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Updated: Jun 18, 2026
Routine Screening Method for Microparticles in Platelet Transfusions
Published on: January 31, 2018
A multicolor flow cytometric assay for measurement of platelet-derived microparticles
Fariborz Mobarrez1, Jovan Antovic, Nils Egberg
1Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, Division of Cardiovascular Medicine, Stockholm, Sweden. fariborz.mobarrez@ki.se
Introduction:
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.
Materials And 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.
Insights
Analyzing frozen platelet-derived microparticles (PDMPs) is now feasible. High-speed centrifugation minimizes cell fragment contamination, enabling reproducible quantification of PDMP markers like P-selectin and tissue factor using MESF values.
Area of Science:
- Hematology
- Biotechnology
- Analytical Chemistry
Background:
- Flow cytometry (FCM) is standard for platelet-derived microparticle (PDMP) detection but lacks standardization and requires fresh samples.
- Analyzing frozen PDMPs would enhance clinical utility, but freezing/thawing introduces cell membrane fragment artifacts.
- Contaminating cell fragments can interfere with accurate PDMP measurements.
Purpose of the Study:
- To develop a standardized method for analyzing PDMPs in frozen samples.
- To assess the impact of freezing/thawing on PDMP integrity and contamination.
- To establish reproducible quantification of PDMP markers using Molecules of Equivalent Soluble Fluorochrome (MESF) values.
Main Methods:
- PDMPs were labeled with antibodies against CD42a/CD62P or CD42a/CD142.
- PDMP identification used forward scatter (FSC) and CD42a expression.
- Molecules of Equivalent Soluble Fluorochrome (MESF) values were derived from Mean Fluorescence Intensities (MFIs) using a standard curve.
- FITC-labeled phalloidin detected cell fragments; high-speed centrifugation reduced contamination.
Main Results:
- Frozen/thawed samples showed significantly more phalloidin-positive particles than fresh samples.
- High-speed centrifugation yielded PDMP samples with low cell fragment contamination.
- Reproducibility (intra/inter-assay CVs) for MESF values was <10%; inter-laboratory variation was <5%.
- PDMP expression of CD142 (tissue factor) and CD62P (P-selectin) was twofold higher in type 1-diabetes patients versus controls (p<0.001).
Conclusions:
- MESF values provide reproducible quantification of P-selectin and tissue factor on PDMPs, enabling inter-laboratory comparisons.
- High-speed centrifugation effectively minimizes cell fragment contamination in frozen/thawed PDMP samples.
- Standardized analysis of frozen PDMPs is achievable, expanding their diagnostic potential.
