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
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).
Last update: July 28, 2013