Analytical validation of a flow cytometric protocol for quantification of platelet microparticles in dogs

Abstract

Calcium induced higher in vitro PMP concentrations, likely due to platelet activation. PMP concentrations were reliably quantified in WB, indicating the potential for clinical applications. PPP analyses were unreliable due to high inter-CV and DL overlap, and not obtainable due to hemolysis and lipemia interference.

Analytical validation of platelet microparticle quantification in cats

“…For AnV‐positive PMPs, there was also no difference between the healthy cats (362,000 PMP/μL, 224,900‐677,100 PMP/μL) and the cats with asymptomatic HCM (383,600 PMP/μL, 39,900‐477,400 PMP/μL, P > .99). The detection limits for MP quantification were calculated in a recently published canine study and are depicted in Figure A. The detection limits in WB were 147,000 MP/μL for the PMPs and 3,500 MP/μL for the AnV‐positive PMPs.…”

“…The cats with HCM underwent the same diagnostic procedures, with the addition of blood pressure measurements. For both groups of cats, exclusion criteria included the presence of systemic disease and the presence of hemolysis and lipemia, the latter due to interference with flow cytometric acquisition …”

“…Lyngby, Denmark) and by the same investigator (SEC). The setup of the MP gate, subgates, voltages, and sample preparations were performed as previously described in dogs . The entire gating hierarchy is provided in Figure S1, in brief: Gating and identification of MP populations: Megamix‐Plus SSC (side scatter) beads (Biocytex, Marseille, France) were used to define the MP gate (events in P1; Figure S1A), and 0.3‐1.0 μm MPs were detected (as per the manufacturer's instructions).…”

“…The CV was calculated as: CV = (standard deviation/mean) × 100%, accepting a CV of maximum 20% . The detection limit was previously established as: Detection limit = mean + (3 × standard deviation), using 10 negative controls from separate days …”

“…In the human literature, PPP remains the most commonly used sample type for PMP quantification. However, in a previous study, the detection of PMPs in canine platelet‐poor plasma (PPP) by flow cytometry was found to be unreliable due to high inter‐assay variation and a high detection limit . Interestingly, whole blood (WB) samples could be analyzed reliably.…”