Imaging flow cytometry documents incomplete resistance of human sickle F-cells to ex vivo hypoxia-induced sickling. (A) HbF in the hemolysate correlates positively with the percentage of F-cells detected by SIFCA. HbF protective effect against sickling is shown by a positive correlation with the percent of cells that remain in normal shape after deoxygenation (B), and an opposite correlation with the percent of cells that become sickled (C). Sample images acquired by the imaging flow cytometer show simultaneous brightfield images (left column) and fluorescence of an anti-HbF-PE antibody (right column) in a normal non-F-cell (Di), sickled non-F-cell (Dii), normal F-cell (Diii), and a sickled F-cell (Div). (E) Transmission electron microscopy of RBCs enriched by fluorescence-activated cell sorting was performed to confirm that sickled cells contained similar fibers corresponding to hemoglobin S polymers regardless of whether they were non-F-cells (top) or F-cells (bottom). Bar = 100 nm. Images acquired with a JEM1400 electron microscope (JEOL) equipped with an AMT XR-111 digital camera (Advanced Microscopy Techniques Corporation). Image Adjust-Levels and Image Adjust Brightness functions in Photoshop Creative Suite 6 software (Adobe Systems Corporation) were used to equalize the densities in both images. (F) Non-F-cells sickled more than F-cells in both patients off hydroxyurea (left side, *P = .0014, paired Student t test) and on HU (right side, *P = .0057, paired Student t test), but cells from treated patients sickle less than from untreated patients (Mann-Whitney test, #P = .0197 for F-cells from patients off HU compared with on HU, P = .0501 for comparison between non-F-cells).