PRF has gained tremendous momentum in recent years as a natural concentration of autologous growth factors capable of stimulating tissue regeneration. Despite its widespread use, to date, very little scientific data exist from studies investigating centrifugation protocols. In 2014, lower centrifugation speeds were proposed as a means to better accumulate growth factors and cells within the upper platelet-rich layers by modifying L-PRF protocols from 700 g down to 200 g . An approximately 20% increase in platelet concentration could be observed following these lower speed centrifugation protocols . While these previous methods based on histological observations allow for a relative estimate of the cells found in the various blood cell layers, the new methodology recently proposed by our group  allows for the precise quantification and concentration of cells in 1 mL incremental layers following centrifugation. This protocol provides researchers with a better ability to understand the events occurring following centrifugation at various protocols.
The aim of the present study was therefore to utilize the previously established method to investigate 24 protocols on a horizontal centrifuge. Table 2 demonstrates the properties of blood cells, including their density, frequency, surface areas and surface volumes. Note that while platelets are the least dense of the group, minor differences occur between leukocytes and RBCs, which greatly impacts the ability to separate blood cell layers based on density. Furthermore, while white blood cells (WBCs) are less dense when compared to RBCs (1055-1085 kg/m3 vs 1095-1100 kg/m3), note that WBCs are generally larger in size when compared to RBCs (surface area 330 vs 140 µm2, radium 5-7.5 vs 4 µm and volume of 200 vs 92 µm3). In addition, RBCs greatly outnumber WBCs by over 1000-fold (5 million RBCs vs 5000 WBCs per µL). These findings greatly influence the ability to separate cell types in a centrifuge based on density.
A horizontal centrifuge was utilized in this study owing to its better ability to separate blood cell layers (up to a 4 times greater yield in platelets/leukocytes) and avoid the grouping of cells along the back distal walls of centrifugation tubes as observed on fixed angle-centrifuges (Figure 8) . Two advantages were previously noted utilizing horizontal centrifugation . First, a completely horizontal position produced from a swing-out bucket allows for the greatest differential between the minimum and maximum radius found within a centrifugation tube (Figure 8). This effect allows for a greater ability to separate cell layers based on disparities between the RCF-min and RCF-max produced within a tube. Second, a fixed-angle centrifuge results in more trauma to cells along the back walls of centrifugation tubes . A recent study demonstrated that fixed-angle centrifugation led to uneven concentrations of cells and growth factors, with the majority of cells found on the back distal side of PRF tubes with uneven concentrations found throughout PRF clots . That paper highlights yet another effective method whereby cell layer separation can be evaluated histologically via sectioning of clotted PRF membranes layer by layer . Once allocated along the back distal tube surfaces, difficulty exists in effectively separating blood cells leading to lower yields of cells . For these reasons, horizontal centrifugation was recently proposed as a means to better separate blood cell layers for the production of PRF when compared to the results from fixed-angle centrifuges commonly utilized to produce solid-PRF protocols .
In general, platelets were evenly distributed throughout a variety of protocols within the upper 3-6 plasma-rich layers (Figures 3-7). Additionally, white blood cells required more pristine fine-tuning to reach adequate harmony within the upper plasma layers. Protocols within the 400-700 g (5-8 minute) range were able to accumulate platelets more evenly distributed throughout the upper layers, whereas slightly better optimization was required to effectively spread leukocytes within the upper plasma layers. Note that even though platelets were evenly distributed throughout the upper plasma layer, white blood cells typically did not fully distribute evenly throughout the upper 4-6 layers, with the greatest concentration remaining at the buffy coat layer (arrows, Figure 4).
The most surprising finding from the present study was the marked difference in cell layer separation that occurred at the various protocols. Notably, inadequate testing of protocols may drastically lead to a poor yield of cells evenly distributed throughout the PRF clot. A 400-700 g protocol for 8 minutes was required to effectively separate the cell layers for solid-PRF protocols. Interestingly, there was also variability reported between patients. As erythrocytes and other blood components also play a role in coagulation, as well as the hematocrit counts vary between patients and affect the size of PRF membranes , the present study also showed that not only the total plasma volume but also the cellular content of the layers were altered (Supplemental Figures 1-3). We have previously shown that both females and patients above the age of 65 typically have larger membranes as a result of their lower hematocrit counts . Future research is needed to better understand how centrifugation speed and time may be further optimized to result in similar final cell concentrations from patients with different starting blood cell counts. In summary, patients with lower cell blood counts would theoretically require lower centrifugation speeds, and further computational research is likely needed to optimize the protocols.
Currently, one standard in the field of PRF is the novel use of injectable i-PRF . Previously, our research group found that only slight increases in platelets and leukocytes were noted with failure to adequately accumulate cells in the upper plasma layer owing to extremely low RCF values (60 g) and centrifugation times (3-4 minutes) . While our group reported a 33% increase in cell concentrations in i-PRF protocols, other groups have more recently reported similar findings . In a study titled “Injectable platelet-rich fibrin: cell content, morphological, and protein characterization”, only a slight increase in platelets (less than 33%) and leukocytes was observed following i-PRF protocols, with decreases in VEGF reported when compared to that in whole blood . Altogether, these studies confirm that previously utilized i-PRF protocols (~60 g for 3-4 minutes on a fixed-angle centrifuge) are inadequately effective at separating blood cell layers owing to their considerable reduction in centrifugation speed and time. We found in the present study, that protocols at 100 g or lower were inefficient at accumulate platelets and leukocytes in the upper plasma layer. It must therefore be highlighted that a limit exists with respect to the ‘low-speed centrifugation concept’, with further research being needed to further optimize fixed-angle centrifugation systems.
In the present study, the use of PRF produced via horizontal centrifugation with the highest concentration of platelets and leukocytes was observed at higher and longer centrifugation speeds and times. By combining an increase in speed and time, up to a 4-fold increase in platelet/leukocyte concentration and/or yield was observed when compared to the results of previously utilized i-PRF protocols produced on a fixed-angle centrifuge . The present study further questions the use of trademarks such as ‘leukocytes and platelet-rich fibrin’ or ‘L-PRF’. As observed in our study, changes in centrifugation speeds and times can alter the final leukocyte concentrations/yields and may even further lead to actual reductions in leukocyte number when compared to that in whole blood (Table 1). Previously, we have shown how the production of L-PRF on a fixed angle-centrifuge actually led to an actual decrease in final leukocyte concentrations when compared to those in whole blood . In the present study, a protocol of 400 g-700 g for 8 minutes produced via horizontal centrifugation led to increases in leukocyte concentrations. To the best of the authors knowledge, these procedures are the only protocols established with a reported increase in leukocyte concentrations found in solid-PRF-based protocols.
One of the principal advantages of utilizing the present methodology to investigate cell layer separations was the ability to locate and quantify cell layer changes accurately over time and at different RCF values. The ability to quantify 960 CBCs favored a better understanding of the cell layer separation of PRF-based protocols. Future research aims to better understand how time versus speed affect blood cell layer changes and how patient variability can be fine-tuned to further optimize the centrifugation protocols for PRF in patients with different hematocrit levels. This study further highlights the need for additional research in the field to further improve the understanding of centrifugation protocols for the production of PRF.