FCM–based leukemic stem cell and aberrant antigen expression analysis. (A) FCM profiles of CD34⁺-gated BM cells showing expression of CD38 (CD38-APC) vs a combination of cell surface antigens (CD7, CD11b, CD22, CD56, CD366, and CD371; LSC-PE) reported as aberrantly expressed on CD34⁺CD38^low/− LSCs. LSC-PE expression was, as indicated, investigated in a gate set strictly on CD38⁻ cells (lower CD38 gate based on erythrocytes that are CD38⁻), as well as on the 10% lowest CD38-expressing CD34⁺ cells for samples in which <10% of CD34⁺ cells were CD38⁻. Representative data from 2 healthy donors (left: young, aged <40 years; right: old, aged >70 years), 2 patients with high-risk MDS/AML, and pre– and post–allo-HSCT samples from 4 patients (also analyzed by ddPCR; see Figure 4A) achieving continuous-CR after allo-HSCT. Percentages shown in FCM plots for the 2 patients with high-risk MDS/AML (left, patient 32; right, patient 33), and the continous-CR pre–allo-HSCT samples indicate the VAF for the most clonal recurrent mutation. For all continuous-CR posttransplantation samples the mutational VAF was below detection level. Percentages for the representative healthy control BM samples show CD34⁺CD38⁻LSC-PE⁺ cells out of total cells. (B) Mean (± SEM) percentage CD34⁺CD38⁻ LSC-PE⁺ cells (left, red) and CD34⁺CD38^low/−LSC-PE⁺ cells (right, green) of total CD45⁺ BM cells. (C) Mean (± SEM) percentage LSC-PE⁺ cells in CD34⁺CD38⁻ (left, red) and CD34⁺CD38^low/− (right, green) gates, of total CD34⁺CD38⁻/CD34⁺CD38^low/− BM cells. Equal symbols for the patients who achieved continuous-CR indicate sequential samples taken before and after allo-HSCT for the same patient. “0” indicates a sample showing no LSC⁺ events within the set gates. For 2 patients with high-risk MDS/AML, >10% of the CD34⁺ cells were CD38− and were therefore not analyzed based on a separate CD38^low/− gate (red symbols in right B and C panels). (D) The same BM samples analyzed in panels A to C were also analyzed for other aberrant antigen expression patterns reported in myeloid malignancies. Each dot represents an individual BM sample. Results are presented as mean (± SEM) percentages of total CD45⁺ BM cells coexpressing indicated (populations 1-8) combinations of antigens. Equal symbols for the patients in continuous-CR indicate sequential samples taken before and after allo-HSCT for the same patient. Blast counts for the patients with high-risk MDS/AML (patients 30-33) were 62.5%, 10%, 43.5%, and 11%, respectively (supplemental Table 1).