Flow Cytometry

Some assays of platelet function are best achieved with flow cytometry, a method that allows both enhanced analytical sensitivity and minimal mechanical manipulation during processing. A flow cytometer device operates as a cell counter that uses a 488 nm argon ion laser as an excitation light source to additional detectors for fluorescence signal measurement. This allows to gather information on the size and internal complexity (forward scatter and side scatter, respectively) as well as markers, identified by staining with fluorescent probes, ususally phycoerythrin (red fluorescence) and fluorescein (green fluorescence).

  • Platelet surface markers: appear on the platelet surface before activation. Some useful are CD41 (GP IIb/IIIa), CD42a (GPIX), CD42b (GPIb), and CD61 (avb3, vitronectin receptor).
  • Platelet activation markers: appear on the platelet surface during activation. Some useful are PAC-1 (activated IIb/IIIa), CD62P (P-selectin), CD31 (PECAM) and CD63.

Once cells or tissues have been collected, flow cytometric procedure starts through staining and washing, continue through acquisition of flow data on the cytometer, and culminate in data analysis and the reporting of results. Each of these components is subject to quality assessment evaluation and quality control procedures. The cytometer itself is set up and monitored with a quality control program involving the use of alignment, reference, and calibration standards.

Left: a Becton-Dickinson FACScan Flow Cytometer, equipped with a Mac computer with suitable software. Right: a scheme of the flow cytometry procedure using labelled antibodies. Click on pictures to enlarge. (For image uses, please see Use of Content at Legal Information).

Bibliography

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Braunwald E, Angiolillo D, Bates E, Berger PB, Bhatt D, Cannon CP, Furman MI, Gurbel P, Michelson AD, Peterson E, Wiviott S. Antiplatelet therapy and platelet function testing. Clin Cardiol. 2008; 31: I36. 10.1002/clc.20371.

Hernandez MR, Galan AM, Lozano M, Bozzo J, Cases A, Escolar G, Calls J, Ordinas A. Platelet-leukocyte activation during hemodialysis detected with a monoclonal antibody to leukocyte integrin CD11b. Nephron. 1998; 80: 197-203. 10.1159/000045167.

Lopez-Vilchez I, Hedner U, Altisent C, Diaz-Ricart M, Escolar G, Galan AM. Redistribution and hemostatic action of recombinant activated factor VII associated with platelets. Am J Pathol. 2011; 178: 2938-48. 10.1016/j.ajpath.2011.02.026.

Jerez-Dolz D, Torramade-Moix S, Palomo M, Moreno-Castano A, Lopez-Vilchez I, Hernandez R, Badimon JJ, Zafar MU, Diaz-Ricart M, Escolar G. Internalization of microparticles by platelets is partially mediated by toll-like receptor 4 and enhances platelet thrombogenicity. Atherosclerosis. 2020; 294: 17-24. 10.1016/j.atherosclerosis.2019.12.017.