Whole Blood Flow Cytometric Quantification of Tissue Factor-Positive Platelets: A Harmonized Multicenter Workflow

Tissue Factor (TF)^pos-platelets represent a subset of the platelet population. Recently, this subset has been shown to predict cardiovascular mortality in patients with coronary artery disease, establishing it as a biomarker useful for thrombotic risk stratification. Accurate quantification of TF^pos-platelets by flow cytometry requires strict standardization of pre-analytical handling, staining procedures, and instrument settings, particularly in multicenter studies where technical variability may affect their measurement. This protocol describes a harmonized workflow for whole-blood flow cytometry assessment of circulating TF^pos-platelets, designed to ensure reproducibility across laboratories with different technical infrastructures. The procedure includes standardized blood collection and whole-blood fixation to preserve the in vivo platelet phenotype. Two different methods for sample preparation are provided according to local laboratory capabilities: shipment of fixed samples to the Core Laboratory for centralized staining, acquisition, and analysis for centers without flow cytometry facilities, or local staining and acquisition for centers equipped with flow cytometry instrumentation and trained personnel. The assessment of the percentage of TF^pos-platelets is achieved by direct labeling with anti-TF Star Fluor 488 and anti-CD41 PerCP-Cy5.5 antibodies to identify the target protein and the platelet population marker, respectively. Flow cytometer harmonization is achieved either through a shared acquisition template for identical cytometer models or through bead-based alignment for different platforms. The workflow further incorporates a standardized gating approach and centralized data analysis to enable reliable comparison of TF^pos-platelet measurements across sites. Representative results demonstrated that this workflow supports reproducible quantification of TF^pos-platelets across the validated multicenter acquisition settings. This protocol may facilitate broader application of TF^pos-platelet assessment in thrombotic risk stratification and support wider standardization of platelet flow cytometry in translational and clinical research.