Figure 1.
Intracellular flow cytometric detection of GATA1s cells in TAM and ML-DS is comparable to NGS. (A) Antibody directed against GATA1 N terminus (green) detects only GATA1fl. Antibody directed against GATA1 C terminus (brown) detects both GATA1fl and GATA1s. Left, a normal cell (red circle) expresses both GATA1fl and GATA1s. Right, TAM or ML-DS cells (blue circle) only express GATA1s. (B) PB and BM samples were subject to both iFC (top) and NGS sequencing of GATA1 exon 2 and 3 (bottom). Top right, schematic of iFC plot of wild-type cells (red gate) and GATA1s-only expressing cells (blue gate) immunostained with anti GATA1 N′ terminus (y-axis) and anti-GATA1 C′ terminus (x-axis). (C) Linear regression analysis showing correlation between GATA1s VAF (x-axis) by NGS and percentage of GATA1s+ cells detected by iFC, as a percentage of live cells (y-axis). Samples tested: PB DS neonates screened by NGS and found to have no GATA1 mutations (red open circles), PB TAM (blue open circles), ML-DS BM (purple open circles), and ML-DS postchemotherapy treatment BM (brown open circles). Dotted lines show 95% confidence intervals. R2 = 0.75; P < .0001; n = 42. For samples in which more than 1 mutation was identified, the biggest VAF value was used. Bottom, samples with 1% to 10% VAF and 1% to 10% GATA1s+ cells are magnified to illustrate data more clearly. (D) Linear regression analysis showing correlation between GATA1s VAF (x-axis) by NGS and percentage of cells in the live, CD45lowCD117+CD7+ gate (y-axis) by flow cytometry. Dotted lines show 95% confidence intervals. R2 = 0.61; P < .0001; n = 42. The rest of the panel is as in panel C. (E) Representative flow cytometry plots of PB DS neonate, PB TAM, and BM ML-DS before and after chemotherapy. Cells within the CD45low CD117+ (left) were plotted in the CD117 vs CD7 (middle) or in the GATA1 N terminus (GATA1 N′) vs GATA1 C terminus (GATA1 C′) (left). CD117+CD7+ cells (orange box) were overlaid (as orange dots) on the GATA1 N′ and GATA1 C′ axis. Figures indicate the mean percentage ± 1 SD of cells within the gates. Number of samples tested: 7 PB DS neonate samples, 26 PB TAM samples, and 6 and 3 BM ML-DS samples before and after chemotherapy, respectively.

Intracellular flow cytometric detection of GATA1s cells in TAM and ML-DS is comparable to NGS. (A) Antibody directed against GATA1 N terminus (green) detects only GATA1fl. Antibody directed against GATA1 C terminus (brown) detects both GATA1fl and GATA1s. Left, a normal cell (red circle) expresses both GATA1fl and GATA1s. Right, TAM or ML-DS cells (blue circle) only express GATA1s. (B) PB and BM samples were subject to both iFC (top) and NGS sequencing of GATA1 exon 2 and 3 (bottom). Top right, schematic of iFC plot of wild-type cells (red gate) and GATA1s-only expressing cells (blue gate) immunostained with anti GATA1 N′ terminus (y-axis) and anti-GATA1 C′ terminus (x-axis). (C) Linear regression analysis showing correlation between GATA1s VAF (x-axis) by NGS and percentage of GATA1s+ cells detected by iFC, as a percentage of live cells (y-axis). Samples tested: PB DS neonates screened by NGS and found to have no GATA1 mutations (red open circles), PB TAM (blue open circles), ML-DS BM (purple open circles), and ML-DS postchemotherapy treatment BM (brown open circles). Dotted lines show 95% confidence intervals. R2 = 0.75; P < .0001; n = 42. For samples in which more than 1 mutation was identified, the biggest VAF value was used. Bottom, samples with 1% to 10% VAF and 1% to 10% GATA1s+ cells are magnified to illustrate data more clearly. (D) Linear regression analysis showing correlation between GATA1s VAF (x-axis) by NGS and percentage of cells in the live, CD45lowCD117+CD7+ gate (y-axis) by flow cytometry. Dotted lines show 95% confidence intervals. R2 = 0.61; P < .0001; n = 42. The rest of the panel is as in panel C. (E) Representative flow cytometry plots of PB DS neonate, PB TAM, and BM ML-DS before and after chemotherapy. Cells within the CD45low CD117+ (left) were plotted in the CD117 vs CD7 (middle) or in the GATA1 N terminus (GATA1 N′) vs GATA1 C terminus (GATA1 C′) (left). CD117+CD7+ cells (orange box) were overlaid (as orange dots) on the GATA1 N′ and GATA1 C′ axis. Figures indicate the mean percentage ± 1 SD of cells within the gates. Number of samples tested: 7 PB DS neonate samples, 26 PB TAM samples, and 6 and 3 BM ML-DS samples before and after chemotherapy, respectively.

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