Cell and platelet composition assays by flow cytometry: basis for new platelet-rich fibrin methodologies

Abstract

Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are selective blood fractions obtained by cen¬trifugation. They act locally on inflammation and immunity as adjuvant homeostatic modulators during tissue regeneration. In recent years, many methods for achieving these blood concentrates have emerged, whose parameters of time and force of centrifugation presented themselves as critical, conflicting, and poorly understood points. Thus, the present study aimed to evaluate the effect of different centrifugal experimental parameters on the concentration of cells and platelets in samples of anticoagulated blood. Blood samples were centrifuged by forces of 200, 400 and 800 x g for 5, 10 and 15 minutes of centrifugation times to obtain three fractions: a) platelet-poor plasma (PPP), b) leukocyte-rich plasma (L- PRP) and c) red blood cell sed¬iment (RBC). The leukocyte and platelet content of each centrifuged fraction was measured by automated flow cytometry associated with the peroxidase reaction for differential leukocyte count. The application of 200 x g generated a more significant dispersive content of leukocytes and platelets in the supernatant fraction of PPP when compared to the other two strength ranges. However, it presented the highest concentration of platelets in the sediment (P <0.05 ANOVA), representing a loss of total mass during processing. The 400 and 800 x g forces showed leukocytes and platelets condensed in the L-PRP fraction and lower levels in the sedi¬ment, demonstrating the greater effectiveness of buoyancy in the resuspension of these sedimented elements. Our experimental data showed that the concentration and organization of leukocytes and platelets in the centrifuged blood matrices are very sensitive to variations in g force and centrifugation time, thus generating products with different biological composition and characteristics, and with specific potential therapeutic effects. The present study did not focus on comparing authoring methods, but on presenting the impact of methodological variations on the biological nature of centrifuged blood matrices. Further in vivo studies are needed to assess the specific clinical effect of each methodological change.

Keywords: centrifugation; g-force; leukocytes; platelet rich-fibrin; platelets.