Abstract TP20: Protocol to Isolate Lymphocytes for Flow Cytometry From BACTRAC Samples

Abstract

Introduction: Since 2015, mechanical thrombectomy is the standard treatment for emergent large vessel occlusion stroke. Using standard techniques during mechanical thrombectomy, the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683) isolates distal blood within the artery immediately downstream from the clot, peripheral blood proximal to the clot, and the thrombus. BACTRAC is the first protocol utilizing the thrombectomy technique to collect local whole blood samples during brain infarction. We aimed to augment the current collection protocol to reproducibly obtain and study local leukocyte populations during human stroke. Methods: We started with the established BACTRAC protocol previously published (PMID: 30064997). We modified the tissue collection protocol to isolate lymphocytes for flow cytometry and to bank the clot and plasma. We performed a flow cytometry panel that identifies CD3, 4, 8, 11b, 11c, 14, 19, 31, 45, 66b, 161, 183 markers to investigate populations of B-cells, T-cells, dendritic cells, NK cells, macrophages/monocytes, granulocytes, endothelial cells, and progenitors, respectively. Results: The protocol was first established in healthy controls (n=9) by drawing venous blood and simulating a vial of distal blood and a vial of proximal blood. In healthy subjects, we were able to isolate on average 2x10 6 leukocytes/ml with a viability of 97.85%. Banked lymphocytes are also viable upon reconstitution and for flow cytometry. This protocol successfully isolates leukocytes in proximal and distal blood samples in subjects undergoing mechanical thrombectomy, with identification and analysis by flow cytometry currently ongoing. Conclusions: This modification to the existing BACTRAC protocol provides the opportunity, for the first time, to study local leukocyte populations in the arteries undergoing ischemic stroke in the human condition. Efficient processing of these samples will provide insight into the neuroinflammatory microenvironment of the occlusion and accelerate translational stroke research towards new methods of diagnosis and treatment.