L-PRF clot (marked in blue = the face part), membrane & plug.
SEM figure: shows a 3D fibrin network that entraps leukocytes and platelets.
Courtesy Jize Yu
STEP 1: venipuncture:
STEP 2: centrifugation (fast coagulating):
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place the tubes in the centrifuge within 1 minute after blood collection and start centrifugation; due to this time restriction, the centrifuge is loaded 2 by 2, allowing previously loaded tubes to be centrifuged while new ones are being filled with blood),
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the original L-PRF protocol prescribes a centrifugation for ≥ 12 min at a g-force of 408 (see below); however, if the patient is taking anticoagulants, the centrifugation time needs to be extended to 18-20 minutes,
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the centrifuge rotates at very high speeds, requiring optimal rotor stability; therefore, the tubes must always be placed opposite each other to ensure the centrifuge remains balanced; see special guidelines for centrifugation (Quirynen et al. 2025a).
STEP 3: collect the L-PRF clots:
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after centrifugation, 3 distinct layers are formed in the blood tube, with the L-PRF clot located in the middle,
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use tweezers to carefully grab the clots (which have a consistency similar to that of a snail),
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immediately remove the red blood cells; do not cut the red part of the clot away with scissors; instead, carefully scrape away the red blood cells, as beneath them lies a high concentration of white blood cells and platelets.
step-by-step flow chart
Blood is collected in "specialized" tubes with an inner surface that promotes blood coagulation. After ≥ 12 minutes of centrifugation, 3 distinct layers are formed: an acellular plasma at the top, the L-PRF clot in the middle, and the red blood cell fraction at the bottom. The L-PRF clot can be grabbed with tweezers and gently compressed into membranes. These membranes measure 3 by 1 cm in size and 0.5 mm in thickness. The area marked with a blue line is the biologically most active region (the face portion).
STEP 4: prepare the L-PRF membranes:
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gently compress the clots into membranes using a special compression kit,
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the glass plate, as shown in the video, is used solely to demonstrate the amount of exudate released from the clot during the compression; this exudate is beneficial for several applications.
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after 5 minutes, the "strong" L-PRF membranes are ready for use.
When L-PRF plugs (cylindrical form) are preferred (e.g., to fill extraction sockets), the clots must be compressed in the white cylinders of the compression kit.
Equipment
The equipment includes useful hand instruments and a specialized compression box. The compression plate (indicated by the blue arrow) and the box cover facilitate gentle compression. The white cylinders are used to shape the clots into plugs; with compression achieved using a piston until it is leveled with the border of the cylinder.
Collect blood in specialized tubes that promote coagulation. Rotate the tubes immediately after drawing the blood to increase contact between the inner tube surface and the blood. Place the first 2 tubes of blood in the centrifuge immediately after collection, positioning them opposite to each other, and start centrifugation. Collect the 3rd and 4th tubes of blood, pause the centrifuge, add the new tubes, and restart the centrifugation. Repeat this procedure for all remaining tubes.
After centrifugation, remove the L-PRF (fibrin) clots from the tubes and carefully separate them from the red blood cells. Do not cut away the red (face) portion of the clot, as it is the most biologically active part. Gently compress the clots using the light metal plate and cover of the compression box as weight to form L-PRF membranes (5 minutes). The liquid that leaks out of the clots is called L-PRF exudate. Plugs can be prepared using the white cylinders.
Characteristics of L-PRF membrane
1: Histology.
This drawing shows a detailed view of an L-PRF clot, pointing out where red blood cells (RBC), white blood cells (WBC), and platelets are located in the face, body, and tail sections. The face (right side) appears red due to the presence of residual RBCs. Directly beneath, there is a dense zone of WBCs, followed by a slightly deeper zone of blood platelets. The remainder of the clot consists of a dense 3-D fibrin matrix with platelets and only a few WBC.
2: Biological capacities.
L-PRF is rich in fibrin, platelets, white blood cells, growth factors, cytokines, and other components that promote tissue repair. L-PRF membranes are stable, resilient, strong, adhesive, and malleable. Within the L-PRF membrane, platelets are tightly merged, and the enmeshed leukocytes remain alive and functional. These membranes possess attractive biochemical properties, including hemostatic, angiogenic, osteogenic, anti-inflammatory, anti-microbial, pain-inhibitory, and wound-healing characteristics. When you compare the makeup of the patient's whole blood to that of an L-PRF membrane (which is only 3% of the blood's weight), the levels of platelets and WBCs are 20–25 times higher (for more information, see Blanco et al. 2025).
3: Mechanical strength.
L-PRF membranes are relatively strong and can be used as a barrier membrane. A single membrane, previously being blood, can support up to 500 g in weight. Their tensile strength and E-modulus are relatively high (for more details see Ockerman et al. 2022).
This video shows osseodensifying burs pushed into a single L-PRF membrane. Despite the slight bend in the instrument's arms, the membrane remains intact.
4: Release of growth factors.
The release of growth factors from an L-PRF membrane varies by region, with the face part (indicated in blue) and the body part (indicated in orange) showing different profiles. Key growth factors such as PDGF-AB (Platelet-Derived Growth Factor-AB), VEGF (Vascular Endothelial Growth Factor), TGF (Transforming Growth Factor), and BMP-1 (Bone Morphogenetic Protein-1) are released over a period of up to 14 days, with the majority being released during the first 72 hours. Notably, the face part releases higher concentrations of growth factors.
paper in preparation
Key factors for success:
A: Choice of blood tubes
The inner surface of the blood tube significantly impacts blood coagulation and, consequently, the formation of an L-PRF clot.
The left tube has an inner surface (silica coating) that promotes blood coagulation during centrifugation, thereby enhancing the formation of an L-PRF clot. Conversely, the right tube has an inert inner surface that prevents coagulation; this tube is used to prepare liquid fibrinogen or i-PRF (injectable PRF).
For the preparation of L-PRF membranes, clots, or plugs, it is essential to use a tube with a coagulation-stimulating inner surface, either a tube with a silica coating on the inside (left tube) or an entire glass tube (three tubes on the right).
It is important to note that not all glass tubes have the same impact on the coagulation cascade. Some glass surfaces promote rapid coagulation, while others result in slower coagulation. For surfaces that promote faster coagulation, centrifugation should begin promptly. For surfaces that cause slower coagulation, you can collect all the blood tubes, wait an additional 5 minutes, and then start the centrifugation process.
In some tubes the L-PRF clot may adhere to the inner surface and must be loosened with a spatula.
The tubes have an expiration date that indicates when the vacuum is guaranteed to be effective.
B: Choice of centrifuge
A centrifuge separates substances of different densities in a liquid by rotating at a certain speed (measured in revolutions per minute, RPM). The force applied during centrifugation is called relative centrifugal force (RCF). This force causes denser substances and particles to move outward in the radial direction, settling at the bottom of the tube, while low-density substances move to the top. The separation (layer formation in the tube) during centrifugation is influenced by several factors, including: (i) time of spinning, (ii) the speed (RPM), and (iii) the g-force (also referred to as the relative centrifugal force, RCF).
The final g-force can be calculated by the following formula: RCF = 11.18 x r x (RPM/1000)². In this formula, r is the radius (in centimeters) from the center of the rotor to the blood tube (particularly to the area where the L-PRF clot is formed). In other words, the speed is influenced by the size of the rotor; the wider the centrifuge, the higher the g-force at a given RPM.
To achieve a g-force of 408 (original protocol for L-PRF), the RPM for an IntraSpin centrifuge is 2,700, whereas for a DuoQuatro centrifuge it would be 2,180 due to its wider radius.
C: Conditions for a successful centrifugation
To achieve an optimal separation between the different layers in the blood tubes, the following prerequisites should be considered:
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due to the high speeds at which the centrifuge spins, it must be placed on a steady, flat surface; if the operator notices the centrifuge sliding or the counter underneath sagging, the centrifuge should be relocated to a more stable location,
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all tubes must have equal mass; ensure the tubes are filled to the mark indicating the correct fill level, or wait until the blood begins to drip instead of flowing.
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when the tubes require a short time between blood draw and centrifugation, and 2-by-2 centrifuge loading is needed, it is crucial to place the tubes opposite each other to maintain rotor balance during centrifugation,
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when spinning an uneven number of blood tubes, an extra tube filled with water at the same volume must be prepared and positioned opposite to the blood tube to maintain balance in the rotor.
Important Notice
What is the difference between PRP, PRGF and L-PRF?
Abbreviations: PRP = platelet-rich plasma, PRGF = plasma rich in growth factors, L-PRF = leukocyte- and platelet-rich fibrin, PPP = platelet-poor plasma, PPGF = plasma poor in growth factors, WBC (white blood cells).
Autologous platelet concentrates (APCs) have evolved significantly since their inception. This illustration highlights the differences in preparation between first-generation (PRP, PRGF) and second-generation (L-PRF) autologous platelet concentrates (APCs), along with their respective concentrations of platelets and leukocytes when compared to whole blood. The first generation of APCs emerged in the late 1990s. Their preparation is relatively complex and requires the use of anticoagulants and coagulation factors. In contrast, the second-generation APCs, L-PRF, introduced by Choukroun et al. in 2001, are easier to prepare and do not require the use of either anticoagulants or coagulation factors, ensuring the final product is still 100% autogenous (for more details see Quirynen et al. 2025).
What is the difference between L-PRF, A-PRF, A-PRF+, T-PRF, H-PRF, or CGF?
Several recent modifications in the preparation of Leukocyte- and Platelet-Rich Fibrin (L-PRF) have been introduced, including Concentrated Growth Factors (CGF), Advanced PRF (A-PRF), Advanced Plus PRF (A-PRF+), Titanium-Prepared Platelet Rich-Fibrin (T-PRF, using titanium tubes), and L-PRF prepared with a horizontal centrifuge (H-PRF). These modifications involve minor adjustments in centrifugation time, speed, tube type, and/or the angle of the tubes within the centrifuge.
To date, laboratory data regarding the beneficial effect of these centrifugation modifications on cellular content and/or growth factor release have been contradictory. Furthermore, the "clinical" benefits of these so-called "improved" clots and membranes remain to be confirmed. Randomized controlled trials (RCTs) comparing these modifications directly to each other are highly desirable.
Interesting references
Several videos and/or cases on this webpage are discussed more in detail in the following book: Quirynen M & Pinto N 2022. Leukocyte- and Platelet-Rich Fibrin in Oral Regenerative Procedures. Quintessence Publishing;
ISBN: 978-1-78698-105-9