Blood platelet activation evaluated by flow cytometry: optimised methods for clinical studies.

Area of Science:

• Hematology
• Biomedical Engineering
• Clinical Diagnostics

Background:

• In vivo blood platelet activation is crucial for understanding thrombotic and inflammatory conditions.
• Existing flow cytometry methods for platelet activation require optimization for clinical settings.
• Accurate and stable assessment of platelet activation markers is essential for reliable clinical studies.

Purpose of the Study:

• To develop and optimize flow cytometry techniques for evaluating in vivo blood platelet activation in clinical studies.
• To enhance the stability and accuracy of platelet activation marker measurements.
• To extend the preanalytic storage time for various platelet function assays.

Main Methods:

• Preloading EDTA vacuum blood sampling tubes with paraformaldehyde (PFA) to stabilize whole blood samples.
• Assessing platelet CD62P (P-selectin) expression using flow cytometry with phycoerythrein-conjugated anti-CD62P.
• Utilizing Monovette citrate tubes preloaded with buffer to extend storage for PAC-1 and fibrinogen binding assays.

Main Results:

• PFA preloading enabled assessment of CD62P expression with <0.2% activated platelets, requiring no washing or neutralization steps.
• Basal and stimulated CD62P expression remained stable for at least 48 hours post-sampling.
• Assay conditions proved suitable for evaluating other platelet markers (CD41, CD42a, CD61, CD63) and microparticle/aggregate analysis.
• Preanalytic storage for PAC-1 and fibrinogen binding was extended to at least 2 hours.

Conclusions:

• Optimized paraformaldehyde preloading offers a robust, stable, and clinically applicable method for assessing platelet activation via CD62P expression.
• The developed techniques improve the reliability and extend the feasibility of platelet function testing in clinical research.
• These advancements facilitate more accurate evaluation of platelet behavior in various clinical conditions.