Wright stained peripheral blood pictures from 2 index patients who had normal α- and β-globin genes. In P1 (A), a marked hypochromic microcytosis with anisopoikilocytosis, acanthocytes, and numerous nucleated red blood cells was observed; in P5 (B), a blood picture with numerous fragmented red blood cells with schistocytes and numerous nucleated red blood cells was seen, similar to nonspherocytic hemolytic anemia. Both smears were performed after splenectomy in both patients and were free from blood transfusion. Peripheral blood features in other patients are available in supplemental Figure 1. (C) IEF study of embryonic globins identified in a patient with KLF1 mutations. Comparing hemoglobin profiles from the patient, P2, with control human embryonic stem cell (hES)-derived hematopoietic cells reveals 3 distinct abnormal hemoglobin protein bands separated at a more cathodic position than HbA and HbF. These hemoglobin species were similar to those of patients (P2, in triplication), as they were separated to the same isoelectric points. These hemoglobin bands were subsequently identified by mass spectrometry to be Hb Portland-1 (ζ₂γ₂) and Hb Bart’s (γ₄). Of note, a different level of embryonic protein expression during erythroid differentiation from embryonic to fetal erythropoiesis in hES cells from day 6 to day 10 was observed (Right). Moreover, a fast-moving hemoglobin specie of Hb Gower 2 (α₂ε₂) was identified in this erythroid cell model but not from the patient. The standard hemoglobin controls are shown on the far left lane.