154 patients underwent MT and 868 passes was performed which resulted in 263 thrombus fragments retrieval. The analysis of thrombus components per pass showed higher RBC, lower fibrin and platelet composition in the pass 1 and 2 when compared to pass 3 and passes 4 or more combined (P values <0.05). There were no significant differences between thrombus fragments retrieved in pass 1 and pass 2 in terms of RBC, WBC, fibrin, and platelet composition (P values >0.05). Similarly, when each composition of thrombus fragments retrieved in pass 3 and passes 4 or more combined were compared with each other, no significant difference was noted (P values >0.05). CONCLUSION: Our findings confirm that thrombus fragments retrieved with each pass differed significantly in histological content. Fragments in the first passes were associated with lower fibrin and platelet composition compared to fragments retrieved in passes three and four or higher. Also, thrombus fragments retrieved after failed pass were associated with higher fibrin and platelet components.

The administration of rtPA before mechanical thrombectomy in acute ischemic stroke patients is associated with a significant reduction of the retrieved clot area but it does not influence revascularization outcome.

Both intravenous thrombolysis (IVT) and mechanical thrombectomy (MT) are evidence-based treatments for acute ischemic stroke (AIS) in selected cases. Recanalization may occur following IVT without the necessity of further interventions or requiring a subsequent MT procedure. IVT prior to MT (bridging-therapy) may be associated with benefits or hazards. We studied the retrieved clot area and degree of recanalization in patients undergoing MT or bridging-therapy for whom it was possible to collect thrombus material. We collected mechanically extracted thrombi from 550 AIS patients from four International stroke centers. Patients were grouped according to the administration (or not) of IVT before thrombectomy and the mechanical thrombectomy approach used. We assessed the number of passes for clot removal and the mTICI (modified Treatment In Cerebral Ischemia) score to define revascularization outcome. Gross photos of each clot were taken and the clot area was measured with ImageJ software. The non-parametric Kruskal-Wallis test was used for statistical analysis. 255 patients (46.4%) were treated with bridging-therapy while 295 (53.6%) underwent MT alone. By analysing retrieved clot area, we found that clots from patients treated with bridging-therapy were significantly smaller compared to those from patients that underwent MT alone (H1 = 10.155 p = 0.001*). There was no difference between bridging-therapy and MT alone in terms of number of passes or final mTICI score. Bridging-therapy was associated with significantly smaller retrieved clot area compared to MT alone but it did not influence revascularization outcome.

Large Artery Atherosclerotic Clots are Larger than Clots of other Stroke Etiologies and have Poorer Recanalization rates.

OBJECTIVES: There is a paucity of knowledge in the literature relating to the extent of clot burden and stroke etiology. In this study, we measured the Extracted Clot Area (ECA) retrieved during endovascular treatment (EVT) and investigated relationships with suspected etiology, administration of intravenous thrombolysis and recanalization. MATERIALS AND METHODS: As part of the multi-institutional RESTORE registry, the ECA retrieved during mechanical thrombectomy was quantified using ImageJ. The effect of stroke etiology (Large-artery atherosclerosis (LAA), Cardioembolism, Cryptogenic and other) and recombinant tissue plasminogen activator (rtPA) on ECA and recanalization outcome (mTICI) was assessed. Successful recanalization was described as mTICI 2c-3. RESULTS: A total of 550 patients who underwent EVT with any clot retrieved were included in the study. The ECA was significantly larger in the LAA group compared to all other etiologies. The average ECA size of each etiology was; LAA=109 mm2, Cardioembolic=52 mm2, Cryptogenic=47 mm2 and Other=52 mm2 (p=0.014*). LAA patients also had a significantly poorer rate of successful recanalization (mTICI 2c-3) compared to all other etiologies (p=0.003*). The administration of tPA was associated with a smaller ECA in both LAA (p=0.007*) and cardioembolic (p=0.035*) groups. CONCLUSION: The ECA of LAA clots was double the size of all other etiologies and this is associated with a lower rate of successful recanalization in LAA stroke subtype. rtPA administration prior to thrombectomy was associated with reduced ECA in LAA and CE clots.

Per-pass analysis of acute ischemic stroke clots: impact of stroke etiology on extracted clot area and histological composition.

BACKGROUND: Initial studies investigating correlations between stroke etiology and clot composition are conflicting and do not account for clot size as determined by area. Radiological studies have shown that cardioembolic strokes are associated with shorter clot lengths and lower clot burden than non-cardioembolic clots. OBJECTIVE: To report the relationship between stroke etiology, extracted clot area, and histological composition at each procedural pass. METHODS: As part of the multi-institutional RESTORE Registry, the Martius Scarlett Blue stained histological composition and extracted clot area of 612 per-pass clots retrieved from 441 patients during mechanical thrombectomy procedures were quantified. Correlations with clinical and procedural details were investigated. RESULTS: Clot composition varied significantly with procedural passes; clots retrieved in earlier passes had higher red blood cell content (H4=11.644, p=0.020) and larger extracted clot area (H4=10.730, p=0.030). Later passes were associated with significantly higher fibrin (H4=12.935, p=0.012) and platelets/other (H4=15.977, p=0.003) content and smaller extracted clot area. Large artery atherosclerotic (LAA) clots were significantly larger in the extracted clot area and more red blood cell-rich than other etiologies in passes 1-3. Cardioembolic and cryptogenic clots had similar histological composition and extracted clot area across all procedural passes. CONCLUSION: LAA clots are larger and associated with a large red blood cell-rich extracted clot area, suggesting soft thrombus material. Cardioembolic clots are smaller in the extracted clot area, consistent in composition and area across passes, and have higher fibrin and platelets/other content than LAA clots, making them stiffer clots. The per-pass histological composition and extracted clot area of cryptogenic clots are similar to those of cardioembolic clots, suggesting similar formation mechanisms.

Characterizing blood clots using acoustic radiation force optical coherence elastography and ultrasound shear wave elastography.

Thromboembolism in a cerebral blood vessel is associated with high morbidity and mortality. Mechanical thrombectomy (MT) is one of the emergenc proceduresperformed to remove emboli. However, the interventional approaches such as aspiration catheters or stent retriever are empirically selected. An inappropriate selection of surgical devices can influence the success rate during embolectomy, which can lead to an increase in brain damage. There has been growing interest in the study of clot composition and using a priori knowledge of clot composition to provide guidance for an appropriate treatment strategy for interventional physicians. Developing imaging tools which can allow interventionalists to understand clot composition could affect management and device strategy. In this study, we investigated how clots of different compositions can be characterized by using acoustic radiation force optical coherence elastography (ARF-OCE) and compared with ultrasound shear wave elastography (SWE). Five different clots compositions using human blood were fabricated into cylindrical forms from fibrin-rich (21% red blood cells, RBCs) to RBC-rich (95% RBCs). Using the ARF-OCE and SWE, we characterized the wave velocities measured in the time-domain. In addition, the semi-analytical finite element model was used to explore the relationship between the phase velocities with various frequency ranges and diameters of the clots. The study demonstrated that the wave group velocities generally decrease as RBC content increases in ARF-OCE and SWE. The correlation of the group velocities from the OCE and SWE methods represented a good agreement as RBC composition is larger than 39%. Using the phase velocity dispersion analysis applied to ARF-OCE data, we estimated the shear wave velocities decoupling the effects of the geometry and material properties of the clots. The study demonstrated that the composition of the clots can be characterized by elastographic methods using ARF-OCE and SWE, and OCE demonstrated better ability to discriminate between clots of different RBC compositions, compared to the ultrasound-based approach, especially in clots with low RBC compositions.

High-resolution scanning electron microscopy for the analysis of three-dimensional ultrastructure of clots in acute ischemic stroke.

BACKGROUND: Characterization of acute ischemic stroke (AIS) clots has typically focused on two-dimensional histological analysis of the thrombus. The three-dimensional (3D) architecture and distribution of components within emboli have not been fully investigated. The aim of this study was to examine the composition and microstructure of AIS clots using histology and serial block-face scanning electron microscopy (SBFSEM). METHODS: As part of the multi-institutional STRIP registry, 10 consecutive AIS emboli were collected from 10 patients treated by mechanical thrombectomy. Histological and immunohistochemical analysis was performed to determine clot composition. SBFSEM was used to assess the ultrastructural organization of the clots and specific features of individual components. RESULTS: Quantification of Martius Scarlett Blue stain identified fibrin (44.4%) and red blood cells (RBCs, 32.6%) as the main components. Immunohistochemistry showed a mean platelet and von Willebrand factor content of 23.9% and 11.8%, respectively. The 3D organization of emboli varied greatly depending on the region analyzed. RBC-rich areas were composed mainly of tightly packed RBCs deformed into polyhedrocytes with scant fibrin fibers interwoven between cells. The regions with mixed composition showed thick fibrin fibers along with platelets, white blood cells and RBC clusters. Fibrin-rich areas contained dense fibrin masses with sparse RBC. In three cases, the fibrin formed a grid-like or a sponge-like pattern, likely due to thrombolytic treatment. Segmentation showed that fibrin fibers were thinner and less densely packed in these cases. CONCLUSIONS: 3D-SEM provides novel and potentially clinically relevant information on clot components and ultrastructure which may help to inform thrombolytic treatment and medical device design.

Storage of blood clots for histological analysis: How long is too long in saline and paraformaldehyde?

To investigate the composition of blood clots following mechanical thrombectomy, it is essential to ensure optimum storage for highest quality histological and immunofluorescence analysis. We investigated for how long clots can be stored in paraformaldehyde (PFA), saline and heparinised saline before the tissue integrity is compromised. Whole blood and fibrin-rich clot analogues were made under dynamic flow conditions. Clots were stored in 4% PFA, saline or heparinised saline for timepoints ranging from 1 hour to two months. Five µm sections were stained with Martius Scarlet Blue to visualise red blood cells (RBCs), white blood cells (WBCs) and fibrin. Semi-quantitative analysis of the integrity of clot components used a scoring system (0: Poor; 1: Sub-par; 2: High). Quantitative analysis used Orbit Image Analysis software. Autofluorescence was assessed using a relative scale. Clots stored in PFA for up to two months were qualitatively similar to those stored for all shorter periods (median score: 2 per component). Clots stored in saline/heparinised saline for one week showed degradation of RBCs and WBCs, but fibrin remained intact (median score: 1, 1, 2 respectively). Degradation of the samples stored in saline/heparinised saline made accurate quantification using Image Analysis software difficult from 24h. Samples stored in PFA for up to two weeks showed an edging autofluorescence effect, which became more evident with prolonged storage. For optimum histology, ideally clots should not be stored in saline before fixation and should ideally be stored in formalin for less than one month to minimise the impact of autofluorescence on immunofluorescence.