Accurate Quantification of Platelets and Leukocytes in Platelet-Rich Fibrin Using Direct Fibrinolytic Digestion: Effects of Centrifugation Protocols on Cellular Composition and Fibrin Architecture

Background/Objectives: Platelet-rich fibrin (PRF) is widely used in regenerative medicine and dentistry because of its ability to deliver growth factors and cellular components that support tissue healing. However, accurate quantification of platelets and leukocytes within PRF matrices remains challenging, as indirect subtraction-based methods may not reliably reflect the true cellular composition. This study aimed to apply a direct fibrinolytic digestion–flow cytometry approach for precise cellular quantification and to systematically evaluate the effects of different centrifugation protocols on PRF cellular composition and fibrin architecture. Methods: PRF clots were generated using high-speed protocols (leukocyte- and platelet-rich fibrin [L-PRF] and concentrated growth factors [CGF]) and low-speed protocols (advanced PRF [A-PRF] and A-PRF+). Cellular content was quantified using a fibrinolytic digestion-based approach with tissue plasminogen activator, followed by flow cytometry analysis. Histological evaluation was performed to assess fibrin architecture and cellular distribution. Results: L-PRF and CGF exhibited significantly greater platelet enrichment than A-PRF. T-lymphocyte counts were elevated across all PRF groups, with significantly higher levels observed in L-PRF and CGF than in A-PRF. Monocyte and neutrophil levels were reduced following PRF preparation; however, A-PRF retained significantly higher neutrophil levels than CGF, whereas B-lymphocyte levels showed a moderate increase without significant intergroup differences. Histologically, high-speed protocols produced pronounced cellular stratification and denser fibrin networks, whereas low-speed protocols demonstrated a more homogeneous leukocyte distribution within a porous fibrin matrix. Conclusions: Centrifugation protocols significantly influence PRF cellular composition and fibrin architecture. The direct fibrinolytic digestion–flow cytometry approach provides a reliable method for accurate cellular quantification and may facilitate the standardization and optimization of PRF preparation protocols for regenerative applications.