Analytical validation of platelet microparticle quantification in cats

Abstract

Background: Cardiogenic embolism (CE) in cats is a devastating condition primarily associated with hypertrophic cardiomyopathy (HCM). Hypercoagulability may pose a risk for thrombus formation; however, no single test can predict CE development. Platelet microparticles (PMPs) released from platelet membranes are associated with thrombosis in humans.

Objectives: The aims were to validate flow cytometric PMP quantification in cats analytically and, in a pilot study, evaluate the procoagulant annexin V (AnV) positive PMP concentration in healthy cats and cats with asymptomatic HCM.

Methods: With CD61 as a platelet marker, CD61⁺ AnV⁺ PMPs (0.3-1.0 μm) were quantified in citrated whole blood (WB) and platelet-poor plasma (PPP) using flow cytometry. Analyses were performed in 6 healthy cats and 5 cats with asymptomatic HCM. The coefficient of variation (CV) for duplicate (intra-assay) and parallel (inter-assay) analyses were calculated.

Results: PMP concentrations were quantified with acceptable intra-assay CV for WB (CD61⁺ /AnV^- ; 2.4%, 0.2%-8.4% (median, range), CD61⁺ /AnV⁺ ; 3.8%, 0.1%-12.5%) and PPP (CD61⁺ /AnV^- ; 5.0%, 0.7%-12.8%, CD61⁺ /AnV⁺ ; 7.4%, 0.5%-15.3%), and acceptable inter-assay CV for WB in 10/11 cats (CD61⁺ /AnV^- ; 6.2%, 1.4%-13.3%, CD61⁺ /AnV⁺ ; 6.4%, 0.7%-17.2%), but unacceptable for PPP (CD61⁺ /AnV^- ; 15.6%, 5.8%-42.7%, CD61⁺ /AnV⁺ ; 27.8%, 8.4%-77.1%). For WB PMP concentrations, the pilot data demonstrated no differences between healthy cats and cats with asymptomatic HCM (4/5 with left ventricular outflow obstruction) for either the CD61⁺ /AnV^- or the CD61⁺ /AnV⁺ PMPs.

Conclusions: Only WB PMP concentrations could be quantified reliably in cats in a clinical setting. PMP concentrations did not differ between healthy and asymptomatic HCM cats in this pilot study.

Keywords: Annexin; CD61; cardiac; microvesicles; precision.