Splenectomy partially reestablishes RBC baseline characteristics as well as RBC morphology and functionality–related parameters. (A-L) RBCs from patients (P; 1 color, 1 family), either splenectomized (spl; filled circles or semifilled circles for intrafamily comparison) or not (nonspl; open circles), and RBCs from healthy controls (CTLs; light and dark blue dotted lines for child and adult donor ranges, respectively; or subtraction from P values [ΔP–CTL]) were compared for morphology (A-D), baseline characteristics (E-G), and functionality (H-L). (M-N) Based on these different parameters, a RBC alteration score was calculated. Statistics are indicated above the patient cohorts for the comparison with CTL values and above a horizontal line for comparison between the 2 patient cohorts, respectively. (A-D) RBC morphology determined by electron or light microscopy on RBCs in suspension. The relative abundance of discocytes (A), spherocytes (B), stomatocytes (C), and echinocytes (D) was evaluated and expressed as percentage of the global RBC population (mean of 1-5 independent experiments per patient; Kruskal-Wallis tests followed by Dunn post hoc for the comparison of the 3 cohorts). (E) RBC distribution width (mean of 1-7 independent measurements per patient; Mann-Whitney tests to compare the 2 patient cohorts). (F-G) Hemi-RBC membrane area and area-to–mean corpuscular volume (MCV) ratio. (F) Hemi-area of RBCs spread on poly-L-Lysine (PLL)–coated coverslips. (G) Ratio of values provided in panel F to the MCV provided in supplemental Figure 1G (mean of 4-23 independent measurements per patient for panel F; Kruskal-Wallis tests followed by Dunn post hoc for the comparison of the 3 cohorts). (H) RBC osmotic fragility determined in increasingly hypotonic media. The osmolarity required to lyse 50% of RBCs (Half maximal effective concentration [EC₅₀]) was calculated using hemolysis curves (mean of 1-5 independent experiments per patient; Kruskal-Wallis test followed by Dunn post hoc). (I) RBC cryohemolysis (mean of 1-6 independent experiments per patient; Mann-Whitney test). (J) EV abundance in plasma samples determined by Nanoparticle tracking analysis (mean of 1-3 independent experiments per patient; Kruskal-Wallis test followed by Dunn post hoc). (K) Intracellular calcium content. RBCs were labeled with the nonfluorescent Fluo4-AM, which is transformed in RBCs into the fluorescent Fluo4 after de-esterification and interaction with calcium ions. Labeled RBCs were analyzed by fluorimetry, and data were normalized to the Hb content (mean of 1-11 independent experiments per patient; Kruskal-Wallis test followed by Dunn post hoc). (L) Intracellular ATP content determined with a kit based on the activity of the firefly luciferase in the presence of ATP and emitted light in the presence of luciferin. ATP levels were normalized to Hb (mean of 1-8 independent experiments/patient; Kruskal-Wallis test followed by Dunn post hoc. (M) RBC morphology, functionality and biological parameters considered to establish the RBC functionality alteration score. These parameters were associated with a scale ranging from 0 to 1 when the parameter was nearly unaffected for most patients or from 0 up to maximum 8 when different degrees of affection for a parameter were observed in patient cohorts. The different scores corresponding to the different parameters were then added and the sum divided by the maximal score that could have been obtained to determine the RBC global functionality alteration score for each patient. The closer the score to 1, the more affected the RBCs by the disease. (N) RBC alteration score (Kruskal-Wallis tests followed by Dunn post hoc). MCHC, mean corpuscular Hb concentration; ns, not significant; RDW, red cell distribution width.