Blood Collection and Preparation

The study was approved by the Medical Ethics Committee of UCLouvain and University Medical Center Utrecht (Study 17450). After informed consent, blood from the patient and 4 healthy volunteers was collected by venipuncture into K⁺/EDTA-coated tubes at the University Medical Center Utrecht. Whenever possible, healthy donors were selected to be age- and gender-matched. After collection, the tubes were transferred to the research laboratory at UCLouvain. Two splenectomised healthy donors were included in the study. Before experiments, RBCs were collected through 10-fold blood dilution in a glucose- and HEPES-containing medium [Dulbeccos modified eagle medium (DMEM), Invitrogen]. Diluted blood was centrifugated at 200 g for 2 min, the supernatant removed and RBCs suspended in medium. RBCs were washed a second time by centrifugation at 200 g for 2 min and resuspended, as in Cloos et al. (2020). The number of RBCs used for experiments were as follows: (i) Hb release, 12.5 10⁶; (ii) intracellular ATP content, 5 10⁶; (iii) calcium and ROS contents, 15 10⁶; (iv) lipidomic and FA analyses, 2.56 10⁸; (v) PS surface exposure, 0.5 10⁶; (vi) cholesterol assay, 37.5 10⁶; and (vii) fluorescence imaging, 12.5 10⁶.