Human ITP serum–triggered macrophage phagocytosis of platelets in vitro. (A) Serum from patients with ITP were evaluated for their potential to trigger THP-1-CD16A macrophage phagocytosis of platelets. Each serum was evaluated over 4 to 5 independent experiments. As a negative control, sera from healthy laboratory donors (NHS) were used to opsonize platelets (4 unique NHS donors used over 9 independent experiments). Phagocytosis was performed blinded to patient clinical information and all characterization of ITP sera to avoid bias. Only ITP sera negative for MHC class I alloantibodies (n = 19) are displayed. Phagocytosis was performed as described in the “Methods” and quantified from fluorescent photomicrographs. Phagocytic index: number of phagocytosed platelets per 100 counted macrophages. Dashed line indicates the phagocytic index mean of NHS plus 2 SD . Error is presented as mean ± 1 SD. (B) Example photomicrographs of THP-1-CD16A macrophages after incubation with human platelets incubated human ITP serum. White arrows point toward example macrophages with phagocytosed platelets. Platelets were labeled with the cytoplasmic dye CMFDA (green, top row). External (nonphagocytosed) platelets were identified after phagocytosis using an AlexaFluor 647 (AF647)-conjugated anti-GPIX antibody (red, middle row). Merged fluorescence and DIC images are shown in the bottom row. Areas of red and green colocalization in the merge appear as yellow-orange. Scale bar: 10 μM. Photomicrographs were taken by spinning-disk confocal microscopy under 63× objective oil immersion (numerical aperture 1.47) with DIC and laser fluorescence (488, 647) channels on a Quorum multimodal imaging system equipped with 50 μm pinhole spinning disk and ORCA-Flash 4.0 V2 PLUS sCMOS camera. At least 4 images at distinct locations were taken near the center of each coverslip with Z-stacking every 0.33 μm. Z-stacked images were 3D reconstructed for analysis using Imaris v8.0.2.