A prospective cohort study to identify clinical diagnostic and prognostic markers of primary immune thrombocytopenia in dogs.

BACKGROUND: Primary immune thrombocytopenia (pITP) in dogs presents a diagnostic challenge, and clinical markers of severity are lacking. OBJECTIVES: Identify clinicopathologic features that differentiate pITP from secondary ITP (sITP) and markers related to bleeding severity, transfusion, and survival of dogs with pITP. ANIMALS: Ninety-eight thrombocytopenic dogs (58 pITP and 40 sITP). METHODS: Client-owned dogs with platelet counts <50 000/µL were enrolled in a prospective, multi-institution cohort study. History and treatment information, through a maximum of 7 days, was recorded on standard data forms. Bleeding severity was scored daily using a bleeding assessment tool (DOGiBAT). At-admission blood samples were collected for CBC, biochemistry, C-reactive protein concentration, and coagulation panels, and to measure platelet surface-associated immunoglobulin G (PSAIg) and expression of platelet membrane proteins and phospholipids. Dogs with evidence of coincident disease were classified as sITP. RESULTS: No definitive pITP diagnostic test was found. However, pITP cases were characterized by lower platelet counts, D dimer concentrations, and platelet membrane protein expression than sITP cases. Differentiation between pITP and sITP was further enhanced using logistic regression modeling combining patient sex, coagulation profile, platelet count, D dimer, and PSAIg. A second model of pITP severity indicated that low hematocrit and high BUN concentration were associated with non-survival. Low hematocrit at admission, but not platelet count or DOGiBAT score, was associated with transfusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Pending validation studies, models constructed from at-admission clinicopathologic findings may improve differentiation of pITP from sITP and identify the most severe pITP cases at the time of presentation.

Preliminary evaluation of a flow cytometric assay with microsphere controls for the detection of platelet-bound antibodies in canine immune thrombocytopenia.

BACKGROUND: Canine immune thrombocytopenia (ITP) ranges from a mild to severe bleeding disorder, and platelet counts do not reliably predict clinical disease course. The detection of platelet autoantibodies may further define the disease phenotype, but variability in assay configurations and a lack of well-characterized controls limit the diagnostic utility of anti-platelet antibody assays. OBJECTIVES: We aimed to develop control reagents to facilitate the characterization of canine platelet surface-associated immunoglobulin (PSAIg) in flow cytometric assays. METHODS: Silica microspheres were coated with canine IgG and IgM to assess the reactivity of goat and rabbit origin anti-canine immunoglobulin reagents. They were also used as positive controls in the PSAIg assay. Preliminary assay evaluation and determination of sample stability used PRP isolated from seven healthy dogs and 26 dogs newly diagnosed with thrombocytopenia. RESULTS: Blood sample stability was established for up to a 48-hour storage time. The conjugated positive control microspheres demonstrated stable fluorescent labeling over a 2-year observation period. Rabbit and goat origin anti-dog IgM fluorescent antibody labels reacted nonspecifically with canine IgG. Rabbit origin anti-dog IgG antibody demonstrated greater class specificity for canine IgG than a goat origin antibody. Thrombocytopenic dogs had a broad range of membrane-bound immunoglobulin. Median PSAIgG for dogs with primary or secondary ITP (18.4%, 34.1%, respectively) were significantly higher than controls (3.8%, P < .05). CONCLUSIONS: The described assay reagents and procedures provide positive controls and allow consistent thresholding to define a positive test result, suitable for any flow cytometer. A rabbit anti-dog IgG fluorescent label demonstrated specificity for canine IgG and was useful for the detection of PSAIgG in thrombocytopenic dogs.