Validation of flow cytometric detection of platelet microparticles and liposomes by atomic force microscopy.

Background: Platelet microparticles (PMPs) are a promising prognostic marker for thrombotic disorders because of their release during platelet activation. The use of flow cytometry for the enumeration of PMPs in plasma has generated controversy due to their size, which is below the stated detection limits of conventional flow cytometry instruments. The potential impact of this is an underestimation of PMP counts.

Objectives/methods: To address this possibility, we used a combination of fluorescence-activated cell sorting (FACS) and atomic force microscopy (AFM) to determine the size distribution of PMPs present in plasma from acute myocardial infarction (AMI) patients and normal volunteers, and PMPs generated by expired platelet concentrates and washed platelets treated with agonists such as thrombin and calcium ionophore (A23187).

Results: According to AFM image analysis, there was no statistically significant difference in height or volume distributions in PMPs from thrombin-activated, calcium ionophore-activated, expired platelet concentrates and plasma from healthy volunteers and AMI patients. Based on volume, expired platelets released the greatest proportion of exosomes (< 1.0 × 10(-22) L(3) in volume) in relation to the entire PMP population (29.7%) and the smallest proportion of exosomes was observed in AMI patient plasma (1.8%). Moreover, AFM imaging revealed that PMPs from expired platelets exhibited smooth surfaces compared with other PMP types but this was not statistically significant.

Conclusions: We confirm that flow cytometry is capable of analyzing PMPs from plasma by using AFM to perform nanoscale measurements of individual PMP events isolated by FACS. This method also provided the first quantitative nanoscale images of PMP ultrastructure.