Macroscopic evaluation of the PRF clots produced with the 4 different centrifuges: original Intra-Spin L-PRF system (A) , A-PRF system (B) , Salvin centrifuge (C) and LW centrifuge (D) . Obvious differences can be observed in terms of size and aspect, the original L-PRF (A) being always denser and heavier (and in most cases larger) than the others. 

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... and cell and growth factors contents of the L-PRF are key characteristics of an original L-PRF clot/membrane as characterized in the literature [19] , and any modification of the material and protocol can lead to a different biological signature and clinical result [18] . The objective of this series of 3 articles was to point out the impact of the centrifuge characteristics and centrifugation protocol on the cell, growth factors and fibrin architecture of a L-PRF clot and membrane. In the first article, the mechanical vibrations (both radial and vertical) appearing during centrifugation were evaluated in 4 models of commercially available table centrifuges frequently used to produce L-PRF. It was proven that the original L-PRF centrifuge (Intra-Spin) was by far the most stable machine. At the classical speed of production of L-PRF, the level of undesirable vibration on this centrifuge is between 4.5 and 6 times lower than with other centrifuges. Moreover, Intra-Spin always remains under the threshold of resonance, unlike the 3 other tested machines. In this second article, the exact macroscopic and microscopic (photonic and scanning electron microscopy) characteristics and the cell composition of the L-PRF clots and membranes produced with these 4 different machines were evaluated. As a secondary objective, the impact of the vibration parameter on the architecture and cell content of the L-PRF clots was discussed. The study was conducted in accordance with the Helsinki Declaration (2000) and approved by the Medical Ethics Committee of the University of the Andes (UANDES). All volunteers provided signed informed consent. In this study, 4 different centrifuges, found on the market and used to produce L- PRF, were tested. The country of manufacture being used by some companies as a claim for quality, the country of manufacture of each centrifuge and its main components was checked. The 4 selected centrifuges were purchased from their manufacturers (or distributors). The first centrifuge was the original centrifuge used during the early development of the L-PRF open-access method and is nowadays marketed under the name Intra-Spin L-PRF centrifuge (Intra-Lock International, Boca-Raton, FL, USA; Made in Germany). It is actually the only CE marked and FDA cleared system for the preparation of L-PRF clots. The 3 other centrifuges are not CE/FDA cleared for L-PRF, but they can be found relatively frequently available on the market for this use (mostly because they are much cheaper): centrifuge A-PRF 12 (Advanced PRF, Process for PRF, Nice, France; Country of manufacture not indicated on the label, components inside show “Made in China”), centrifuge LW - UPD8 (LW Scientific, Lawrenceville, GA, USA; Components made in China, assembled in the USA) and centrifuge Salvin 1310 (Salvin Dental Specialties, Charlotte, NC, USA; Made in China). Blood samples were collected at the San Bernardo University of the Andes Health Center from 8 healthy volunteers (age range 25-35 years, ASA 1), with no history of recent aspirin intake or any medication neither disease correlated with the coagulation process. For each volunteer, nine tubes of blood were obtained from the antecubital vein. One tube with 2,5ml of anticoagulant was used for whole blood analysis as a control for normal blood parameters. Eight plastic glass-coated tubes were taken without anticoagulant (with BD Vacutainer Serum 10.0ml tubes, Becton Dickinson, Franklin Lakes, NJ, USA) for the production of L-PRF clots and membranes. The blood was collected quickly (22 seconds mean value, less 25 seconds per tube) and immediately (before 1 minute) centrifuged at 400g during 12 minutes in the four different centrifuges (two tubes were distributed per centrifuge in a randomized way) at room temperature. To standardize exactly the protocol and isolate only the centrifuge vibration parameter, the 400g centrifugation force used in the original L-PRF method (corresponding to 2700 rpm in the original Intra-Spin centrifuge) was used with all centrifuges, and rpm were adjusted accordingly for each centrifuge, i.e. 2400 rpm for the A-PRF machine and 2300 rpm for the LW centrifuge. Salvin centrifuge has only one preset possible speed (3400 rpm), which lead to a centrifugation force higher than 400g. The temperatures of the surface at the center of the tubes were registered before and after centrifugation with an infrared thermometer (HVACPro, Fluke, Everett, WA, USA). A total of 64 L-PRF clots/membranes were obtained: 32 membranes were prepared for Scanning Electron Microscopy (SEM) analysis and 32 membranes were prepared for light/photonic microscopy. After centrifugation the L-PRF clot was removed from the tube using sterile tweezers and a smooth spatula to gently release the red blood cells clot inside the tube ( Figure 1A ). The L-PRF fibrin clot obtained was placed on a sterile microscope slide ( Figures 1B, 1C ) placed in an individual tray for weight and size measurements ( Figure 2 ). The supernatant and red blood cells clot remaining in the tube were also weighted to get the L-PRF fibrin clot / whole blood ratio per tube. Each sterile microscope slide had in every corner a 1mm rubber stop ( Figure 1C) to allow the compression of the clot with another microscope slide using 100 grams constant pressure for two minutes. This standardized method allowed to obtain from each clot 1mm-thick L-PRF membranes, which were weighted and measured individually ( Figure 3 ). From each volunteer, two membranes were obtained per each centrifuge and after macro analysis (weight, size measurements) were prepared for histologic procedures. One membrane was prepared for SEM evaluation and the second one for light-microscopy analysis. The membranes were kept between the microscope slides during fixation to avoid distortions. The membranes were fixed in 10% neutrally buffered formalin for 24 hours at room temperature for paraffin inclusion. Successive sections of 4 microns were performed along the center of the long axis of the membranes and were stained with hematoxylin-eosin. Each section was divided in three areas of equal size: Proximal (Head & Face), Center (Body), Distal (Tail). Each area of these sections was observed through light microscopy and analyzed by counting the visible cell bodies (marked in dark purple, mostly leukocytes) in the center of each area observed with a 40X magnification. The total numbers of counted cell bodies were used to correlate their distribution among the three areas of the membrane (head & face, body and tail). Most of the cells were concentrated in the proximal area (head & face). A morphologic evaluation of the L-PRF membranes was done with a scanning electron microscope. The membranes were fixed in 2.5% glutaraldehyde for 24 hours at 4oC and treated for gradual desiccation. The specimens were sputter-coated with 20nm gold (Edwards S-150, Crawley, UK) and examined in a scanning electron microscope (JEOL JSM- 6380LV, JEOL Ltd, Tokyo, Japan). Photographs were taken with 15 to 20kV using 2,000 to 5,000X magnifications. This study was mainly descriptive. All the macroscopic results are presented in the Table . The numeric values are clearly illustrated by the observation of the clots and membranes in the Figures 2 and 3 . For the temperature of the tubes, Intra-Spin allowed to keep the lowest temperature among the 4 tested machines. A-PRF and Salvin were both associated with a significant increase of temperature in the tube. For the clot and exudate weights, Intra-Spin produced by far the heaviest clot and quantity of exudate among the 4 techniques. Salvin remains high but far behind. Finally A- PRF and LW produced very light and small clots. For the membranes weights, Intra-Spin and Salvin presented similar weight. The A-PRF and LW membranes were significantly lighter. In terms of clot and membrane length and width, the clots and membranes from Intra-Spin and Salvin presented similar sizes. The A-PRF and LW clots and membranes were significantly shorter and more narrow. Finally, the Intra-Spin L-PRF clot was the heaviest clot to be produced with an initial blood harvest of ...