PB0869 - Single platelet function in thrombocytopenia patients measured with droplet microfluidics

Background: Platelet functional heterogeneity has been a topic of interest in several studies. However, only a droplet-based method can study pure intrinsic heterogeneity without the influence of adjacent platelets. This method has previously shown to detect novel subtypes of differently responsive platelets that contribute to the total response of platelets as a collective.

Aims: To adapt and apply the single platelet droplet microfluidic assay to detect subtypes of platelets in different patient groups suffering from thrombocytopenia.

Methods: The innovative droplet microfluidic protocol involves compartmentalising platelets in monodisperse droplets with agonists while excluding paracrine signalling. High frequency encapsulation (250 Hz) is coupled with flow cytometry for high throughput quantification of platelet responses to multiple agonists. The ideal intake of platelets is in platelet rich plasma at a concentration of 5x109/L and consumption is 60 μL/hr, so for mildly thrombocytopenic patients no adaptation is required and for severely thrombocytopenic patients a slow centrifugation of the platelet rich plasma can increase the concentration as required.

Results: Platelets were individually encapsulated in water-in-oil droplets with a mean diameter of 25 μm. In healthy individuals, it is shown that there exist hyperresponsive platelet subpopulations visible after addition of low concentration agonist that are not detectable outside droplets and drive the collective response. Furthermore, at high concentrations of agonists we can detect hyposensitive platelets that don’t respond with submaximal agonist activation. We hypothesize that these are increased in leukaemia patients where there is a functional defect in addition to the low counts and that the hyperresponsive population is enhanced in immune thrombocytopenic patients where a compensatory mechanism prevents bleeding even at very low counts.

Conclusion(s): This study demonstrates the value of a high throughput droplet microfluidics and flow cytometry workflow for measuring platelet heterogeneity and its application to thrombocytopenic patients of different origins.