Intracellular flow detection of GATA1s⁺ cells is highly sensitive and can be used to measure MRD in ML-DS. (A) Representative flow cytometry panels of ML-DS samples before (top) and after chemotherapy (bottom) in which additional markers previously described as aberrant (CD235A, CD41, CD7, CD36, and CD34) were examined in GATA1s⁺ cells (blue gate) and GATA1^WT (red gate). (B) Bar graph showing the proportion of GATA1s⁺ (blue), GATA1^WT (red) and GATA1 negative (light green) cells within the CD45^lowCD117⁺ gate in 5 prechemotherapy and 3 postchemotherapy ML-DS samples. (C) Violin plots showing the percent of cells positive for CD235A, CD41, CD36, CD34, and CD7 in GATA1s⁺ cells before chemotherapy (blue), GATA1^WT after chemotherapy (red), and GATA1⁻ cells after chemotherapy (light green). (D) Flow cytometry plots of dual immunostaining with antibodies against the N′ and C′ termini of GATA1 in: OCI-AML3 cells (left), a GATA1 negative cell line; K562 cells (middle), a cell line expressing both GATA1fl and GATA1s; and CMK (right), a GATA1s⁺ cell line derived from a ML-DS patient. (E) Schematic representation of a serial dilution of CMK cells. A total of 500 000 CMK cells were serially diluted 11 times into OCI-AML3/K562 cells mix and iFC performed on tubes 1, 3, 5, 7, 8, 9, 10, and 11. Expected frequency of CMK cells is shown below each tube that was tested by iFC. (F) Representative flow cytometry panels of serially diluted GATA1s⁺ CMK cells (blue gate) in a cell mixture of CMK/OCI-AML3/K562. A total of 500 000 live events were recorded per tube but only a fraction of events is shown for clarity. Each gate contained more than 300 events. Percent of cells in the blue gate from 2 replicates is shown. (G) Top, 3 flow cytometry plots showing the percentage of GATA1^WT and GATA1s⁺ cells in the live CD45^lowCD117⁺ cell compartment in 3 postchemotherapy ML-DS samples in which no GATA1s mutations were identify by NGS. Below, graph of the percentage of GATA1s⁺ cells live CD45^lowCD117⁺ gate in the isotype controls (4 samples), DS neonates (7 samples), and 3 ML-DS postchemotherapy samples.