Protocol for assessing and visualizing cell microaggregate formation in whole blood by imaging flow cytometry

Abstract

Blood cell aggregates are clinically useful biomarkers in a number of medical disorders. This protocol provides accurate and quantitative analysis of cell aggregates using a small volume of whole blood and imaging flow cytometry. We describe steps for sample collection, staining, and measurement. We then detail gating procedures and analysis of cell morphology. Sample preparation artifacts, activation, and morphological changes of cells are mitigated by omitting erythrocyte lysis and leukocyte isolation while maintaining high-throughput accurate imaging of leukocytes and platelets.

Keywords: Clinical Protocol; Flow Cytometry; Immunology; Microscopy.

Graphical abstract

Figure 1

Gating of IFC data, WB-Protocol, unstimulated and unfixed (A) The acquisition template is designed to gate out most RBCs via negative selection. The left figure displays the available populations for reference, the right figure displays the actual acquisition gate used for the experiments. Only events in this gate are saved for future analysis. (B) The analysis is done in Ideas 6.3, proprietary software which combines bright-field imaging and fluorescent intensities to provide high throughput image-based analysis. First, small particles and focusing beads (Amnis SpeedBead) were excluded. After gating on in-focus events, we analyze aggregates containing WBCs or only PLTs. (B I) To detect WBC-PLT aggregates and WBC-WBC aggregates, we use following gating strategy: WBC-WBC aggregates are detected via symmetry and circularity analysis. For WBC-PLT aggregates, double positive events for CD45+ and CD41a+ were selected. To exclude coincidental events (platelets and WBCs share one frame but are not adherent to each other) we used the internalization mask. The actual aggregates can now be analyzed using various morphological features. (B II) To detect PLT-PLT aggregates, PLT activation and shape following gating strategy was used: All WBC-PLT aggregates were excluded. To remove remaining RBCs we gate on CD31-CD41a double positive events. This population is analyzed for PLT-PLT aggregates, platelet activation and platelet morphology.

Figure 2

Expected results of the WB protocol, IFC pictures of various cell aggregates (A, C, D, and E) Depict the composite images of the channels for CD41a (red), CD31 (yellow) and CD45 (violet). (A) Features PLT-WBC-aggregates (Figure 1, gate BI6). The scale bar measures 7 micrometers. (B) Displays PLT-PLT aggregates (Figure 1, gate BII5a). Here each channel and the bright-field image are displayed individually. (C) Highlights additional composite images of platelet-platelet aggregates. (D) Shows leukocyte-leukocyte aggregates (Figure 1, gate BI4a). (E) Displays coincidental events (Figure 1, gate BI5). Additional populations are shown in Figure S1 e.g., erythrocyte-platelet-aggregates.

Figure 3

Ratios of cell aggregates and surface marker intensities – WB protocol vs. TRAP6-stimulated positive control (A–E) The unstimulated WB protocol was compared with a WB sample stimulated for 1 min with TRAP6 as positive control, n = 25. The median intensity or the percentage of the selected population is displayed. The platelet marker CD62P is used to assess platelet activation. Additionally, morphological changes of platelets, which are a sign of early platelet activation (pseudopods, see Figure S1F), are being compared, based on gate B-II-6. Cell aggregates were determined using the mentioned gating protocol. A Kruskal-Wallis with Dunn’s Post hoc was performed, where ∗ : p < 0.05, ∗∗ :p < 0.005, ∗∗∗: p < 0.0005. (F and G) Spearman correlation analysis conducted between cell concentrations of platelets and leukocytes obtained using the WB protocol and those measured by a validated automated hematology analyzer from the routine diagnostics laboratory (Sysmex XN-350). Cell concentrations were automatically determined by the IFC. PLT-WBC aggregate concentrations are based on gate B-I-6, Platelet concentrations are based on gate B-II-4, WBC concentrations on gate B-I-3. The Spearman correlation coefficient (r) and its corresponding p-value are annotated on the plot. (H) Kernel Density Estimation of absolute WBC counts of CD45+ cells of all measured unstimulated samples. The X-axis depicts the counts, the Y-axis the respective density. The dashed line indicates the mean WBC counts of all samples.

Figure 4

Determination of optimal anticoagulant selection and measurement timing The anticoagulants citrate 3.2%, natrium-heparin (17 IU/mL blood), and EDTA (1.8 mg/ml blood) were compared at basal conditions (without stimulation) over 6 h for healthy controls (n = 5). The time from the blood draw to the start of the experimental protocol was recorded. The whiskers express the 1st and 4th IQR, lines connect the mean. Percentages of the selected population in relation to the total population or the median marker intensities are displayed. (A and B) The ratio of cell-aggregates of their parent population are compared. (C) The combined intensities of CD69 and HLADR on the surface of WBCs (gate B-I-3) are compared over time. (D) Platelet activation was evaluated by the ratio of PLTs with surface expression CD62P. (E) The PLT concentration (based on gate B-II-4) was compared over time. (F) The intensity of CD31 on PLTs was compared over time.

Figure 5

Comparison of WB and RBC lysis protocols The WB protocol was compared with an RBC lysis protocol, n = 17. The median intensity or the ratio of the selected population is displayed. We performed a Kruskal-Wallis with Dunn’s Post hoc where ∗ : p < 0.05, ∗∗ :p < 0.005, ∗∗∗: p < 0.0005. (A) The ratio of positive CD62P expression on the PLT surface based on gate B-II-5b was compared. (B) The intensity of CD62P on PLT-WBC-aggregates. (C–E) The ratio of cell aggregates compared to the respective parent populations are displayed. Large PLT-leukocyte aggregates are defined by gate B-I-7. (F) The size of PLT aggregates determined by the CD41a expression area, based on gate B-I-6 are being compared. (G and H) The concentration of cell aggregates in WB and lysis are being compared.

Figure 6

Catching large PLT-PLT aggregates in the PLT-WBC gate A sample stimulated with TRAP6 as described includes large PLT-PLT aggregates which can be confused with PLT-WBC aggregates. Abnormally high CD62P intensities give these aggregates away.