Richard J. Miron - Understanding Platelet-Rich Fibrin

57.Miron RJ, Dham A, Dham U, Zhang Y, Pikos MA, Sculean A. The effect of age, gender, and time between blood draw and start of centrifugation on the size outcomes of platelet-rich fibrin (PRF) membranes. Clin Oral Investig 2019;23:2179–2185.

58.Zeng SM, Yankowitz J, Widness JA, Strauss RG. Etiology of differences in hematocrit between males and females: Sequence-based polymorphisms in erythropoietin and its receptor. J Gend Specif Med 2001;4:35–40.

59.Billett HH. Hemoglobin and hematocrit. In: Walker HK, Hall WD, Hurst JW (eds). Clinical Methods: The History, Physical, and Laboratory Examinations, ed 3. Boston: Butterworths, 1990.

3

Horizontal Versus Fixed-Angle Centrifugation of PRF: Optimization of C-PRF

Contributors

Richard J. Miron

Masako Fujioka-Kobayashi

Tomoyuki Kawase

Yufeng Zhang

Chapter Highlights

Advantages of horizontal centrifugation versus fixed-angle centrifugation

Systematic evaluation of horizontal centrifugation using 24 different protocols

Optimization of H-PRF in both liquid and solid formulations

Histologic evaluation of L-PRF versus H-PRF

Optimization of C-PRF using horizontal centrifugation

Video 3-1

The first two chapters of this textbook discussed the history of platelet concentrates and their commercial and biologic evolution. The aim of this chapter is to discuss centrifugation parameters that may best optimize the production of PRF. A great deal of work has been conducted at both the preclinical and clinical level to maximize the regenerative potential of PRF, and this chapter provides a more detailed overview of these methods and takes a closer look at the cell types found in PRF. It also discusses the work conducted by our laboratories to improve PRF using horizontal centrifugation by evaluating a range of 24 different protocols in a systematic and standardized way. Thereafter, comparative histologic evaluation compares PRF membranes produced following fixed-angle versus horizontal centrifugation. Lastly, data from the previous chapter is expanded upon to explain the concept of C-PRF.

The previous chapter provided a brief overview of the advantages of horizontal centrifugation of PRF. Previously, publications by our group found that horizontal centrifugation was superior at accumulating platelets and leukocytes when compared to standard fixed-angle centrifugation utilized to produce PRF. 1Both solid-based and liquid-based PRF matrices were obtained with up to a fourfold increase in platelet/leukocyte numbers and/or concentrations. 1

This chapter discusses in greater detail the ability of horizontal-PRF (H-PRF) to better separate cell layers in blood based on their density. As previously shown (see Table 2-1), platelets are the lightest of the blood cells, with white blood cells (WBCs) and red blood cells (RBCs) similar in size and density ( Fig 3-1). Therefore, the optimization of centrifugation parameters becomes extremely relevant should the clinician desire to maximize the harvest of cell types following centrifugation.

Fig 3-1SEM showing the relative size of RBCs, platelets, and WBCs (leukocytes). Note the much larger size of RBCs and WBCs when compared to platelets as well as the similarity in size between WBCs and RBCs.

When cells are separated in a blood collection tube based on their density, it is important to understand that their ability to separate is based on differences in g-force produced at the RCF-min versus RCF-max of the tube (as reviewed in greater detail in chapter 4). Note that the greater the angulation of the tube, the greater the difference in g-force differential that exists between the RCF-min of the tube versus the RCF-max ( Fig 3-2).

Fig 3-2Graphic demonstrating the difference between the RCF-min and RCF-max on both fixed-angle and horizontal centrifugation systems. Note that because the tubes are completely horizontal on a horizontal centrifugation system, the gradient difference between the RCF-min and RCF-max is much greater, resulting in much better layer separation.

One avenue that is extremely relevant to the production of PRF has been the effect of the larger radius from the rotor found when centrifugation is carried out on a horizontal versus fixed-angle centrifuge. To illustrate this point further, an overlapping image of a centrifugation tube is provided in Fig 3-3. If a certain g-force can be expected at the RCF-clot, a much smaller RCF-max will be produced on a fixed-angle centrifuge ( Fig 3-3a) when compared to a horizontal centrifuge ( Fig 3-3b). While certain commercial entities such as the L-PRF system will report the g-force values specifically at the RCF-clot, note how in Fig 3-3b, the same RCF-clot value on both fixed-angle and horizontal systems displays a much greater RCF-max value if the tube is horizontal. This allows for higher g-force productions at the RCF-max on a horizontal centrifuge, which means faster and better ability for the heavier cells (ie, RBCs) to be pulled down to the bottom of the tube more effectively.

Fig 3-3 (a and b) Demonstration that even at the exact same RCF-clot value on either fixed-angle or horizontal centrifugation, the final RCF-max value on a fixed-angle centrifugation system is much smaller than that observed on a horizontal system due to the greater distance from the centrifuge rotor. (c) The introduction of 13-mL tubes has led to claims that they are better able to concentrate cells. While this is true compared to 10-mL tubes, if centrifugation remains carried out on a fixed-angle device, the difference between the RCF-min and RCF-max remains inferior to that on a horizontal centrifuge, even if a 10-mL tube is used.

More recently, an introduction of longer tubes (13-mL tubes versus 10-mL) has been proposed as a way to further concentrate cells ( Fig 3-3c). While this concept is true, note that should these tubes be utilized on a fixed-angle centri-fugation device, the difference between the RCF-min and RCF-max will still equate to less than that obtained with a 10-mL tube in a horizontal centrifuge. These longer tubes still pose the main issue related to cells accumulating on the back distal surface of PRF tubes without proper cell layer separation, as reviewed in the previous chapter.

Larger Radius, Higher RCF, Shorter Spin Time

While not peer-reviewed in the same way as our published studies, the Internet is rife with support for horizontal centrifugation. Drucker Diagnostics, for example, clearly demonstrate how tubes centrifuged in a horizontal manner experience a much larger radius, resulting in a higher RCF and more efficient pull-down forces ( https://druckerdiagnostics.com/horizontal-vs-fixed-angle/). Furthermore, they have shown that the time required for complete centrifugation on a horizontal rotor is only two-thirds of the time required on a fixed-angle centrifuge ( Fig 3-4). This is particularly important because PRF is subject to clotting over time, so saving a few critical minutes by switching to horizontal centrifuge can translate to better layer separation and greater accumulation of cells following the protocol. 2