Homozygous CALR mutations lead to MPO and EPX deficiency. Analysis of 2 peripheral blood neutrophil granulocyte populations with distinct MPO protein levels in patient 1. (A) Detection of MPO protein by confocal microscopy in neutrophil granulocytes of patient 1 with normal (top) and reduced (bottom) MPO protein. Scale bar, 5 μm. (B) Purification of the 2 neutrophil granulocyte populations according to the MPO protein level determined by flow cytometry. The numbers indicate the percentage of cells in the gate. DNA was isolated from each fraction and fragment analysis was performed. The respective histograms are shown on the right. Fragment length for CALR WT = 260 bp and for CALR type 2 mutation = 265 bp. MUT, mutated allele. (C) MPO immunocytochemistry of peripheral blood neutrophil granulocytes in patient 1. Brown precipitates represent MPO-positive granules. The ratio of MPO-positive to MPO-negative granulocytes was ∼1:2 (200 neutrophil granulocytes counted manually). Scale bar, 4 μm. (D) Peripheral blood ADVIA dot plots from a patient with MF and normal MPO activity (left), patient 2 with a homozygous CALR mutation and MPO/EPX deficiency (middle), and patient 6 with a JAK2-V617F mutation and MPO deficiency only (right). LUC (light blue), neutrophil granulocytes (purple), monocytes (green), lymphocytes (dark blue), eosinophil granulocytes (orange), and platelets/lysed red blood cells (black) are shown. MPO/EPX, MPO, and EPX-positive cells, respectively. (E) MPO DNA sequences of patient 6. Two mutations were observed in the MPO gene. The base change is denoted below the chromatogram, and the corresponding amino acid change is displayed to the right of the chromatogram. Both mutations have previously been reported to cause hereditary MPO deficiency.¹⁹