A multicolor flow cytometric assay for measurement of platelet-derived microparticles.
BACKGROUND
Flow cytometry (FCM) is the most commonly used method for detection of platelet-derived microparticles (PDMPs), but it is poorly standardized and mainly used for "bedside" analyses in fresh samples. If PDMPs could be analyzed in previously frozen samples it would increase the usefulness of the method. However, cell membrane fragments from contaminating cells created during freezing/thawing may cause artifacts and disturb measurements.


METHODS
PDMPs were labeled with monoclonal antibodies directed against CD42a and CD62P, or CD42a and CD142. The PDMP gate was determined using forward scatter (FSC) and CD42a expression. The mean fluorescence intensities (MFIs) of CD62P or CD142 positive particles were translated into MESF -values (Molecules of Equivalent Soluble Fluorochrome) using a standard curve. FITC-labeled phalloidin (which binds to intracellular actin) was used to detect destroyed cells/cell fragments.


RESULTS
Phalloidin-positive particles were significantly more common in supernatants of frozen/thawed platelet rich and platelet poor plasma samples compared with supernatants of platelet free plasma. High-speed centrifugation was then used to obtain PDMP samples with low contamination of cell fragments. Electron microscopy showed that these samples contained numerous round stained particles with cellular membranes of a size of 100-700 nm. Reproducibility experiments using plasma samples from healthy individuals showed that the coefficients of variation (CVs) of MESF values of CD62P and CD142 (both intra- and interassay) were <10%, and the variation between two cytometers in two different laboratories was <5%. We also found that PDMP expression of CD142 (i.e. tissue factor [TF]) and CD62P (i.e P-selectin) was around two times higher in samples from type 1-diabetes patients compared with those from healthy controls (p<0.001).


CONCLUSIONS
The use of MESF values to quantify PDMP expression of P-selectin and TF yields reproducible data and enables comparison of data between laboratories. If high-speed centrifugation is performed, contamination of cell fragments is low in frozen/thawed samples.
Mobarrez
Fariborz
F
Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, Division of Cardiovascular Medicine, Stockholm, Sweden. fariborz.mobarrez@ki.se
Antovic
Jovan
J
Egberg
Nils
N
Hansson
Mona
M
Jörneskog
Gun
G
Hultenby
Kjell
K
Wallén
Håkan
H
D000911:Antibodies, Monoclonal / Q000276:immunology; D001681:Biological Assay / Q000379:methods*; D001792:Blood Platelets / Q000276:immunology*; D055252:Cell-Derived Microparticles / Q000276:immunology*; D002498:Centrifugation; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005615:Freezing; D006801:Humans; D019007:P-Selectin / Q000276:immunology; D010590:Phalloidine / Q000276:immunology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology; D012015:Reference Standards
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D019007:P-Selectin; D019038:Platelet Glycoprotein GPIb-IX Complex; D010590:Phalloidine; D016650:Fluorescein-5-isothiocyanate