Premium
Analytical validation of a flow cytometric protocol for quantification of platelet microparticles in dogs
Author(s) -
Cremer Signe E.,
Krogh Anne K. H.,
Hedström Matilda E. K.,
Christiansen Liselotte B.,
Tarnow Inge,
Kristensen Annemarie T.
Publication year - 2018
Publication title -
veterinary clinical pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.537
H-Index - 51
eISSN - 1939-165X
pISSN - 0275-6382
DOI - 10.1111/vcp.12605
Subject(s) - flow cytometry , platelet , phosphatidylserine , coefficient of variation , annexin , chemistry , microbiology and biotechnology , calcium , andrology , chromatography , immunology , medicine , biology , biochemistry , phospholipid , membrane , organic chemistry
Background Platelet microparticles ( PMP s) are subcellular procoagulant vesicles released upon platelet activation. In people with clinical diseases, alterations in PMP concentrations have been extensively investigated, but few canine studies exist. Objectives This study aims to validate a canine flow cytometric protocol for PMP quantification and to assess the influence of calcium on PMP concentrations. Methods Microparticles ( MP ) were quantified in citrated whole blood ( WB ) and platelet‐poor plasma ( PPP ) using flow cytometry. Anti‐ CD 61 antibody and Annexin V (AnV) were used to detect platelets and phosphatidylserine, respectively. In 13 healthy dogs, CD 61 + /AnV − concentrations were analyzed with/without a calcium buffer. CD 61 + /AnV − , CD 61 + /AnV + , and CD 61 − /AnV + MP quantification were validated in 10 healthy dogs. The coefficient of variation ( CV ) for duplicate (intra‐assay) and parallel (inter‐assay) analyses and detection limits ( DL s) were calculated. Results CD 61 + /AnV − concentrations were higher in calcium buffer; 841,800 MP /μL (526,000‐1,666,200) vs without; 474,200 MP /μL (278,800‐997,500), P < .05. In WB , PMP were above DL s and demonstrated acceptable (<20%) intra‐assay and inter‐assay CV s in 9/10 dogs: 1.7% (0.5‐8.9) and 9.0% (0.9‐11.9), respectively, for CD 61 + /AnV − and 2.4% (0.2‐8.7) and 7.8% (0.0‐12.8), respectively, for CD 61 + /AnV + . Acceptable CV s were not seen for the CD 61 − /AnV + MP . In PPP , quantifications were challenged by high inter‐assay CV , overlapping DL s and hemolysis and lipemia interfered with quantification in 5/10 dogs. Conclusions 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.