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Updated: May 25, 2026
Routine Screening Method for Microparticles in Platelet Transfusions
Published on: January 31, 2018
Red cell microparticle enumeration: validation of a flow cytometric approach
Z Xiong1, T B Oriss, J P Cavaretta
1Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
Background And Objectives:
There is growing interest in the clinical application of red blood cell (RBC) microparticle (MP) enumeration as they have been postulated to be effectors of coagulation and inflammation following transfusion and in sickle cell disease. No uniform approach in MP enumeration exists and a key limitation is the lack of an internal validation process. We present and validate a flow cytometric approach where an internal standard is utilized.
Materials And Methods:
Glycophorin A(+) Annexin V(+) events were enumerated using MPs isolated from RBC units or plasma samples obtained from volunteers. A mixture of absolute counting (7·6 μm) and calibration beads (0·5, 0·9 and 3 μm) at a fixed ratio was added to each sample.
Results:
RBC MPs were initially selected based upon a fluorescence threshold, and the 0·5- and 0·9-μm beads defined the upper and lower light scatter distribution of MPs. The ratio of 7·6:3-μm bead events was used as an internal standard to validate the precision of MP enumeration across samples (coefficient of variation = 2·5-7·2%) and remained constant in both platelet-rich plasma (PRP) and platelet-free plasma (PFP). RBC MP counts increased in both PRP and PFP obtained from whole blood stimulated with ionophore and increasing calcium concentrations, with PRP showing higher MP counts than PFP at every concentration studied.
Conclusion:
This method is a useful strategy to detect RBC MP counts across bio-samples provided that the flow cytometer can reliably discriminate the size of the calibration beads.
Insights
Researchers developed a flow cytometry method using an internal standard to accurately count red blood cell microparticles (RBC-MPs). This validated approach improves precision for enumerating RBC-MPs in various samples, aiding research in transfusion and sickle cell disease.
Area of Science:
- Hematology
- Biotechnology
- Analytical Chemistry
Background:
- Red blood cell microparticles (RBC-MPs) are implicated in transfusion reactions and sickle cell disease.
- Current methods for enumerating RBC-MPs lack standardization and internal validation.
- Accurate RBC-MP quantification is crucial for understanding their role in coagulation and inflammation.
Purpose of the Study:
- To present and validate a novel flow cytometric approach for enumerating RBC-MPs.
- To introduce an internal standard for improving the precision and reliability of RBC-MP counts.
- To establish a standardized method for RBC-MP analysis in clinical and research settings.
Main Methods:
- Enumeration of Glycophorin A(+) Annexin V(+) events from isolated RBC microparticles (MPs).
- Utilized a mixture of absolute counting beads (7.6 μm) and calibration beads (0.5, 0.9, 3 μm) as an internal standard.
- Validated precision using the ratio of 7.6:3-μm bead events across samples.
Main Results:
- The 0.5- and 0.9-μm beads helped define the light scatter distribution of MPs.
- The internal standard (7.6:3-μm bead ratio) demonstrated high precision (CV 2.5-7.2%) across samples.
- RBC MP counts increased with ionophore and calcium stimulation, with higher counts in platelet-rich plasma.
Conclusions:
- The developed flow cytometry method provides a validated strategy for RBC-MP enumeration.
- Reliable discrimination of calibration bead sizes by the flow cytometer is essential for method accuracy.
- This approach enhances the precision of RBC-MP detection in various biological samples.
