Anti-D triggers macrophage-mediated erythrocyte membrane loss consistent with macrophage trogocytosis. (A) Binding of RhD-specific pIgG (anti-D, WinRho SDF) to RhD⁺-human RBCs (RhD⁺ hRBCs) was performed by incubation of 5 × 10⁶ RhD⁺ hRBCs with 50 μL of anti-D solutions yielding anti-D concentrations from 1770 ng/mL to 0.86 ng /mL (0.885-0.0004 IU), or with a mix of human IgG subclasses as an isotype control. Later, the RBCs were incubated with AF647-labeled anti–human IgG and analyzed by flow cytometry. MFI of the RBCs was determined. (B) THP-1-CD16A macrophages were stimulated with RhD⁺ hRBCs sensitized with anti-D, with the isotype control or nonsensitized for 30 minutes at 37°C and 5% CO₂. External, nonphagocytosed RBCs were removed by hypotonic lysis. Brightfield microscopy images of macrophages from each condition were captured. The phagocytic index was calculated as the number of RBCs engulfed per 100 macrophages. Data represents the mean ± SEM of 4 independent experiments with the total number of data points per group equaling n = 5 to 11. (C) RhD⁺-human RBCs were fluorescently labeled with PKH67 (PKH67⁺ RhD⁺ hRBCs) and sensitized with different concentrations of anti-D as indicated. Nonsensitized PKH67⁺RhD⁺ hRBCs (0 ng/mL) or sensitized with isotype control were used as controls. Sensitized and nonsensitized PKH67⁺RhD⁺-hRBCs were incubated with or without THP-1-CD16A macrophages for 30 minutes and 3 hours at 37°C and 5% CO₂. Later, RBCs were recovered from each condition and the residual external RBCs attached outside of macrophages were lysed. MFI of the PKH67 signal of the PKH67⁺RhD⁺-hRBC recovered after incubation in the presence vs absence of macrophages were analyzed by flow cytometry. The quantity of RBC membrane loss was measured as a percentage of the MFI of the PKH67 signal from RBCs that had been incubated with macrophages to the MFI of RBCs without macrophages. Data represent the mean ± SEM of 3 independent experiments with 2 replicates per condition. (D-E) THP-1-CD16A macrophages incubated with nonsensitized vs sensitized RBCs were also analyzed by flow cytometry, and (D) the percentage of the PKH67⁺ macrophages and (E) the MFI of PKH67 signal in the PKH67⁺ macrophage gate was determined. The dotted and dashed lines represent background fluorescence of macrophages incubated with isotype control sensitized RBC at 30 minutes and 3 hours, respectively. (F) THP-1-CD16A macrophages were seeded on coverslips for 2 days. Later, macrophages were stimulated with PKH67⁺RhD⁺-hRBCs sensitized with anti-D at the indicated concentrations, nonsensitized, or sensitized with isotype control. After 3 hours of incubation, excess RBCs were washed away, and external RBCs were lysed followed by fixation. Images from each condition were taken on a Quorum multimodal imaging system (spinning disk confocal) at original magnification ×63 objective oil immersion (numerical aperture 1.47). Scale bar = 10 μm. Statistical analyses of the data represented in the graph of panel B were performed by Kruskal-Wallis test with Dunn multiple comparisons post hoc test, by two-way ANOVA with Sidak multiple comparisons post hoc test for data in the graph of panel C, and Dunnett multiple comparisons post hoc test for the graph of panels D and E. Significant differences to the control group (nonsensitized) at 30 minutes and 3 hours are indicated with ∗ and ^& respectively. (∗P = .05; ∗∗P = .01; ∗∗∗P = .001; ∗∗∗∗P = .0001; ^&P = .05; ^&&&P = .001; ^&&&&P = .0001).