Flow Cytometry

Flow-cytometric measurement of platelet-surface glycoproteins in unfixed whole blood is a rapid, sensitive, and quantitative method that enables simultaneous analysis of multiple aspects of platelet biology to be conducted on large numbers of single platelets in a short time (29). It can be carried out in small samples of whole blood, and thrombocytopenic samples can be analyzed. It enables 100% of the platelet population to be studied, including giant platelets, platelet-derived microparticles, and platelet-leukocyte aggregates. Analysis of unfixed blood samples allows investigation of the platelet response to agonist stimulation. Finally, platelets are studied in autologous plasma and in the presence of the other blood cells, which can contribute to the overall platelet response through the release of soluble mediators.

Usually, the platelets are labeled with two monoclonal antibodies (MAbs) conjugated to fluorophores with different emission spectra (30). Platelets are identified by the fluorescence of the platelet identifier. The fluorescence emitted by the second MAb is directly proportional to the density of the epitope of interest and is evaluated by acquiring several thousand platelet events. If the expression of the epitope of interest changes with time (e.g., by trafficking ofmembrane glycoproteins), platelets can be fixed before staining (30). Platelets can also be fixed after staining if immediate access to a flow cytometer is delayed.

Many fluorescently conjugated antibody- and nonantibody-based probes are available to label platelet epitopes. In addition to MAbs that bind glycoproteins on resting platelets, there are activation-dependent MAbs that detect conformational changes in glycoproteins (e.g., PAC1 for GpIIb/IIIa) or the expression of granule membrane proteins (e.g., anti-P-selectin). Nonantibody probes include PKH lipophilic dyes of membranes; the protein label biotin, which is detected by fluorescently conjugated streptavidin; and fluorescently conjugated annexin V, which detects exposure of the procoagulant surface after platelet activation (31).

Clinically, flow cytometry is useful in assessing the extent of platelet activation ex vivo and the sensitivity of platelets to added agonists in vitro. It has been used to show activated platelets in patients with unstable angina, acute myocardial infarction (AMI), stable coronary disease, and coronary angioplasty (32-34). It has also been used to assess differences between anticoagulation regimen in patients with non-ST-elevation acute coronary syndrome (ACS) (35). The randomized study ARMADA (n = 141) identified markers of blood cell activation that independently predict outcomes at 1 mo. Enoxaparin, dalte-parin, and unfractionated heparin (UFH) were compared in terms of efficacy, safety, and effects on any such markers. Using multivariate analysis, increased plasma levels of vWF, decreased platelet levels of GpIb/IX complexes, and monocyte tissue factor expression were identified as independent predictors of adverse outcomes at 1 mo. Enoxaparin and

Fig. 4. Mechanical assessment of platelet function (36).

dalteparin reduced the release of vWF compared with UFH. Enoxaparin had a more favorable effect on GpIb/IX complexes than dalteparin or UFH.

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