Phosphoflow cytometry and barcoding in blood platelets: Technical and analytical considerations

Platelet function is regulated by finely tuned phosphoprotein signals. Subtle aberrations in signaling can cause platelet hyperactivity and severe cardiovascular events. Mapping phosphorylation profiles in health and disease could accelerate antiplatelet discovery and enhance cardiovascular management, but traditional assays are ill-suited to clinical application as they are laborious and low throughput. Recent advances in multiplex flow cytometry (barcoding) allow the rapid acquisition of highly batched samples with standard laboratory equipment. However, many assays have not been standardized, and success is largely dependent on protocol/reagent selection. Accordingly, we review the technical steps that are key to success with an emphasis on fixation, permeabilization, staining, controls, and data visualization.