Multicolor flow cytometry in clinical samples for platelet signaling assessment

Area of Science:

• Hematology
• Immunology
• Biochemistry

Background:

• Multichannel cytometers and specific antibodies enable flow cytometry for simultaneous assessment of intracellular platelet signaling pathways.
• This method is suitable for clinical purposes, even with small blood volumes or low platelet counts.

Purpose of the Study:

• To describe a multicolor flow cytometry technique with fluorescent barcoding.
• To screen signaling pathways downstream of major platelet agonists (ADP, thrombin, thromboxane, collagen).

Main Methods:

• Selected target phosphoproteins (AKT, P38MAPK, LIMK, SPL76) and optimized phosphoflow barcoding conditions by comparison with immunoblotting.
• Conducted a clinical study on whole blood from patients with bleeding of unknown origin, von Willebrand disease, and specific platelet disorders.

Main Results:

• Established fluorescence intensity ranges, kinetics, and distributions for agonist-target protein combinations.
• Principal component analysis revealed correlations in phosphoprotein response to different agonists, but not vice versa.
• Clustering analysis identified heterogeneity in individual responses, with storage pool deficiency patients showing the lowest responses.

Conclusions:

• This study introduces a novel approach for rapid platelet signaling profiling.
• The technique complements existing functional tests for enhanced clinical diagnostics.