Figure 1
Hematological parameters, clinical data, BM evaluation, and in vitro sensitivity assay of erythroid progenitors to EPO. (A) Hematological and clinical analysis of the propositus and his family members revealed erythrocytosis, plethora, and palpable spleen (splenic length of 12 cm based on ultrasound measurement) in the patient. ND, not done. (B) Patient’s BM aspirate (May-Grünwald-Giemsa staining, top) and BM biopsy (glycophorin C staining specific for erythroid lineage, brown color, bottom) showed hypercellularity, erythroid hyperplasia, normal granulopoiesis, and abnormal megakaryopoiesis (for details, see supplemental Figure 1). The images were visualized with an Olympus BX41 light microscope (Hamburg, Germany) and acquired with an Olympus DP73 camera driven by CellSens Entry software. Images were labeled using Adobe Photoshop software (Adobe Systems, San Jose, CA). Top: magnification, ×100; scale bar, 100 µm. Bottom: magnification, ×200; scale bar, 200 µm. (C) The EPO dose-response curves derived from the patient, patient’s brother, patient’s parents, normal controls, and PV patients. The growth of BFU-E colonies at the indicated concentrations of EPO was expressed as a percentage of maximal EPO stimulation (represented by EPO concentration of 1 U/mL). Erythroid progenitors from the patient (red curve) were hypersensitive to EPO; there was a relatively higher number of BFU-E colonies in comparison with healthy controls (black curve) in low EPO concentrations. The in vitro growth of the erythroid progenitors of both of the patient’s parents (blue and green curve, respectively) also showed slightly increased sensitivity to EPO when compared with normal controls (black curve). The progenitors of the patient’s brother (purple curve) showed normal growth. Two PV patients, positive for the V617F mutation (brown curve), were used as positive controls for hypersensitivity and formation of EPO-independent colonies (EECs). The table below the graph shows statistical evaluation of the BFU-E colony number at individual concentrations with respect to normal controls. P values were calculated using Origin 6.1 software (OriginLab Corporation, Norhampton, MA, USA). *P < .05; **P < .01. NS, not significant.

Hematological parameters, clinical data, BM evaluation, and in vitro sensitivity assay of erythroid progenitors to EPO. (A) Hematological and clinical analysis of the propositus and his family members revealed erythrocytosis, plethora, and palpable spleen (splenic length of 12 cm based on ultrasound measurement) in the patient. ND, not done. (B) Patient’s BM aspirate (May-Grünwald-Giemsa staining, top) and BM biopsy (glycophorin C staining specific for erythroid lineage, brown color, bottom) showed hypercellularity, erythroid hyperplasia, normal granulopoiesis, and abnormal megakaryopoiesis (for details, see supplemental Figure 1). The images were visualized with an Olympus BX41 light microscope (Hamburg, Germany) and acquired with an Olympus DP73 camera driven by CellSens Entry software. Images were labeled using Adobe Photoshop software (Adobe Systems, San Jose, CA). Top: magnification, ×100; scale bar, 100 µm. Bottom: magnification, ×200; scale bar, 200 µm. (C) The EPO dose-response curves derived from the patient, patient’s brother, patient’s parents, normal controls, and PV patients. The growth of BFU-E colonies at the indicated concentrations of EPO was expressed as a percentage of maximal EPO stimulation (represented by EPO concentration of 1 U/mL). Erythroid progenitors from the patient (red curve) were hypersensitive to EPO; there was a relatively higher number of BFU-E colonies in comparison with healthy controls (black curve) in low EPO concentrations. The in vitro growth of the erythroid progenitors of both of the patient’s parents (blue and green curve, respectively) also showed slightly increased sensitivity to EPO when compared with normal controls (black curve). The progenitors of the patient’s brother (purple curve) showed normal growth. Two PV patients, positive for the V617F mutation (brown curve), were used as positive controls for hypersensitivity and formation of EPO-independent colonies (EECs). The table below the graph shows statistical evaluation of the BFU-E colony number at individual concentrations with respect to normal controls. P values were calculated using Origin 6.1 software (OriginLab Corporation, Norhampton, MA, USA). *P < .05; **P < .01. NS, not significant.

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