OC 03.1 - Detailed assessment of the microenvironment of stroke thrombi using imaging mass cytometry

Background: Assessment of the cellular and molecular components of thrombi retrieved from stroke patients has advanced our understanding of thrombosis, pathology and treatment. However, limitations of approaches to date include the restricted number of matrix and cellular components that are assessed simultaneously, with analysis typically confined to histology or limited antibodies in immunohistochemistry and immunofluorescence.

Aims: To gain a detailed overview of the composition of ischaemic stroke thrombi using imaging mass cytometry (IMC) and to correlate thrombus matrix and cellular components with clinical characteristics.

Methods: Thrombi and clinical information were collected from ischemic stroke patients who underwent thrombectomy. Thrombus sections were stained using 20 metal-conjugated antibodies. For detection, an area was laser ablated and metals detected by cytometry time-of-flight. Cells were segmented using ilastik and CellProfiler software and populations were extracted using single-cell data.

Results: We identified the expected heterogeneous thrombus composition, with all antibodies against matrix components detected simultaneously including DNA, red blood cells, platelets (Fig.1A-D), platelet activation markers, fibrin(ogen) and von Willebrand factor (VWF). We identified correlations between platelet content and VWF (r=0.53, P=0.034), and fibrin(ogen) (r=0.73, P=0.001). Antiplatelet medication prescribed at the time of stroke had no impact on CD62P+ activated platelet content.
We identified seven cell populations which mapped to thrombi accurately, but acellular components were also detected (Fig.1E). Leukocyte phenotype contribution was heterogeneous but differed with clinical information including a greater contribution of all leukocytes in cardioembolic aetiology. Patients who did not receive alteplase had a larger neutrophil contribution to the thrombus.

Conclusion(s): We implemented a novel technique, IMC, to simultaneously image 20 thrombus components, with the potential to expand this to include up to 50 leukocyte and platelet activation markers and coagulation components simultaneously. Such an approach will help understand the matrix and cellular contributions to thrombus progression and treatment of thrombus-related diseases including stroke.
British Heart Foundation funded.