Characterization of homozygous SAO. (A) Pedigree of the family showing HBA1, HBA2, HBB (coding for α- and β-globin), and SLC4A1 genotype. Genetic analyses indicated that the child (II1) inherited a heterozygous 3.7-kb α-globin deletion from the mother, a heterozygous β-globin variant “La Désirade” (resulting from HBB c.389 C>T) from the father, and homozygous SAO. He has not inherited his mother sickle cell trait (HBB c.20C>T). Hb La Désirade is asymptomatic in heterozygotes and could not account for the early severe anemia because β-globin is not significantly expressed at 22 weeks gestation.5 Informed consent was provided according to the Declaration of Helsinki. Approval was obtained from the Bicêtre institutional review board for these studies. (B) Red cell sample from the transfused child contained donor cells as well as large ovalocytes. (C) May Grunwald Giemsa stain of a proband bone marrow sample showed marked dyserythropoisesis, mostly on late erythroblasts, with binuclearity, carrhyorexis, enlarged erythroblasts, and large reticulocytes and macrocytes. Myeloid and megacaryocyte lineages were normal. (D) Confocal microscopic imaging using selected antibodies could differentiate normal transfused cells and SAO red cells in a proband blood sample. Labeling with a polyclonal anti-band 3 antibody (AE1 pAb, red) that binds an intracellular epitope gave stronger fluorescence in the SAO cells than the donor cells (left). This effect was probably attributable to the fragility of the SAO cells, causing the SAO cells to be more readily permeabilized than the donor cells. In contrast, the donor cells could be seen to fluoresce strongly using the BRIC6 monoclonal antibody and a green fluorescent conjugated secondary antibody (middle). An overlay of the 2 images clearly differentiates the homozygous SAO cells from the donor cells, indicating that some SAO cells could survive in the circulation and that SAO band 3 was present at the red cell membrane, even in the absence of wild-type band 3 (right). Scale bar represents 10 μm.
