GPI-AP deficiency and PIGA mutations in each cell population of PB from PNH patients. (A) Gating strategy to analyze and sort B1 cells and other B-lineage cells from PNH patients. CD19 cells were enriched using microbeads and gated as shown. Doublets were excluded based on forward scatter (FSC) profiles, and dead cells and T cells were excluded by gating 7-aminoactinomycin D–negative (7-AAD⁻) and CD3⁻ cells, respectively. B-cell subsets were defined as: B1 cells, CD19⁺CD20⁺CD27⁺CD43⁺CD38^lo/int; naive B cells, CD19⁺CD20⁺CD27⁻CD43⁻CD38^lo/int; and memory B cells, CD19⁺CD20⁺CD27⁺CD43⁻CD38^lo/int. (B) Representative histograms of FLAER staining in granulocytes, monocytes, B1 cells, naive B cells, memory B cells, T cells, and natural killer (NK) cells from PNH06. Percentages indicate FLAER⁻ cells. (C) Percentages of FLAER⁻ cells in the indicated PB-cell populations in the first and second samples from the 6 PNH patients. Second samples were obtained from PNH01, PNH02, and PNH03. (D) Absolute numbers of B1 cells. The absolute numbers of B1 cells were calculated by multiplying the number of CD19⁺ cells by the percentage of B1 cells in CD19⁺ cells (supplemental Table 4). (E) Absolute numbers of FLAER⁻ B1 cells. The absolute numbers of FLAER⁻ B1 cells were calculated by multiplying the number of B1 cells by the percentage of FLAER⁻ B1 cells (supplemental Table 4). (F) Sanger sequences of the PIGA gene indicating the 2 mutations in PNH01 and 1 mutation in PNH02. The tables below show the percentages of the PIGA-mutated allele frequencies in each cell population measured by digital PCR. (G) Results of sequencing of bacterial colonies transfected with plasmids carrying genomic DNA from PNH01. FSC-A, FSC–area; FSC-H, FSC–height; Mut, mutated; SSC-A, side scatter–area; WT, wild type.