Light microscopic, non-immunologic demonstration of iron-binding proteins in hematopoietic cells

Iron-binding proteins were localized by their saturation with iron using iron nitrilotriacetate (FeNTA), maintenance of protein-iron-binding at specific values of pH, and visualization of the iron with acid ferrocyanide (AF). Human neutrophilic cells showed strong blue granular and diffuse cytoplasmic staining. Human mid- and late-stage erythroblasts showed moderate diffuse cytoplasmic staining. Monocytes and macrophages showed reactions similar to those seen with AF technique alone. Other hematopoietic cells showed minimal or no stain positivity. Nuclear positivity was not observed in any cells. Concanavalin A (ConA) treatment of purified neutrophils reduced their FeNTA-AF positivity; supernatants from these cells showed precipitin lines of identity with anti-lactoferrin (Lf) stainable with FeNTA and AF. Cellulose acetate electrophoresis of crude neutrophil extracts treated with [59Fe]NTA showed multiple protein bands; one band co-migrated with purified Lf and showed autoradiographic positivity. Rabbit heterophils and rat neutrophils showed less FeNTA-AF positivity, consistent with less Lf in these cells than in human neutrophils. Washing smears with 0.1 M citrate, pH 6.0, between FeNTA and AF treatments eliminated only erythroblast positivity; 0.1 M citrate, pH 4.0, ablated neutrophil staining as well. Ferritin-hemosiderin staining was preserved at both values of pH. These results indicate that FeNTA-AF technique specifically visualizes neutrophil Lf, and suggest that the observed erythroblast positivity is due to transferrin (Tf).