Measurement of monocyte-platelet aggregates by imaging flow cytometry

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets express P-Selectin (CD62P) on the cell membrane and bind to P-Selectin glycoprotein ligand 1 expressing monocytes, influencing them toward a pro-adhesive and inflammatory phenotype. It is well established that elevated circulating monocyte-platelet aggregates (MPAs) are linked to atherothrombosis in high risk patients. However, whole blood flow cytometry (FCM) has recently shown that circulating MPAs may also occur in the absence of platelet activation, particularly in healthy children. A potential limitation of conventional FCM is the potential for coincident events to resemble monocyte platelet aggregates. Here we report a novel imaging cytometry approach to further characterize monocyte-platelet aggregate formation by P-Selectin dependent and P-Selectin independent mechanisms and distinguish circulating MPAs from coincidental events. Monocytes were identified by expression of the lipopolysachharide receptor (CD14 BV421), while platelets were identified by expression of the glycoprotein Ib (CD42b APC). Differentiation of P-Selectin dependent and P-Selectin independent binding was achieved with AF488 labeled CD62P. Overall analysis of circulating and in vitro generated MPAs by conventional and imaging cytometry methods showed very strong correlation (r(2)  = >0.99, P < 0.01). The Bland-Altman bias of -1.72 was not significantly different to zero. However, when measuring only P-Selectin negative MPAs, a lack of correlation (r(2)  = 0.27, P = n.s.) likely reflects better discrimination of coincidence events using imaging cytometry. Our data demonstrate that IFC is more accurate in enumerating MPAs than conventional FCM, which over-estimates the number of MPAs due to the presence of coincident events.