Understanding Platelet-Rich Fibrin. Richard J. Miron

      It was certainly ironic that low levels of leukocytes were found in the PRF clot of L-PRF when compared to normal blood, considering the working name leukocyte PRF, which implies that the PRF clot would be leukocyte rich.

      While platelet concentrations were also lower in L-PRF vs A-PRF, it was revealed that the actual differences were not as drastic as previously reported.⁵¹ Our study demonstrated more specifically that the platelets and leukocytes are in fact found precisely at the junction between the yellow plasma and RBC interface. Previous studies likely failed to collect all the liquid within the yellow plasma layer, and as a result, extremely low platelet and leukocyte values may have been reported. As clearly shown in the present study, failure to do so, especially at a g-force of ~700g (RCF-max) or greater, results in extremely low concentration values because the upper 4 mL of plasma is practically devoid of cells. Therefore, we demonstrated in this study that L-PRF protocols are in fact quite rich in platelets (~80–90% total), but these cells are found precisely within a 1-mL layer directly above the RBCs within the buffy coat. It also demonstrates the effectiveness of the present methodologic protocol for evaluating PRF protocols.

In this study, the A-PRF protocol resulted in a more evenly distributed upper platelet layer throughout the PRF plasma layer, further validating the LSCC (Fig 2-18). Tubes at lower g-forces centrifuged for less time consistently resulted in a better distribution of platelets throughout the PRF matrix in the upper 4 to 5 mL, whereas an uneven distribution of cells was found using the 700g by 12-minute protocol. It is therefore clinically recommended to avoid utilizing original L-PRF protocols for membrane fabrication because all the cells are entirely found in a thin layer at the base of the PRF clot. However, low leukocyte yields were observed utilizing these protocols.

Fig 2-18 Summary of the findings comparing L-PRF and A-PRF protocols. While neither was typically able to collect leukocytes (reviewed later in the chapter), the lower centrifugation speeds using the A-PRF protocol allowed for the more even distribution of platelets in the upper layers. (Reprinted with permission from Miron et al.⁵²)

      This study also led to the observation that the manufacturer’s recommended protocol for i-PRF (~60g for 3 minutes) was not adequately effective at separating cell types or producing high yields of platelets/leukocytes. Figure 2-17 demonstrates minimal change in cell layer changes following this short centrifugation cycle at low RCF values. Based on the data obtained within this study, a paradigm shift in our understanding of platelet concentrates with respect to the LSCC was noted. We now know that too-low RCF values/times will produce ineffective separation of blood layers, as demonstrated in these i-PRF protocols.

      Based on these findings, we now know that too-low RCF values/times will produce ineffective separation of blood layers.

      Pitfalls in i-PRF protocols

      Several interesting findings were observed more recently with respect to the original i-PRF protocols. In 2016, when the first PRF textbook was written, Miron and Choukroun wrote the following: “Interestingly however, total growth factor release of PDGF-BB, VEGF, and TGF-β1 were significantly higher in PRP when compared to i-PRF. Methods to further understand these variations are continuously being investigated in our laboratory as well as others. It may be hypothesized that the differences in spin protocols are suggested to have collected slightly different cell populations and/or total growth factors responsible for the variations in release over time.”

      Back then, it remained puzzling to our research team why higher GF content was found in PRP despite the protocols utilized. Based on the results from the newly developed quantification method, it was revealed very clearly that the reason for these lower levels of GFs released from i-PRF was in fact owing to its inability to fully shift cells to their correct blood layers because centrifugation was carried out at too low a speed/time. Figure 2-18 demonstrates that only roughly 30% of total platelets are in fact accumulated in i-PRF, with 70% remaining in the lower RBC layer.⁵² Furthermore, a separate study conducted by an independent group found only minimal improvements in platelet numbers following i-PRF protocols (less than 50% increase), with an actual reduction in leukocyte concentration as well as VEGF release.⁵³ Therefore, it must clearly be noted that with respect to the LSCC, data has now demonstrated that it is definitely possible to centrifuge too slowly and too little a time for effectiveness. It became clear that improvements could be made to these i-PRF protocols.

      Optimization of i-PRF into C-PRF

Based on our findings that following L-PRF protocols, the majority of cells were massively accumulated at the buffy coat directly above the RBC layer (within 1 mL) with very few cells found throughout the upper four 1-mL layers,⁵⁰ we hypothesized that if we could specifically collect this 1-mL layer, we would create a much more liquid-PRF formulation rich in cells and GFs (Fig 2-19). In a study titled “A novel method for harvesting concentrated platelet-rich fibrin (C-PRF) with a 10-fold increase in platelet and leukocyte yield,”⁵² we addressed two specific questions: (1) In what total volume were the majority of these cells located above the RBC layer within the buffy coat? and (2) What final concentration could be harvested by collecting only the cells found within this precise buffy coat region when compared to conventional i-PRF protocols?

Fig 2-19 Proposed method to harvest C-PRF. Based on the finding that following L-PRF protocols all cells are accumulated within 300–500 µL above the RBC, this proposed method would collect this 0.3–0.5 mL of liquid C-PRF directly above the RBC junction for a highly concentrated liquid of platelets, leukocytes, and monocytes. (Reprinted with permission from Miron et al.⁵²)

Unlike the previous study, we decided to quantify PRF using 100-µL sequential layers (ie, 0.1 mL) to precisely investigate the exact location of cells (Fig 2-20). Because we had previously observed a massive cell accumulation within the buffy layer in a 1-mL sample range following the L-PRF protocol, we aimed to investigate precisely the volume in which these cells were located within this 1-mL layer. As such, we developed a novel methodologic approach whereby 100-µL sequential layers were pipetted starting from about 1.2–1.5 mL above the buffy coat down to the RBC layer (see Fig 2-20, depicted as +1 to +12 layers). Additionally, three layers were harvested within the RBC layer to determine the number of cells incorporated within this layer as well (see Fig 2-20, depicted as –1 to –3 layers). Each of these 100-µL layers was sent for CBC analysis.

Fig 2-20 A second methodologic illustration depicting the sequential harvesting technique. Because the majority of cells accumulated within the 1-mL within the buffy coat following L-PRF protocols, we sought to investigate precisely the total volume of liquid (mL) above the buffy coat in which cells are concentrated. For the L-PRF protocols, 3.5 mL were removed followed by sequential 100-µL layers pipetted followed by CBC analysis. Three layers in the RBC layer were also harvested. In comparison, all plasma layers of the i-PRF protocol were also harvested in 100-µL sequential layers. Three RBC layers (100 µL each) were also collected. (Reprinted with permission from Miron et al.⁵²)

      The second tube from each group was utilized to determine the final concentration from the liquid version