Phospho-flow demonstrates the induction of multiple signaling pathways in CD34⁺ cells from patients with MPN. (A) Representative histogram profiles of phospho-ERK (top row), phospho-STAT5 (middle row), and phospho-STAT3 staining (bottom row) in a control and an MF patient (left and middle panels, respectively) are shown. Note that the median florescence intensity (MFI) of the isotype control (green) is different between each of the 2 patient and control samples. Therefore, for each patient sample, a relative value, the -fold increase, was calculated by dividing the MFI of the test antibody (red) by the MFI of the isotype control (green). The bar charts on the right show the results from all patient samples represented as the -fold increase ± SEM. No significant difference in signaling was noted between the 3 different MPNs. However, significant activation of basal signaling was seen for ERK pathway in ET and MF patients compared with controls. (B) A signaling heat-map table is shown for all control and MPN samples analyzed. For each individual in our cohort, the results of the signaling analysis are shown graphically. The patient and control numbers correspond with those shown in the histograms in panel A. Where the patient is JAK2 V617F positive, we have also documented the allele burden of the CD34⁺/CD38⁻ compartment. A degree of signaling heterogeneity is demonstrated, even between patients within the same disease/control phenotype.