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Autoantibodies were solubilized from washed platelets by three successive freeze thaw cycles followed by sonication for 15 seconds at maximum intensity (MSE Mk 2). The disrupted platelets were centrifuged at 30,000g for 20 minutes and the supernatant was heated at 56°C for 30 minutes followed by centrifugation at 3,000g for 5 minutes. An aliquot of the supernatant was diluted with 0,01M phosphate buffered saline pH 7.4 to give a final concentration equivalent to about 5 × 106 platelets/ml. Fifty microlitres of the diluted extract was adsorbed onto the wells of a microtitre plate by incubation at room temperature for two hours. The wells were then washed three times with 0,01M phosphate buffered saline pH 7.4 containing 0,05% Tween (Tween-PBS) and the adsorbed antiplatelet IgG reacted with 50μl of an iodine-125 labelled affinity isolated goat antihuman (heavy chain specific) IgG, specific activity ≃ 50μCi/ μg (California Antibodies) for one hour at room temperature followed by three washes with Tween-PBS. A standard curve was constructed and run in parallel, in the dose range 0,l-100ng using purified human IgG (Miles Laboratories). The separated wells were counted in an automatic gamma spectrometer (Packard 3003) and the results calculated on a desk-top computer (Hewlet-Packard 9800) using a Rodbart weighted Logit-Log transformation (f = 0,996). A relatively poor arithmetic correlation was found between whole blood platelet counts and measured platelet bound IgG (r = -0,42), whereas a better correlation was obtained using a log-log plot (r = - 0,84).  The results in ng/106 platelets were - Known ATP 133,2 ± 154, other autoimmune diseases with thrombocytopenia 212,0 ± 202, non-immune thrombocytopenia 18,6 ± 4,8, normal healthy controls 11,4 ± 3.7

A Rapid And Sensitive Method For The Quantitation Of Platelet Autoantibodies