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Routine Screening Method for Microparticles in Platelet Transfusions
Published on: January 31, 2018
Detection of microparticles from human red blood cells by multiparametric flow cytometry
Giulia Grisendi1, Elena Finetti2, Daniele Manganaro3
1Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.
Background:
During storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as "storage lesions". These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC.
Material And Methods:
RBC units (n=10) were stored under blood bank conditions for up to 42 days. Samples were tested at different time points to detect microparticles and determine the haemolysis rate (HR%). Microparticles were identified by flow cytometry combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V and anti-glycophorin A antibody.
Results:
We demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected.
Discussion:
Multiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies.
Insights
Multiparametric flow cytometry effectively quantifies microparticle release from stored red blood cells (RBCs). This method enhances detection of microparticles, crucial for understanding transfusion safety and improving cell-based therapies.
Area of Science:
- Hematology
- Biotechnology
- Analytical Chemistry
Background:
- Stored red blood cells (RBCs) undergo storage lesions, leading to loss of integrity, lysis, and microparticle release.
- Microparticles from stored RBCs are implicated in transfusion side effects and pathogen transmission.
- Current methods like flow cytometry are used to quantify microparticles, but improved techniques are needed.
Purpose of the Study:
- To develop and validate a multiparametric staining technique for monitoring microparticle release from stored human RBCs.
- To quantify the dynamic release of microparticles during RBC storage using flow cytometry.
- To assess the sensitivity and precision of the proposed staining method.
Main Methods:
- Human RBC units (n=10) were stored for up to 42 days under blood bank conditions.
- Samples were analyzed at various time points to detect microparticles and determine the hemolysis rate (HR%).
- Flow cytometry was employed, combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V, and anti-glycophorin A antibody for multiparametric staining.
Main Results:
- CFSE successfully labeled closed vesicles with intact membranes, indicating its utility for microparticle detection.
- The combination of CFSE and glycophorin A antibody effectively monitored and quantified microparticle release.
- Double staining with CFSE/glycophorin A increased vesicle detection by up to 4.7-fold compared to glycophorin A/annexin V alone, showing a robust correlation between HR% and microparticle number (R=0.625; p=0.0001).
Conclusions:
- Multiparametric staining using CFSE, glycophorin A antibody, and annexin V provides a sensitive method for detecting and characterizing microparticle release from stored RBCs.
- This approach offers a precise way to monitor microparticle dynamics during storage.
- The developed technique has significant implications for transfusion medicine and broader cell-based therapies.
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