Richard J. Miron - Understanding Platelet-Rich Fibrin

Fig 2-20A 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 of the i-PRF yellow plasma layer. For L-PRF protocols, one tube was utilized to harvest 0.5 mL of a C-PRF (defined as the 0.5-mL buffy coat directly above the RBC layer). This layer was termed concentrated-PRF in reference to the harvesting of this concentrated buffy coat layer (see Fig 2-19). Similarly, 0.3 mL of C-PRF liquid was harvested from this layer as well.

To address our first question regarding the precise volume in the buffy coat in which the increased cell numbers were observed, the first 3.5 mL of the upper plasma layer was removed (acellular layer) from the centrifugation tube (leaving 1.0–1.5 mL of remaining sample above the RBC layer). The sequential pipetting methodology was then utilized with 100-µL layers to accurately determine up to what layer above the RBC the cells were precisely located (see Fig 2-20). Furthermore, 300 µL within the RBC layer was also harvested and quantified in 100-µL sequential layers. In contrast, the entire i-PRF layer was collected starting from the upper 100-µL layer and sequentially pipetted until all plasma layers were collected. Once again, 300 µL was sequentially pipetted in 100-µL layers from the RBC layer.

Figure 2-21demonstrates the results following sequential pipetting of 100-µL layers of the i-PRF protocol. Notice how specifically in layer +1, a threefold increase (from 5 to 15 × 10 9cells/L) is found in leukocytes directly at the buffy coat layer (represented by arrows ). Notice also the five- to sixfold increase in monocytes. The RBCs begin to accumulate at layer +1, and by layer –1 the sample is within the RBC layer. The remaining WBC and platelet levels drop within layer –1 after the yellow-red transition (see Fig 2-21). Following the i-PRF protocol, we observed a 2.5-fold increase (from baseline ~220 to ~550 × 10 9platelets/L) in platelets in the top 13 layers (1.3 mL) following the i-PRF protocol and only a slight increase in leukocytes.

Fig 2-21The concentration of cell types in each 100-µL layer utilizing the i-PRF protocol (800 rpm for 3 minutes; ~60g). Notice the significant increase in leukocytes and monocytes in the buffy coat layer (+1; arrows ). (Adapted from Miron et al. 52)

Figure 2-22demonstrates the results following sequential pipetting of 100-µL layers of the L-PRF protocol. Interestingly, almost all the cells accumulate within the three layers (ie, 300 µL) above the RBC layer. Most surprisingly, within this layer a massive increase in platelets, monocytes, leukocytes, and lymphocytes was observed. For instance, a roughly 225 to 6,000 × 10 9platelets/L increase was observed, representing a > 25-fold increase in platelet concentration, specifically 100 µL above the RBC layer.

Fig 2-22The concentration of cell types in each of the layers (100 µL each) above the RBC layer following the L-PRF protocol. Notice the massive increase in platelets (roughly a 20-fold increase) specifically at the buffy coat layer between the yellow plasma and RBC layers. Interestingly, all cells seemed to accumulate within the three to five layers (300–500 µL) above the RBC layer. (Adapted from Miron et al. 52)

Based on these results, we assumed that a 0.3- to 0.5-mL layer of C-PRF could be preferentially collected within this buffy coat directly above the RBC layer. Figure 2-23a demonstrates that while the i-PRF protocol increases leukocyte numbers 1.23-fold, a marked and significant increase representing a 4.62- and 7.34-fold increase was observed with both 0.5 mL and 0.3 mL of C-PRF, respectively. Even more pronounced is that while i-PRF protocols have typically been shown to increase platelet yields between 200% and 300%, the C-PRF protocols massively increased platelet yields 1138% and 1687%, respectively ( Fig 2-23b). A similar trend was also observed for monocytes ( Fig 2-23c). The total values following averages from six patients are summarized in Table 2-2.

Fig 2-23Concentration of leukocytes (a) , platelets (b) , and monocytes (c) following centrifugation using i-PRF protocols versus collecting 0.3 to 0.5 mL of C-PRF. Notice that while i-PRF was typically responsible for a 1.2- to 2.5-fold increase in the various cell types following centrifugation, up to a 15-fold increase in platelet concentration could be achieved with C-PRF. An asterisk (*) represents a significant difference when compared to i-PRF; a double asterisk (**) represents a value significantly higher than all groups; P < .05. (Adapted from Miron et al. 52)

Table 2-2Leukocyte, platelet, and monocyte concentrations in whole blood compared to i-PRF and C-PRF

Chapter 3discusses this new method of concentrating liquid-PRF directly from the buffy coat layer in more detail and reports on the marked improvement in cellular activity when compared to original i-PRF protocols.

Horizontal Centrifugation of PRF

Until 2019, the majority of centrifugation carried out for the production of PRF was performed on fixed-angle centrifuges. Horizontal centrifugation, on the other hand, is utilized frequently in research laboratories and in medical hospitals due to its superior ability to separate layers based on their density ( Fig 2-24). In fact, the original PRP systems that date back 20 years were brought to market utilizing horizontal centrifugation for this very reason 54(see chapters 3and 4). In an attempt to investigate cell layer separation utilizing horizontal centrifugation, the same layer-by-layer sequential pipetting (1 mL each) was utilized as previously done with the L-PRF and A-PRF protocols.

Fig 2-24Illustrations comparing fixed-angle and horizontal centrifugation. (a) With fixed-angle centrifuges, a greater separation of blood layers based on density is achieved owing to the greater difference in RCF-min and RCF-max. (b) 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 an even separation. (c) Because of the large RCF values (~200g–700g), cells are pushed toward the outside and downward. On a fixed-angle centrifuge, cells are pushed toward the back of centrifugation tubes and then downward/upward 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 mobility of cells to separate into their appropriate layers based on density, allowing for more optimal cell separation as well as less trauma/shear stress on cells. (Reprinted with permission from Miron et al. 50)