Isolation of GPI-anchored protein–negative (PNH) and GPI-anchored protein–positive (normal) B cells and monocytes. Mononuclear cells were collected by apheresis from PNH patients prior to transplantation. (A) Monocytes were sorted by flow cytometry into GPI-negative (CD64⁺/CD14^–/CD59^–) and GPI-positive (CD64⁺/CD14⁺/CD59⁺) populations using CD64 (non-GPI–anchored protein) to gate on monocytes and CD14 and CD59 as markers for GPI-anchored proteins. (B) B lymphocytes were expanded from pretransplantation apheresis samples using CD40 ligand–transfected NIH3T3 cells. The percentage of GPI-negative and GPI-positive B cells were similar in expanded versus nonexpanded samples. (C) After expansion, cells were flow sorted into GPI-negative (CD19⁺/CD55^–/CD59^–) and GPI-positive (CD19⁺/CD55⁺/CD59⁺) B lymphocytes using CD19 (non-GPI–anchored protein) to identify B cells and CD55 and CD59 as markers for GPI-anchored proteins.