HM01 - A Novel Flow Cytometric Assay to Assess Platelet Desialylation in Canine Immune Thrombocytopenia

Abstract: Background – Immune thrombocytopenia (ITP) is a common hemostatic disorder of dogs leading to significant morbidity and mortality, often from side effects of immunosuppression. In ITP, autoantibodies target platelets for macrophage clearance. Human studies demonstrate autoantibodies also trigger platelet desialylation. Desialylation marks platelets for clearance, thus preventing desialylation could lead to new, safer treatment strategies. Studying desialylation in canine ITP requires a platelet desialylation assay.

Objective – Develop a flow cytometric assay to detect exposed platelet β-galactose residues as a marker of desialylation based on binding of FITC-labeled Ricinus communis agglutinin (RCA).

Animals – 17 healthy, client-owned dogs recruited from a veterinary teaching hospital.

Methods – Platelet-rich plasma (PRP) was isolated from EDTA-anticoagulated blood. Ex vivo desialylated platelets were generated by neuraminidase C treatment (positive control); β-lactose incubated PRP served as negative control. Controls and native PRP were labeled with RCA-FITC and desialylation was quantified flow cytometrically by mean fluorescence intensity/forward scatter (MFI/FSC). Following optimization, analytic precision, performance in serially diluted PRP, and sample stability were assessed.

Results – RCA MFI/FSC remained stable for 48hr post-blood collection at 25°C (p = 0.06) and 4°C (p = 0.24). Precision was acceptable with coefficient of variation=4.8%. RCA MFI/FSC was not influenced by platelet counts ranging from 200,000-10,000 platelets/µL (p = 0.14).

Conclusions and clinical importance – This flow cytometric assay detects desialylated platelets with good analytic performance through a wide range of platelet counts. This assay will enable pathophysiologic investigations of platelet desialylation in canine ITP and set the stage for novel therapeutics.