Isolation and identification of leukocyte populations in intracranial blood collected during mechanical thrombectomy

Abstract

Using standard techniques during mechanical thrombectomy, the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (NCT03153683) isolates intracranial arterial blood distal to the thrombus and proximal systemic blood in the carotid artery. We augmented the current protocol to study leukocyte subpopulations both distal and proximal to the thrombus during human stroke (n = 16 patients), and from patients with cerebrovascular disease (CVD) undergoing angiography for unrelated conditions (e.g. carotid artery stenosis; n = 12 patients). We isolated leukocytes for flow cytometry from small volume (<1 mL) intracranial blood and systemic blood (5-10 mL) to identify adaptive and innate leukocyte populations, in addition to platelets and endothelial cells (ECs). Intracranial blood exhibited significant increases in T cell representation and decreases in myeloid/macrophage representation compared to within-patient carotid artery samples. CD4+ T cells and classical dendritic cells were significantly lower than CVD controls and correlated to within-patient edema volume and last known normal. This novel protocol successfully isolates leukocytes from small volume intracranial blood samples of stroke patients at time of mechanical thrombectomy and can be used to confirm preclinical results, as well as identify novel targets for immunotherapies.

Keywords: Cerebrovascular endothelium; T cells; emergent large vessel occlusion (ELVO); flow cytometry; ischemic stroke.

Conflict of interest statement

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.

Cell yield and viability. (a) Viability of cells is independent of volume processed for 1 mL volumes (squares) and 9 mL volumes (circles). F1,44 = 0.67, p = 0.42 for volume; F1,44 = 0.04, p = 0.84 for injury status; n = 10 controls (white symbols); n = 14 stroke patients (red/blue symbols). (b) Significant viability loss occurred during the cryopreservation process (clear bars) compared to viability prior to banking (hatched bars). * p < 0.05; **** p < 0.0001; n = 12 controls (clear circles), n = 16 stroke (filled circles, systemic; squares, intracranial), n = 12 cerebrovascular disease patients (triangles). One intracranial sample was unreadable using the Nexcelom Cellometer after recovery from cryopreservation but did have viable cells by flow cytometry. (c) Correlation between last known normal and % viability in intracranial blood at time of collection does not show an effect of time to mechanical thrombectomy on leukocyte yield; F1,11 = 1.81, p = 0.21. r2 = 0.14.

Figure 2.

Distribution of tSNE-identified leukocyte populations. (a) tSNE dimensionality reduction and clustering for the general immune panel including innate and adaptive leukocytes. (b) Cell populations identified in A are color coded. (c–f) tSNE overlays of total (grey) and healthy venous control (c), cerebrovascular disease (CVD) control (d), systemic stroke sample (e), and intracranial stroke sample (f). Note the striking differences between systemic and intracranial density localizations, and how they resemble either healthy control or CVD control, respectively.

Figure 3.

Leukocyte changes in intracranial blood prior to thrombectomy. (a–e) All panels show individual healthy subject control venous populations (dashed line), stroke patient systemic arterial stroke samples (red circles; grey bars), intracranial arterial stroke samples (blue squares; grey bars), and cerebrovascular disease (CVD) control patient samples (green triangles; white bar). Subpopulations for (a) B cells, (b) T cells, (c) innate subpopulations, (d) natural killer (NK) populations, and (e) dendritic cells (DC) are shown as the percent of total live CD45+ events and respective subgating on the y axis. *p < 0.05 for within-patient paired t-test; †p < 0.05; ††p < 0.01; †††p < 0.001 from CVD. Control n = 12, stroke n = 16, CVD n = 12. Data also shown as Table 2. (F-H) Results from within-patient Spearman Correlation matrix found association between (f) intracranial CD4+ T cells increasing with edema volume, as well as (g) systemic NK T cells increasing with patient BMI, and (h) systemic classical DCs (cDCs) decreasing with time from last known normal (LKN).

Figure 4.

Thrombus, platelet, and endothelial cell analyses. (a) There is a significant inverse correlation between systemic granulocyte populations and neutrophils per clot field. Representative 40X images of (b) thrombus of Patient 99 (left panel; high systemic granulocytes, but low neutrophil count in thrombus) vs. Patient 127 (right panel; low systemic granulocytes, but high neutrophil count in thrombus). (c) Platelet populations are not significantly different between systemic and intracranial samples. Control venous (black circles), systemic arterial stroke samples (red squares) and intracranial arterial stroke samples (blue triangles). (d) Endothelial cell populations are not significantly different between systemic and intracranial samples. (e) Dot plot of systemic CD31 gating (x axes) for systemic and intracranial blood of Patient 112. Note the lack of CD31+CD45- events in systemic blood (left panel). Patient 112 shows a substantial CD31+CD45- endothelial cell population in intracranial blood (right panel). (f) Patient 112 initial CT angiogram shows vessel occlusion (right panel, black circle). (g) This patient later underwent severe hemorrhagic transformation from Day 1 to Day 6 post-thrombectomy (red elipse).