Imaging flow cytometry in the assessment of leukocyte-platelet aggregates

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets undergo granule exocytosis, resulting in α-granule P-Selectin being expressed on the cell membrane. This allows binding of activated platelets to P-Selectin glycoprotein ligand 1 (PSGL-1) expressing leukocytes, forming leukocyte-platelet aggregates (LPAs). Whole blood flow cytometry (FCM) has demonstrated that elevated circulating LPAs (especially monocyte LPAs) are linked to atherothrombosis in high risk patients, and that activated platelet binding influences monocytes towards a pro-adhesive and pro-atherogenic phenotype. However, a limitation of conventional FCM is the potential for coincident events to resemble LPAs despite no tethering. Imaging cytometry can be used to characterize LPA formation and distinguish circulating MPAs from coincidental events. Platelets and leukocyte subsets are identified by expression of surface markers (e.g. the lipopolysachharide receptor CD14 on monocytes, glycoprotein Ib CD42b on platelets). In conventional FCM, all events with both leukocyte and platelet characteristics are designated as LPAs. However, by using an ‘internal’ mask based on the brightfield image and the fluorescent platelet identifier, imaging flow cytometry is able to distinguish leukocytes with tethered platelets (genuine LPAs) from leukocyte with coincidental, untethered platelets nearby. Mechanisms (e.g. adhesion molecules) or consequences (e.g. signal transduction) can then be separately analysed in platelet tethered and untethered leukocytes. Imaging flow cytometry therefore provides a more accurate approach for both enumeration and analysis of LPAs than conventional FCM.