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

Abstract

BackgroundCharacterization 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).MethodsAs 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.ResultsQuantification 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.Conclusions3D-SEM provides novel and potentially clinically relevant information on clot components and ultrastructure which may help to inform thrombolytic treatment and medical device design.

Association between clot composition and stroke origin in mechanical thrombectomy patients: analysis of the Stroke Thromboembolism Registry of Imaging and Pathology

“…Given the increased utilization of mechanical thrombectomy for the treatment of acute ischemic stroke secondary to large vessel occlusion, there has been growing interest in the analysis of retrieved thrombi 1–7. A number of studies have suggested that histopathological analysis can provide some valuable insights into stroke etiology 8–11.…”

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

“…Furthermore, a recent three-dimensional scanning electron microscopy analysis of AIS emboli showed that rt-PA treatment resulted in thinning of fibrin fibers and disruption of fibrin network which may explain the reduced clot size. 23 A recent study in an animal model reported more intima and medial layer damage after stentriever devices than with aspiration. Several factors may account for this difference, such as the mechanism of clot removal.…”

Clot Composition Analysis as a Diagnostic Tool to Gain Insight into Ischemic Stroke Etiology: A Systematic Review

“…Although H&E staining and machine learning software allowed reproducible quantification of the three major clot components (RBCs, WBCs, and FBR), 48 the more specific MSB staining was used in many studies for the selective quantification of RBCs and FBR. 28,37,39,43,44,[49][50][51][52][53][54][55][56][57][58][59][60][61] The Picro-Mallory stain was used to assess the maturity of FBR in thrombi, based on its age-dependent differential staining. 62 Immunohistochemical staining procedures allowed the best visualization and reliable separate quantification of RBCs (glycophorin A, also designated CD235a antigen), [63][64][65] WBCs (CD45 antigen), [66][67][68][69][70] PLTs (CD41, CD42b, and CD61 antigens), 38,45,49,51,53,56,57,60,63,65,66,[71][72][73] and fibrinogen/FBR.…”

“…28,37,39,43,44,[49][50][51][52][53][54][55][56][57][58][59][60][61] The Picro-Mallory stain was used to assess the maturity of FBR in thrombi, based on its age-dependent differential staining. 62 Immunohistochemical staining procedures allowed the best visualization and reliable separate quantification of RBCs (glycophorin A, also designated CD235a antigen), [63][64][65] WBCs (CD45 antigen), [66][67][68][69][70] PLTs (CD41, CD42b, and CD61 antigens), 38,45,49,51,53,56,57,60,63,65,66,[71][72][73] and fibrinogen/FBR. 49,53,63,65,66,74 Depending on the discriminatory capability of the staining procedures used, the studies expressed the ratios of clot components according to four different classifications, as shown in Table 2.…”