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Routine Screening Method for Microparticles in Platelet Transfusions
Published on: January 31, 2018
Avoiding false positive antigen detection by flow cytometry on blood cell derived microparticles: the importance of
Emerence Crompot1, Michael Van Damme1, Hugues Duvillier2
1Laboratory of Clinical Cell Therapy, Université Libre de Bruxelles (ULB), Jules Bordet Institute, Brussels, Belgium.
Background:
Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results.
Materials And Methods:
We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM).
Results:
Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins).
Conclusion:
We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.
Insights
Characterizing microparticles (MPs) using flow cytometry can yield false positives due to antibody issues. Careful use of negative controls is crucial for accurate MP phenotyping.
Area of Science:
- Biotechnology
- Cell Biology
- Immunology
Background:
- Microparticles (MPs), also known as microvesicles (MVs), are cell-derived fragments.
- Accurate characterization of MPs is vital for understanding their biological roles.
- Current methods for MP analysis lack standardized negative controls, leading to potential inaccuracies.
Purpose of the Study:
- To investigate the reliability of flow cytometry for MP phenotyping.
- To identify sources of false positive results in MP analysis.
- To emphasize the importance of appropriate negative controls in MP characterization.
Main Methods:
- Analysis of MPs derived from platelets, B-cells, T-cells, NK-cells, and monocytes.
- Use of monoclonal antibodies for cell and MP surface marker identification.
- Application of annexin-V staining, latex bead technique, Transmission Electron Microscopy (TEM), and Scanning Electron Microscopy (SEM).
Main Results:
- Platelet-derived MPs (CD41/CD61 positive) were correctly identified.
- Antigens on MPs from other cell types were often undetectable, contradicting existing literature.
- The latex bead technique confirmed some markers but revealed false positives for others due to antibody non-specificity.
Conclusions:
- Direct flow cytometry and latex bead cytometry may not always detect cell-derived antigens on MPs.
- Antibody aspecificity is a significant cause of false positive MP characterization.
- Robust negative controls are essential for reliable phenotypic analysis of microparticles.
