Evaluation of 24 protocols for the production of platelet-rich fibrin

Richard J Miron, Jihua Chai, Masako Fujioka-Kobayashi, Anton Sculean, Yufeng Zhang, Richard J Miron, Jihua Chai, Masako Fujioka-Kobayashi, Anton Sculean, Yufeng Zhang

Abstract

Background: The aim of this study was to evaluate 24 protocols for the production of platelet rich fibrin (PRF) produced via horizontal centrifugation to better understand cell separation following protocols at various times and speeds.

Methods: All protocols were compared utilizing a recent method to quantify cells in PRF in 1 mL sequential layers pipetted from the upper layer downwards until all 10 mL were harvested. In total, 960 complete blood counts (CBCs) were investigated. Both solid and liquid-based PRF protocols were investigated following 24 protocols involving 6 relative centrifugal force (RCF) values (100, 200, 400, 700, 1000 and 1200g) at 4 centrifugation times (3, 5, 8 and 12 min).

Results: In general, platelets could more easily accumulate in the upper 4 layers when compared to leukocytes owing to their lower cellular density. Protocol time seemed to have a greater impact on the final cell layer separation when compared to the effect of speed. Protocols of greater than 8 min at 400g led to no leukocyte accumulation in the upper PRF layers (found specifically within the buffy coat). Protocols at or below 200g were unable to effectively accumulate platelets/leukocytes. The optimal centrifugation speed and time for solid-PRF ranged between 400 and 700g for 8 min. It was noted that variability in patient baseline platelet/leukocyte/erythrocyte counts (hematocrit) significantly affected cell layer separation. This finding was more pronounced at lower centrifugation speeds.

Conclusions: Within the investigated ranges, a protocol of 700g for 8 min presented the highest yield of platelets/leukocytes evenly distributed throughout the upper PRF layers.

Keywords: A-PRF; Advanced platelet-rich fibrin; L-PRF; Leukocyte and platelet-rich fibrin; i-PRF.

Conflict of interest statement

AS is an editorial board member for BMC Oral Health. RJM holds intellectual property for the production of PRF. All other authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Clinical image demonstrating the plasma layer separation for the 24 protocols investigated in this study. Note that while some protocols reveal roughly identical plasma layer separation, the underlying cellular content in the various protocols may be drastically different
Fig. 2
Fig. 2
Illustration demonstrating the proposed novel method to quantify cell types following the centrifugation of PRF. One of the limitations of the current methods utilized to investigate PRF cellular content from the whole plasma layer is the inability to accurately determine where cells migrate following centrifugation. By utilizing the proposed technique using the sequentially pipetting 1 mL layers from the top layer downwards, it is possible to quantify cell numbers in 10 samples from CBC analysis and accurately determine the precise location of each cell layer following centrifugation at various protocols. Note that one layer (in this case, layer 5) will contain both yellow plasma and red blood cells. This effect is figuratively depicted with arrows to demonstrate the location of the buffy coat where a higher concentration of platelets/leukocytes is typically found. Reprinted with permission [16]
Fig. 3
Fig. 3
Evaluation of 24 protocols utilized for the production of PRF. Data includes final volume (mL), total leukocyte and platelet yields (% of the total from 10 mL) as well as concentration of leukocytes and platelets above baseline values (% increase)
Fig. 4
Fig. 4
The concentration of cell types in each layer from 1 mL down to the 10th 1-mL sample utilizing the solid-PRF horizontal centrifugation protocol (700g for 8 min). Notice that most of the platelets are evenly distributed throughout the upper plasma layer. Similarly, white blood cells are primarily distributed throughout the upper 4 layers, though not as even as platelets owing to their slightly higher cellular density (Arrows represent the separation between the plasma and red blood cell layer (buffy coat)). Reprinted with permission [16]
Fig. 5
Fig. 5
Observed variability of the concentration of cell types in each layer from 1 mL down to the 10th 1-mL sample utilizing the same solid-PRF horizontal centrifugation protocol (700g for 8 min) as depicted in Fig. 4 (owing to difference in patient gender and age). Notice that when compared to Fig. 4, more leukocytes are found within the buffy coat layer. This finding is a result of a patient demonstrating lower hematocrit counts (typically occurring in older female patients), thereby resulting in easier cell-layer separation and more pronounced accumulation within the buffy coat layer (arrows represent the separation between the plasma and red blood cell layer (buffy coat))
Fig. 6
Fig. 6
The concentration of cell types in each layer from 1 mL down to the 10th 1-mL sample utilizing a solid-PRF horizontal centrifugation protocol at 700g for 12 min. Notice that when compared to Fig. 4, the higher centrifuge time resulted in more leukocytes concentrated within the buffy coat layer. This finding demonstrates that over time, cells migrate more toward the buffy coat layer with extended periods of time or extended RCF values (Arrows represent the separation between the plasma and red blood cell layer (buffy coat))
Fig. 7
Fig. 7
The concentration of cell types found in each layer from 1 mL down to the 10th 1-mL sample utilizing the liquid-PRF protocol of 200g for 5 min. Notice that while not as many cells are located in the plasma layer, a higher concentration of platelets and leukocytes can be found owing to the reduced plasma volume when compared to that in the solid-PRF protocols demonstrated in Fig. 3 (Arrows represent the separation between the plasma and red blood cell layer (buffy coat))
Fig. 8
Fig. 8
Illustrations comparing fixed-angle and horizontal centrifuges. With horizontal centrifugation, a greater separation of blood layers based on density is achieved owing to the greater difference in RCF-min and RCF-max. Following centrifugation on fixed-angle centrifuges, blood layers do not separate evenly, and as a result, an angled blood separation is observed. In contrast, horizontal centrifugation produces even separation. Owing to the large RCF values (~ 200–700g), the cells are pushed toward the outside and downwards. On a fixed-angle centrifuge, cells are pushed toward the back of centrifugation tubes and then downwards/upwards based on cell density. These g-forces produce additional shear stress on cells as they separate based on density along the back walls of centrifugation tubes. In contrast, horizontal centrifugation allows for the free movement of cells to separate into their appropriate layers based on density, allowing for better cell separation as well as less trauma/shear stress on cells. Modified from Miron et al. [16].

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Source: PubMed

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