Methodological Considerations in Platelet Flow Cytometric Studies

Platelets are anucleate cells that can be studied by benchtop flow cytometers and today there are several types of flow cytometric assays for platelets. Platelet structure studies in clinical flow cytometry mostly target rare congenital platelet glycoprotein deficiencies (e.g., Glanzmann-thrombasthenia, and Bernard-Soulier syndrome) that can be considered as the prototypes for platelet flow analysis, since the identification of complete or partial deficiency of these molecules provides the proper diagnosis. Another area of platelet flow cytometry is the detection of activated platelets. Mostly it means the gold standard platelet P-selectin assay or the investigation of PAC-1 binding-an assay where the activation dependent epitope of the GPIIb/IIIa receptor is investigated-but the measurement of platelet-leukocyte aggregates and microparticle analysis have gained increasing role. A unique platelet subtype is the subgroup designated as coated platelets that are formed by the simultaneous activation with collagen and thrombin. In clinical practice further functional assays are also informative tests: the monitoring of clopidogrel resistance by measuring the intracellular phosphorylated VASP and identifying heparin induced thrombocytopenia (HIT) by the use of patient plasma. The flow cytometric HIT test utilize the detection of phosphatidylserine expression on normal platelets or the formation of microparticles in the presence of the patient's plasma and therapeutic concentration of heparin, thus it seems reasonable to suggest it for the study of the functionality of the HIT antibodies. Nevertheless, there are also limitations to these assays as several of them require fresh samples, thus sample transport to specialized laboratories is not always feasible.