Detection and quantification of platelet-bound antibodies with immunoperoxidase

The use of immunocytochemical techniques enables one to quantify antibodies bound to cell membranes. We have tested the platelets of subjects with immune thrombocytopenic purpura by this method. The washed platelets were incubated in the presence of antiglobulin conjugated with an equimolar amount of peroxidase. The fixed peroxidase was revealed and quantified by an enzymatic reaction using orthodianizidine-H2O2. From the results the number of immunoglobulin molecules per platelet was calculated. The test was calibrated so as to eliminate non-specific fixation of the reagent. Under these conditions the platelets of subjects with idiopathic thrombocytopenic purpura bound a much large amount of detectable immunoglobulin (800-21 000 molecules per platelet) than those of normal subjects (235 +/- 74 sites per platelet) or subjects with non-immune thrombocytopenia (395 +/- 203 molecules per platelet).