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.
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ABSTRACT: Flow cytometry measures multiple characteristic of single cells using light scatter properties and fluorescence properties of fluorescent probes with specificity to cellular constituents. The use of flow cytometry in the veterinary clinical laboratory has become more routine in veterinary diagnostic laboratories and institutions (http://www.vet.k-state.edu/depts/dmp/service/immunology/index.htm), and reference laboratories. The most common applications in small animal medicine includes quantitation of erythrocytes and leukocytes in automated hematology instruments, detection of antibodies to erythrocytes and platelets in cases of immune-mediated diseases, immunophenotyping of leukocytes and lymphocytes in immunodeficiency syndromes, or leukemias and lymphomas. DNA content analysis to identify aneuploidy or replicating cells in tumor preparations has not gained routine acceptance because of the variability of prognostic results. Other applications including cell sorting and multiplexing using microspheres are potential assays of the future once they become validated and the instrumentation footprint becomes more and more compact, less expensive, and easier to use.Veterinary Clinics of North America Small Animal Practice 01/2012; 42(1):53-71. DOI:10.1016/j.cvsm.2011.09.012 · 1.04 Impact Factor
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ABSTRACT: In human medicine, transfusion of ABO-mismatched platelets has been associated with shortened platelet survival and refractoriness to platelet transfusion because of expression of certain blood group antigens on platelets. It remains unknown if canine platelets express dog erythrocyte antigens (DEAs). The aim of this study was to develop a flow cytometric assay for DEA 1.1 and determine whether DEA 1.1 is present on canine platelets. Blood was collected from 172 clinically healthy dogs. Platelets and erythrocytes from each dog were tested for DEA 1.1 by flow cytometry using anti-DEA 1.1 blood-typing sera. Erythrocytes from each dog were also assessed for DEA 1.1 using a standard tube-typing test (T1) and using a second tube method (T2), if the flow cytometric and T1 results differed. Using flow cytometry, DEA 1.1 was detected on erythrocytes of all 110 dogs shown by T1 or T2 testing to be DEA 1.1-positive. Initial results of the T1 test had a diagnostic accuracy of 93% (160 correct/172 tests). The frequency of erythrocyte DEA 1.1 positivity in previously untyped dogs (n = 118) was 56%. DEA 1.1 expression was not detected on platelets from DEA 1.1-positive dogs. Flow cytometry was a reliable method for detection of DEA 1.1 on canine erythrocytes. The absence of DEA 1.1 on platelets from DEA 1.1-positive dogs suggests that their platelets do not express DEA 1.1 and will not induce production of anti-DEA 1.1 antibodies that might lead to platelet refractoriness or reactions to a subsequent transfusion of DEA 1.1-positive erythrocytes.Veterinary Clinical Pathology 12/2011; 40(4):435-43. DOI:10.1111/j.1939-165X.2011.00374.x · 1.21 Impact Factor
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ABSTRACT: Vaccination is often cited as a potential cause of immune-mediated thrombocytopenia (ITP) in dogs. Although an association has been documented in humans, particularly in children, this relationship has not been definitively established in dogs. To identify the presence of an association between recent vaccination and ITP in dogs. Forty-eight client-owned dogs with presumptive idiopathic ITP and 96 age-matched, client-owned dogs with non-immune-mediated disease. Retrospective, case-control study. Dogs were identified through the Veterinary Medical Database (VMDB) and Hospital Information System at Purdue University. The median age at presentation for dogs with ITP was 7 years (range: 2-15 years). The majority of the ITP group was comprised of mixed breed dogs (38%); no pure breed was represented by more than 3 cases. The number of dogs that were vaccinated within 42 days of diagnosis of ITP did not differ significantly (P = .361) between cases of presumptive ITP (4/48, 8%) and the control group (13/96, 14%). This study failed to confirm the presence of an association between presumptive idiopathic ITP in dogs and recent vaccination; however, the possibility of an association cannot be completely ruled out based on the small sample populations and requires further investigation.Journal of Veterinary Internal Medicine 12/2011; 26(1):142-8. DOI:10.1111/j.1939-1676.2011.00850.x · 2.22 Impact Factor