Figure 2. Sorted HLA-DR⁺ PMN from healthy donors have morphologic and biochemical characteristics of neutrophils.

(A and B) Micrographs showing cytospun, FACS-isolated HLA-DR⁻ or HLA-DR⁺ PMN (Diff Quik stain). (C) Quantification of band neutrophils in FACS-isolated HLA-DR⁺ vs. HLA-DR⁻ fractions from healthy blood donors (n = 4; paired Student’s t test). (D) Quantification of the total percentage of band and metamyelocyte (Meta) neutrophil forms in blood smear differential counts from patients with CL compared with endemic healthy controls (unpaired students t test). (E) Correlation plot between the percentage of HLA-DR⁺ PMN vs. the percentage of bands plus metamyelocytes in peripheral blood smears, showing a lack of correlation between the abundance of immature neutrophil forms and the abundance of HLA-DR⁺ PMN in patients with CL (P = 0.732). (F and G) Representative flow cytometry plots showing gating on granulocytes (62.1%) and monocytes (5.2%) in the whole blood of a patient with CL (F), and gating on HLA-DR⁺ and HLA-DR⁻ CD66b⁺ neutrophils in a patient with CL (G). (H) Histogram showing intracellular staining of HLA-DR⁺ PMNs, HLA-DR⁻ PMNs, and monocytes for MPO. Plots are compared with the fluorescence minus one (FMO) staining control lacking MPO Abs. (I) Quantification of G-MFI of HLA-DR⁻ PMNs, HLA-DR⁺ PMNs, and monocytes in patients with CL (statistical analysis, 1-way ANOVA with Tukey’s posttest analysis).