Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied.
BACKGROUND
Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification.


METHODS
Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1,550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 degrees C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 degrees C for 7 and 28 days. A final aliquot was further centrifuged at 10,000g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 microm.


RESULTS
High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p=0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b=0.611, p=0.025). Storage at 4 degrees C did not affect EMP quantification. However, freezing at -80 degrees C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p<0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance >0.487; p<0.05).


CONCLUSIONS
The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols.
van Ierssel
Sabrina H
SH
Department of Critical Care Medicine, Antwerp University Hospital (UZA), University of Antwerp, Edegem, Belgium. sabrina.vanierssel@ua.ac.be
Van Craenenbroeck
Emeline M
EM
Conraads
Viviane M
VM
Van Tendeloo
Viggo F
VF
Vrints
Christiaan J
CJ
Jorens
Philippe G
PG
Hoymans
Vicky Y
VY
D015415:Biomarkers / Q000097:blood; D002498:Centrifugation; D019040:E-Selectin / Q000097:blood; D004727:Endothelium / Q000473:pathology* / Q000503:physiopathology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D052250:Nitric Oxide Synthase Type III; D010641:Phenotype; D010949:Plasma / Q000166:cytology*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000097:blood
D015415:Biomarkers; D019040:E-Selectin; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III