Understanding Platelet-Rich Fibrin. Richard J. Miron
In a study titled “Injectable platelet rich fibrin (i-PRF): Opportunities in regenerative dentistry?”,20 it was demonstrated that at lower centrifugation speeds and times (~60g for 3 minutes), a liquid-PRF (termed injectable-PRF or i-PRF) could be obtained. While these protocols typically produced minimal volumes (~1.0–1.5 mL), it was shown that both platelets and leukocytes were even more highly concentrated when compared to L-PRF or A-PRF (Fig 1-7).40 This liquid-PRF layer could be utilized clinically for approximately 15 to 20 minutes, during which time fibrinogen and thrombin had not yet converted to a fibrin matrix (ie, remained liquid). This has since been utilized for injection into various joints/spaces similar to PRP, however with the reported advantages of a longer GF release time. Furthermore, the concept of “sticky” bone was also developed. Importantly, a different type of tube (plastic) was needed to minimize clotting, as will be discussed in detail in chapter 5.
Fig 1-7 Newer centrifugation protocols allow production of a liquid formulation of PRF found in the top 1- to 2-mL layer of centrifugation tubes following a 3- to 5-minute protocol. This liquid can be collected in a syringe and reinjected into defect sites or mixed with biomaterials to improve their bioactive properties. (Reprinted with permission from Davies and Miron.40)
Snapshot of A-PRF and i-PRF
Original L-PRF protocols were shown to be too fast, leading to all the cells being accumulated only at the buffy coat zone, with the majority of leukocytes found within the red blood cell layer.
The low-speed centrifugation concept was shown in 2014 to favor a higher concentration of cells within PRF membranes.
By further lowering speed and time, a liquid-PRF formulation became available, commonly known as injectable-PRF (or i-PRF).
H-PRF and C-PRF (2019–Present)
Very recently, our research group discovered through a series of basic laboratory experiments that horizontal centrifugation led to significantly greater concentrations of platelets and leukocytes when compared to currently available fixed-angle centrifugation devices most commonly utilized to produce L-PRF and A-PRF. Simply, horizontal centrifuges are routinely utilized in high-end research laboratories as well as in medical hospitals because of their greater ability to separate layers based on density (Fig 1-8; see also chapters 2 and 3). Unlike fixed-angle centrifugation systems whereby the tubes are actually inserted at a 45-degree angle, in horizontal centrifugation systems (often referred to as swing-out bucket centrifugation), the tubes have the ability to swing out to 90 degrees once they are in rotation (Video 1-2). Amazingly, the original PRP systems developed by Harvest and Marx utilized and still use this technology.
Fig 1-8 (a) Clinical photograph of a Bio-PRF centrifuge. (b) Photograph demonstrating the horizontal centrifugation concept. The tubes are inserted vertically (up and down), but once the device begins to rotate, the tubes swing out completely horizontally. This favors better blood cell layer separation with higher platelet and GF concentrations.
In 2019, an article on the topic demonstrated clearly that horizontal centrifugation could lead to up to a four-times greater cell content when compared to fixed-angle centrifugation.41 This represented a marked ability to greatly concentrate cells found within PRF, which were primarily being accumulated on the back distal surfaces of PRF tubes (Fig 1-9). The major disadvantage of fixed-angle centrifugation is that during the spin cycle, cells are typically driven along the back wall of the centrifugation tubes at high g-forces (Fig 1-10). This also exposes cells to higher compressive forces against the back wall, and cells must then separate by traveling either up or down the inclined centrifugation slope based on their respective cell density differences. Because red blood cells are larger and heavier than platelets and leukocytes, they travel downward, whereas lighter platelets travel toward the top of the tube where PRF is collected. This makes it relatively difficult for the small cell types such as platelets and leukocytes to reach the upper layer, especially granted that red blood cells outnumber in particular white blood cells typically by 1,000-fold (see chapter 2). Therefore, it is not possible to reach optimal accumulation of platelets or leukocytes using a fixed-angle centrifuge.
Fig 1-9 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 (~200g–700g), the 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 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.41)
Fig 1-10 Visual representation of layer separation following either L-PRF or H-PRF protocols. L-PRF clots are prepared with a sloped shape, and multiple red dots are often observed on the distal surface of PRF tubes, while H-PRF results in horizontal layer separation between the upper plasma and lower red corpuscle layer.
Furthermore, by utilizing a novel method to quantify cell types found in PRF, it was possible to substantially improve standard i-PRF protocols that favored only a 1.5- to 3-fold increase in platelets and leukocytes. Noteworthy is that several research groups began to show that the final concentration of platelets was only marginally improved in i-PRF when compared to standard baseline values of whole blood.41,42 In addition, significant modifications to PRF centrifugation protocols have further been developed, demonstrating the ability to improve standard i-PRF protocols toward liquid formulations that are significantly more concentrated (C-PRF) with over 10- to 15-times greater concentrations of platelets and leukocytes when compared to i-PRF (see chapters 2 and 3). Today, C-PRF has been established as the most highly concentrated PRF protocol described in the literature.
Snapshot of H-PRF and C-PRF
Horizontal