Immature JMML populations express targetable surface markers. Splenic MNCs from 3 patients were stained with metal-conjugated Ab, fixed with paraformaldehyde, stained with an iridium metallointercalator, and then measured on the cytometry by time-of-flight mass cytometer. (A) Pooled data of 3 separately acquired samples were analyzed in SPADE simultaneously. A minimum spanning tree was constructed based on differentiating surface markers. Putative cell populations were annotated manually based on known coexpression of lineage markers. (B) Nodes representing JMML hematopoietic stem/progenitor cells were identified by CD34, CD38, and the absence of lineage markers (Lin^–), and expression of LSC-associated markers (CD7, CD33, CD44, CD47, CD96, CD123, CD143, CD202b, TIM-3, and CLL-1) were analyzed. (C) Nodes representing JMML hematopoietic stem/progenitor cells were evaluated for pSTAT5 expression. Each dot on the graphs in panels B-C represents the average expression of the analyzed marker in the corresponding nodes. Dotted line represents threshold of expression. ∗∗∗P < .001. AU, arbitrary units; pSTAT5, phosphorylated STAT5.