CD34⁺ cells in NPM1-mutated AML show cytoplasmic expression of nucleophosmin. (A-B) CD34⁺ leukemic cells (brown) from a patient with NPM1-mutated AML showing cytoplasmic expression of NPM (blue; A arrows) and nucleus-restricted positivity for C23/nucleolin (B arrows). (C-D) Another NPM1-mutated patient showing rare CD34⁺ cells (C) that double stain for CD34 (brown) and cytoplasmic NPM (blue; D single arrow); endothelium of a vessel express CD34 (D double arrow). CD34⁺ leukemic cell (brown) from the same case show nucleus-restricted positivity for C23/nucleolin (blue; D inset). (A-B,D) Sequential immunoperoxidase/APAAP staining; no counterstaining. (C) APAAP technique (hematoxylin counterstaining). (A-D) Paraffin sections from bone marrow biopsies. All images were collected using an Olympus B61 microscope and a UPlan FI 100×/1.3 NA oil objective; Camedia 4040, Dp_soft Version 3.2; and Adobe Photoshop 7.0. (E-F) Flow cytometric detection of NPM-cytoplasmic expression (patient 25) in CD34⁺ and CD34⁻ blasts (E), and after additional gating on CD34⁺/CD38⁻ or CD34⁺/CD38⁺ cells, respectively (F).