The diagnosis and management of immune thrombocytopenia requires an effective method for the quantitation of platelet- associated IgG (PAIgG). Although several methods have been recently devised for this purpose, the search continues for a method which will prove to be accurate and reproducible, and yet simple enough to be utilized by the general clinical hemostasis laboratory. We now describe the development of an assay for PAIgG which we believe satisfies these criteria.Human platelets were isolated by differential centrifugation, washed five times in Tris-HCl pH 7.4 or phosphate buffered saline pH 7.4, both containing 5mM EDTA, and disrupted in 0.1M sodium acetate buffer pH 5.0 containing 1% (v/v) Triton X-100. Rabbit antibody specific for γ chains of human IgG (purified IgG fraction from DAKO) was carbamylated such that its isoelectric point was decreased to roughly 5.0. Total PAIgG was measured by electrophoresis at pH 5.0 of Triton X-100 disrupted washed platelets against the carbamylated rabbit anti-human IgG (γ) in 1% agarose gels containing 0.5% Triton X-100.The mean total PAIgG for 8 normals, was 5.7 fg/platelet (range 4.9 to 7.0). This technique is applicable to the quantitation of alloantibodies bound to normal platelets (indirect assay) as well as the quantitation of PAIgG on platelets of individuals with immune thrombocytopenic states, such as ITP.
Thermo-voltage measurements of atomic contacts at low temperature