Phagocytosis of opsonised fluorescent platelets by neutrophils to detect antiplatelet antibody

A platelet immunofluorophagocytosis method was developed to detect the presence of antiplatelet antibody. The method combines the advantages of immunofluorescence of antibody-coated platelets, the ability of neutrophils to phagocytose opsonised platelets, and the property of methylene blue to quench FITC-generated fluorescence of extracellular particles. Paraformaldehyde-fixed equine platelets in suspension were treated with rabbit antiequine platelet serum and FITC-conjugated goat antirabbit IgG. Viable equine neutrophils were incubated with these platelets at 37°C for 15–30 minutes. Phagocytosis was evaluated by fluorescence microscopy immediately after addition of 1% methylene blue to neutrophil platelet suspensions. Extracellular and free platelets were rendered nonfluorescent by methylene blue, whereas intracellular platelets remained unaffected and appeared highly fluorescent depending on the antiserum dilution. Phagocytosis was significantly (P <0.05) increased in the presence of complement. Addition of divalent cations (Ca²⁺ and Mg²⁺) did not increase phagocytosis, whereas EDTA significantly (P <0.05) reduced phagocytosis irrespective of the presence of complement. Sensitivity of the method can be increased by quantifying phagocytosis using flow cytometry.