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. 2020 Aug 27;106(9):2478–2488. doi: 10.3324/haematol.2020.261586

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Copyright© 2021 Ferrata Storti Foundation

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Figure 4. — Expression pattern of Lu/BCAM and ICAM-4 on red blood cells by confocal microscopy and imaging flow cytometry. Confocal microscopy images of red blood cells (RBC) expressing (A) Lu/BCAM and (B) ICAM-4 under non-oxidized and oxidized conditions. (C) Imaging flow cytometry analysis steps of Lu/BCAM expression on the RBC surface. After gating the single cells, gating is set on RBC facing the camera (circular cells), then on those expressing Lu/BCAM. In the final step a modulation feature is applied to measure the intensity range and distribution of Lu/BCAM, and define two main patterns: Low Modulation (Spots) and High Modulation (Patches). (D) Percentage of Lu/BCAM-positive RBC with Spots pattern (left) and Patches pattern (right) (n=10). Wilcoxon test, **P<0.01. (E) Representative flow cytometry plots of Lu/BCAM expression in a non-oxidized (phosphate buffered saline [PBS]) and oxidized (cumene hydroperoxide) AA RBC sample. (F) Percentage of ICAM-4-positive RBC with Spots pattern (left) and Patches pattern (right) (n=8). Wilcoxon test, P=0.8125 and P=0.3828. (G) Representative flow cytometry plots of ICAM-4 expression in a non-oxidized (PBS) and oxidized (cumene hydroperoxide) AA RBC sample. (H) Percentage of Lu/BCAM-positive RBC with Spots pattern (left) and Patches pattern (right) in low-density (LD) and high-density (HD) SS RBC (n=8); Wilcoxon test, **P<0.01, *P<0.05. (I) Representative flow cytometry plots of Lu/BCAM expression in LD and HD RBC from one SS blood sample.