Abnormal appearance of V205GR characterized as spherocytic hemolytic anemia. (A) Macroscopic appearance of 3-day-old neonates. Note that V205GR-A neonate shows (lower pup) marked yellowish skin pallor compared with WT littermate (middle pup), whereas Gata1^G1.05/X (upper pup) shows significant anemia. (B) Plasma color derived from WT (top) and V205GR-A (bottom). (C) Bilirubin concentration in the neonatal plasmas. Ten WT, 4 V205GR-A, and 3 V205GR-B neonates at the age of 2 to 3 days old were used for this experiment (open bars, total-bilirubin; filled bars, indirect-bilirubin; *P < .05, **P < .01). (D) Abnormal red blood cells in the blood smears. Various sizes of red cell fragments were observed in V205GR-A at 2 days old (right panel). The left panel is the WT control. Scale bars correspond to 20 μm. (E) Scanning electron microscopic analysis of erythrocytes prepared from V205GR-A newborn (right panel) and WT control (left panel). Scale bars correspond to 1 μm. (F) Osmotic fragility curves of erythrocytes from V205GR-B (black line) or WT (gray line) pups. Percent hemolysis was calculated for each solution based on the absorbance value obtained from 0.2% concentration. (G) ROS levels in erythrocytes of WT (dotted line) and V205GR-B (solid line) pups without (left panel) or with (right panel) addition of exogenous H₂O₂ (50μM).