Multicolor flow cytometry in clinical samples for platelet signaling assessment

BackgroundAvailability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume blood or low count.ObjectivesTo describe multicolor with fluorescent barcoding technique screening downstream membrane receptors major agonists (adenosine diphosphate, thrombin, thromboxane, collagen).MethodsBy comparison immunoblotting, we first selected the target phosphoproteins, AKT, P38MAPK, LIMK, SPL76; times stimulation; phosphoflow conditions. We then performed study on whole patients without evidence disorder standard biological screening, consulting trivial occasionally provoked bleeds familial antecedent (bleeding unknown origin, n = 23) type-1 von Willebrand disease (n 9). In addition, included group definite disorders (Glanzmann thrombasthenia, δ-storage pool deficiency, immune glycoprotein VI–related granule secretion defect).ResultsThe range, kinetics, distribution fluorescence intensity were established each agonist-target protein combination. Principal component analysis indicates correlation response phosphoprotein (AKT P38MAPK) different but no phosphoproteins same agonist. The heterogeneity individual responses population displayed was analyzed using clustering algorithm. Patients storage deficiency positioned as lowest responders heatmap.ConclusionIn complement functional tests, this introduces approach rapid profiling practice.