Development of a sensitive immunoradiometric assay for detection of platelet surface-associated immunoglobulins in thrombocytopenic dogs

Abstract

To develop a direct assay to measure platelet surface-associated immunoglobulins (PSAIg) in dogs and to determine whether the assay is useful in the diagnosis of immune-mediated thrombocytopenia (IMT). 20 healthy dogs were used to develop reference intervals, and 23 dogs with IMT and 17 with non-IMT were used to evaluate the clinical use of this assay. After optimization of platelet collection and assay conditions, concentrations of PSAIg were measured, using radiolabeled staphylococcal protein A (SpA) and polyclonal antibodies against canine IgG (anti-gamma) and IgM (anti-micro). Concentrations of PSAIg were expressed as the percentage of radiolabeled immunoglobulin detector bound. Cut-off values (mean + 3 SD) were as follows: SpA, 1.1%; anti-gamma, 1.3%; and anti-micro, 3.5%. Values greater than these cut-off values were considered positive. Values determined by use of radiolabeled SpA for all dogs with IMT were greater than the cut-off value; values were considered high positives (> 5 times cut-off value) for 22 of these 23 dogs. Although 9 of 17 dogs with non-IMT also had PSAIg concentrations greater than the cut-off value, values were considered high positives for only 3 of these 9 dogs. The immunoradiometric assay developed is a reliable and sensitive method to detect PSAIg in dogs. However, to obtain accurate results, optimum temperature, time, and storage conditions must be used. Detection of increased concentrations of PSAIg in dogs presumed to have non-IMT should alert clinicians to reconsider an immune-mediated basis for the thrombocytopenia.