Diagnostic and Prognostic Role of Clot Analysis in Stroke Patients

BACKGROUND AND RATIONALE OF THE STUDY

The analysis of the composition of the clot constitutes a promising tool for investigating the possible pathogenetic mechanisms underlying ischemic stroke. This analysis was made possible thanks to the numerous mechanical thrombectomy operations which have now become routine.

Several studies have attempted to explore the possible relationship between the primary site of thrombus formation and clot composition, reporting that cardioembolic stroke may have a higher percentage of platelet-rich areas than noncardioembolic thrombosis. However, the data are conflicting and do not seem to support an association between clot histology and etiology. Furthermore, thrombus composition appears to influence thrombolysis and the efficacy of thrombectomy. For example, fibrin-rich thrombi appear to reduce the effectiveness of thrombolytic treatment and require more steps with mechanical thrombectomy treatment.

Primary ENDPOINT:

Evaluate how clot composition relates to stroke etiology according to the TOAST classification.

Secondary ENDPOINT:

• relationship between different clot components and the degree of thrombectomy recanalization as defined by treatment modified cerebral ischemia score (mTICI).
• relationship between the different components of the clot and the number of steps required to achieve recanalization.
• relationship between the different clot components and outcome indicators (NIHSS score and mRS score).

TARGET POPULATION Patients with ischemic stroke with occlusion of large intracranial vessels will be included in the study if deemed suitable for mechanical thrombectomy therapy in accordance with national and international guidelines.

INCLUSION CRITERIA

• age > 18 years;
• Patients diagnosed with large vessel occlusion stroke in the emergency room CT Angio-study, undergoing mechanical thrombectomy procedure.
• Recovered thrombus available for analysis

EXCLUSION CRITERIA

● Lack of written informed consent.

MATERIALS AND METHODS The clot will be portioned. Part of the sample will be fixed in a 10% formalin solution (3.7% formaldehyde), part will be frozen in liquid nitrogen. Within 24-48 hours of fixation, formalin-fixed thrombi will be dehydrated by increasing the concentration of ethanol (70%, -80%, -95%, 100%) and paraffin-embedded allowing good preservation of tissue morphology and easy long-term storage. The included samples will be sectioned along the major axis of the thrombus, in slices with a thickness between 4 and 5 µm.

Base staining will be used to visualize RBC, PLT and fibrin.

• Hematoxylin and Eosin (H&E) will allow visualization of general thrombus structures and identification of aggregates of fibrin/platelets (colored pink), red blood cells (red), and nucleated cells (dark blue).

• Martius Scarlet Blue (MSB), selectively stains fibrin (dark pink/red), red blood cells (yellow) and collagen (blue

• Mallory-Azan for collagen and phosphotungstic acid hematoxylin for fibrin.

• immunohistochemistry to detect the presence of

• Platelets (CD42-Gp-Ib+, CD41a-Gp-IIb/IIIa+, CD61-GpIIIa),

• white blood cells (CD45+, common leukocyte antigen), or monocytes/macrophages (CD14+, CD1a+, CD68+),

• T lymphocytes (CD3+, CD8/CD4+), or natural killers (CD16+, CD56+), or mobile premature endothelial cells and blood progenitors (CD34+), or neutrophils (CD45+, CD16+), or fibrinogen or von Willebrand factor.