Principles and Methods of Preparation of Platelet-Rich Plasma: A Review and Author's Perspective

BACKGROUND

Platelets are cytoplasmic fragments of megakaryocytes, formed in the marrow[12] and approximately 2 μm in diameter. They contain more than 30 bioactive proteins, many of which have a fundamental role in hemostasis or tissue healing.[3]

Seven fundamental protein growth factors that are actively secreted by platelet initiate all wound healing process [Table 1].[4] PRP also includes three proteins in blood known to act as cell adhesion molecules: Fibrin, fibronectin and vitronectin.[5]

Table 1 Platelet growth factors and their specific characteristics[4]

Activation causes the granules present in platelets to fuse to its cell membrane (also called degranulation) where the secretory proteins (e.g. PDGF, TGF-β etc.)[6] are transformed to a bioactive state by the addition of histones and carbohydrate side chains.[3] The active proteins are then secreted, binding to transmembrane receptors of target cells, which include mesenchymal stem cells, osteoblasts, fibroblasts, endothelial cells and epidermal cells. These agonists bound transmembrane receptors then activate an intracellular signal protein that causes the expression of a gene sequence that directs cellular proliferation, matrix formation, osteoid production, collagen synthesis etc. thus provoking tissue repair and tissue regeneration.[345678]

The active secretion of these growth factors by platelets begins within 10 min after activation, with more than 95% of the pre-synthesized growth factors secreted within 1 h.[9]

Marx proposed that platelet count of 10 lakh/ml in 5 mL of PRP, as a working definition of PRP, based on the scientific proof of bone and soft tissue healing enhancement.[9] There is a scarcity of studies stating the concentration at which optimal stimulation occurs. Rughetti et al.[10] studied the relationship between the concentration of platelets in platelet gel and changes in the functional activity of human endothelial cells. The proliferation of endothelial cells and its migration and the invasion of endothelial cells occurred in a bell-shaped manner. The authors found that the stimulation for proliferation of endothelial cells peak at 1.25 × 10⁶ and angiogenesis at 1.5 × 10⁶ platelets/mL, respectively. This signifies the fact that a PRP platelet count 1 million/mL has become the working definition for therapeutic PRP and also reasons out the criticism on not getting the expected best results of PRP, which might be due to lower concentrations of platelets.

CLASSIFICATION

The literature on PRP is considerable, but the published results are often contradictory. It is very difficult to sort and interpret the available data, due to a large number of preparation techniques, terminologies, forms of these materials, and the endless list of potential applications.

According to the classification proposed by Ehrenfest et al. (2009), four main families of preparations can be defined, depending on their cell content and fibrin architecture.[11]

1. Pure Platelet-Rich Plasma (P-PRP) or leucocyte-poor PRP products are preparations without leucocytes and with a low-density fibrin network after activation.

2. Leucocyte- and PRP (L-PRP) products are preparations with leucocytes and with a low-density fibrin network after activation. It is in this family that the largest number of commercial or experimental systems exist. Particularly, many automated protocols have been developed in the last years, requiring the use of specific kits that allow minimum handling of the blood samples and maximum standardization of the preparations.

3. Pure platelet-rich fibrin (P-PRF) or leucocyte-poor platelet-rich fibrin preparations are without leucocytes and with a high-density fibrin network. These products only exist in a strongly activated gel form, and cannot be injected or used like traditional fibrin glues.

4. Leucocyte- and platelet-rich fibrin (L-PRF) or second-generation PRP products are preparations with leucocytes and with a high-density fibrin network.

This classification system was largely cited, advocated, and validated by a multi-disciplinary consensus conference published in 2012.[12]

PREPARATION OF PRP

PRP is obtained from a sample of patients’ blood drawn at the time of treatment. A 30 cc venous blood draw will yield 3-5 cc of PRP depending on the baseline platelet count of an individual, the device used, and the technique employed. The blood draw occurs with the addition of an anticoagulant, such as citrate dextrose A to prevent platelet activation prior to its use. The authors employ a specialized ‘table top cold centrifuge’ device. Preparation-related costs are significantly lower than with commercial kits.

PRINCIPLES OF PRP PREPARATION

PRP is prepared by a process known as differential centrifugation. In differential centrifugation, acceleration force is adjusted to sediment certain cellular constituents based on different specific gravity.

There are many ways of preparing PRP. It can be prepared by the PRP method or by the buffy-coat method.

In the PRP method, an initial centrifugation to separate red blood cells (RBC) is followed by a second centrifugation to concentrate platelets, which are suspended in the smallest final plasma volume. In Figure 1, flow chart describes a double centrifugation process of PRP. WB (whole blood) is initially collected in tubes that contain anticoagulants. The first spin step is performed at constant acceleration to separate RBCs from the remaining WB volume. After the first spin step, the WB separates into three layers: an upper layer that contains mostly platelets and WBC, an intermediate thin layer that is known as the buffy coat and that is rich in WBCs, and a bottom layer that consists mostly of RBCs. For the production of pure PRP (P-PRP), upper layer and superficial buffy coat are transferred to an empty sterile tube. For the production of leucocyte rich PRP (L-PRP), the entire layer of buffy coat and few RBCs are transferred. The second spin step is then performed. ‘g’ for second spin should be just adequate to aid in formation of soft pellets (erythrocyte-platelet) at the bottom of the tube. The upper portion of the volume that is composed mostly of PPP (platelet-poor plasma) is removed. Pellets are homogenized in lower 1/3rd (5 ml of plasma) to create the PRP (Platelet-Rich Plasma).

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Figure 1

Flowchart describing preparation of PRP

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In the buffy coat method, whole blood (WB) is centrifuged at a ‘high speed’ with subsequent collection of the buffy coat. A buffy coat contains high concentration of leucocytes.

From small volume of WB (10 mL), a very thin layer of a buffy coat can be produced. The difficulty lies in separating this thin buffy coat layer that contains mainly white blood cells (WBCs) and platelets, from the underlying RBC layer.

COMMERCIALLY AVAILABLE PRP KITS

There are many PRP systems commercially marketed, which facilitate the preparation of ready to apply platelet-rich suspensions in a reproducible manner. All operate on a small volume of drawn blood (20-60 mL) and on the principle of centrifugation. These systems differ widely in their ability to collect and concentrate platelets depending on the method and time of its centrifugation. As a result, suspensions of different concentration of platelets and leucocytes are obtained. Differences in the concentrations in platelets and WBCs influence the diversity of growth factors concentration. It is difficult to assess which kit for PRP preparation is better and which is worse.

PRP devices can be usually divided into lower (2.5-3 times baseline concentration) and higher (5-9 times baseline concentration) systems. The high-yielding devices include Biomet GPS II and III (platelet count 3-8×); Harvest SmartPRep 2 APC+ (4-6×); ArterioCyte-Medtronic Magellan (3-7×). The lower concentration systems include Arthrex ACP (2-3×), Cascade PPR therapy (1-1.5×), and PRGF by Boitech Institute Vitoria, Spain (2-3×).Regen PRP (Regen Laboratory, Mollens, Switzerland).[616171819]

COMPARISON OF STUDIES OF PREPARATION

PRP is prepared by centrifugation varying the relative centrifugal force, temperature and time. It has been seen that the two-step procedure renders the highest output.

Preparation procedures are also relevant, as shown by studies of the chondro-inductive and osteo-inductive potential of PRP.[20]

There are numerous protocols in the current literature that describe the optimal conditions for centrifugation [Table 2]. However, these various protocols have been optimized with respect to different variables of the process, such as volume and sampling of processed WB, number of spins, time period of centrifugation, and range of centrifugal acceleration. Considering the complexity of an autologous product such as PRP and the need for quality control in clinical applications, it is crucial to demonstrate procedure's ability to reproduce consistent results.

Table 2 Comparison of various protocols for platelet yield

Despite these variations, all protocols follow a generic sequence that consists of blood collection, an initial centrifugation to separate RBCs, subsequent centrifugations to concentrate platelets, and other components and an activation of the sample by adding a platelet agonist.

Amable et al.[21] studied variations in relative centrifugal force (RCF), temperature, and time for optimizing conditions for platelet isolation and quantification of cytokines and growth factors in PRP before and after platelet activation.

Peripheral blood from 22 healthy male and female volunteer donors (20-54 years old) was collected using 4.5 mL blood collection tubes containing 0.5 mL citrate solution (VacutainerW, Ref: 369714; BD Biosciences). Blood samples were run by choosing RCF from 240 to 400×g, time from 8 to 19 min and temperature from 8° to 16°C. All steps were performed in a refrigerated centrifuge (certified Jouan Br4i, Saint-Herblain, Loire-Atlantique, France).

The best performance was obtained using parameters of 300×g for 5 min at 12°C and 240×g for 8 min at 16°C for 1^st spin. The second spin of 700 × g for 17 min was chosen since it allowed a lower platelet loss into the PPP fraction and produced a pellet that was easily resuspended.[21]

Amanda et al.[22] demonstrated that the processing of 3.5 mL of blood at 100×g for 10 min (1^st spin), 400×g for 10 min (2^nd spin) and withdrawing 2/3 of remnant plasma, promoted high platelet recovery (70%-80%) and concentration (5×) maintaining platelet integrity and viability. Authors believe that time and acceleration are the fundamental parameters that define the composition of the PRP sample after the first spin step. Longer time periods slightly increased platelet recovery and decreased the concentrations of WBC in the upper layer. Therefore, time could be a control parameter when low levels of WBC, such as granulocytes and lymphocytes, are required in the PRP sample.

Various factors contribute to platelet concentration gradient such as the size of platelets, the biological difference among individuals and hematocrit variability.

However, this gradient is more critical after the second spin step because some erythrocytes are inevitably present in the volume that was transferred from the first spin. The presence of these remaining RBCs can generate a pellet at the bottom of the tube, which adsorb platelets and WBCs on its surface. The manual mixing for a short period of time is insufficient to completely resuspend the platelets, and a large variability in platelet counting is observed. Approximately 20% of platelets remained adsorbed in the RBC pellet.[22]

Kahn et al.[23] determined that a centrifugal acceleration of 3731×g for a period of 4 min was the optimal condition for obtaining the highest platelet concentration from 478 mL of WB.

The highest platelet recovery efficiency obtained by Slichter and Harker[24] was 80%, using a sample of 250-450 mL of WB centrifuged at 1000×g for a period of 9 min. It was observed that a subsequent centrifugation step of 3000×g for a period of 20 min decreased the platelet viability.

Landesberg et al. obtained PRP samples that had approximately 3.2 times the concentration of the WB baseline. The centrifugation procedure processed 5 mL of WB for two spins at 200×g for 10 min per spin.[25]

Jo et al. examined the effect of the centrifugation time and gravitational force (g) on the platelet recovery ratio of PRP. Two-step centrifugations for preparing PRP were used in 39 subjects. WB was centrifuged from 500×g to 1900×g at 200×g increments for 5 min and from 100×g to 1300×g at 200×g increments for 10 min. In the step 2, platelets in the separated plasma were concentrated at 1000×g for 15 min, 1500×g for 15 min, 2000×g for 5 min and 3000×g for 5 min. They achieved better efficiency (92%) by applying an acceleration of 900×g for 5 min for the first spin step. A total of 9 mL of WB were processed, and the platelet concentration was measured to be 310.7 ± 78.5 × 10³ /mm³. The maximum efficiency for the second spin step (84%) was obtained by applying 1500×g for 15 min. The platelet concentration was 633.2 ± 91.6 × 10³ /mm³, which was 4.2 times greater than the baseline concentration.[26]

Bausset et al. found that a centrifugation of 130×g or 250×g for a period of 15 min was optimal when performing a procedure that involved two spins. A platelet concentration factor of 3.47 was obtained from the 8.5 mL WB processed, and 2.0 mL of plasma was processed in the second spin step.[27]

Tamimi et al.[28] compared two methods for obtaining PRP: Double centrifugation (ACE system; Surgical Supply and Surgical Science Systems, Brockton, MA, USA) and single centrifugation (Nahita System; Nahita, Navarra, Spain). Three test tubes of 8.5 mL WB each were introduced into an ACE centrifuge machine and subjected to a force of 160×g (1300 rpm) for 10 min. For second centrifugation, 400×g force (2000 rpm) for 10 min was applied. For Nahita system blood was extracted into 3.5-mL citrated tubes (Venojet; Terumo MR, Tokyo, Japan) containing 0.5 mL of trisodium citrate, citrate, and ACD as anticoagulants. Test tubes were centrifuged with a 280×g force (1500 rpm) for 7 min. Platelet concentration from the ACE and Nahita systems were (336%) and (227%), respectively.

Mazzocca et al.[29] analyzed three protocols for preparing PRP samples with different compositions: A low platelet (382 × 10³ /mm³) and low WBC (0.6 × 10³ /mm³) process with one spin step at 1500 rpm for 5 min (10 mL WB); a high platelet (940 × 103/mm³) and high WBC (17 × 103/mm³) process with one spin step at 3200 rpm for 15 min (27 mL WB); and a double-spin process (1500 rpm for 5 min and 6300 rpm for 20 min) that produced a higher platelet concentration (472 × 103/mm³) and lower WBC (1.5 × 103/mm³).

Anitua et al.[30] used only one centrifugation spin step and collected the volume immediately above the erythrocyte layer. Blood was collected on sterile tubes (4.5 mL) containing 3.8% (w/v) trisodium citrate, then centrifuged at 460×g for 8 min (PRGF System1, B.T.I. Biotechnology Institute, Vitoria-Gasteiz, Spain). This protocol obtained a platelet concentration factor of 2.67 above the baseline value.

Dugrillon et al.[31] reported that the number of platelets is not always proportional to the growth factors’ quantity, so more attention should be given to the quality of PRP than to the number of platelets concentrated. Their study proved that the TGF-β1 and platelet concentration are proportionally related to the centrifugation forces when the forces are less than 800×g. TGF-β1 becomes inversely related to the centrifugal force when forces are above 800×g.

Araki et al.[32] concluded that optimized protocol for PRP preparation was centrifugation of WB at 230-270×g for 10 min, which also contained a low number of WBCs (4.1%-5.8% of WB). WBCs appeared to be precipitated when the centrifugation force was ≥840×g.

In their protocol, WB (40-72 mL) was drawn by venipuncture, collected, and divided into 7.5-mL aliquots in 15-mL conical tubes and centrifuged conical tubes (15 mL; BD Falcon). Tubes were centrifuged at 20°C in a refrigerated centrifuge (Kubota 5900; Kubota Co.). For the second spin step, an acceleration of 2300×g for 10 min was applied. The platelet concentration factor was 7.4 times greater than the baseline after removing approximately 1/10 of the PPP and adding ethylenediaminetetraacetic acid (EDTA) as an anticoagulant.

Many other studies[3334] specified centrifugal accelerations in rotations per minute (rpm) instead of in ×g, complicating the task of comparing and reproducing their results.

In a recent study by Kececi et al.,[35] the first spin (soft spin) protocol was chosen as 250-270×g for 10 min. At the second spin, centrifugation force was varied from 300×g, 500×g, 750×g, 1000×g, 1500×g and 2000×g for 10 min. The platelet concentration factor increased as the centrifugal force of the second spin increased as 1.92-fold, 2.16-fold, 2.80-fold, 3.48-fold, 3.67-fold, and 3.76-fold after a 10-min second centrifugation at 300×g, 500×g, 750×g, 1000×g, 1500×g and 2000×g, respectively. The authors opined that obtaining a definite platelet concentration might be possible by adjusting centrifugation force individually according to the personal baseline value.

AUTHORS’ PERSPECTIVE

There are various protocols described in the literature for preparation of PRP. Centrifugal acceleration, number of spins, time and distance between the particles and the rotor to the volume of processed WB are critical for optimization of PRP. Each laboratory must standardize its protocol. A handmade standard PRP could be prepared reliably and cost effectively without using a commercial kit. The platelet concentration factor can be changed with the centrifugation force applied in PRP preparation.

HELPFUL TIPS FOR STANDARDIZATION OF PRP PROCEDURE

The clinician needs to determine how much PRP he would need to generate for the particular procedure.

As previously stated, many factors influence PRP yield. The following points are helpful in obtaining an optimum PRP concentrate.

1. Draw blood in anticoagulant vacutainer tubes using BD Eclipse™ blood collection needle (Ref: 368607; BD Biosciences, India) [Figure 2]. Set apart 1-2 mL for baseline cell counting including RBCs, platelets, WBCs and hematocrit. Donor percent hematocrit (the ratio of the volume of RBCs to the total volume of blood) is relevant to plasma yield optimization. Blood with a low percent hematocrit has more plasma available thereby diluting the concentration of platelets in plasma.

2. Blood samples that are collected in ACD tubes should be inverted 5-10 times for proper mixing of the anticoagulant and blood. If the tube is not mixed, small fibrin clots may form, causing a falsely decreased platelet count.

3. After 1^st spin, measure the platelet count in RBCs and the supernatant to ensure optimal separation of platelets from the WB.

4. If this does not happen, change the parameters like rpm and time.

5. After first spin, if large volume of plasma is obtained, then it would need higher ‘g’ to concentrate platelets at the bottom of the tube (law of velocity).

6. After 2^nd spin, measure the platelet count in PPP and PRP after adequately racking the tubes.

7. If higher concentration of platelets in platelet poor plasma (upper layer) or lower concentration in PRP (bottom layer) is observed, then the parameters are not optimal.

8. PRP must be separated from the PPP soon after centrifugation because the concentrated platelets will slowly diffuse into the PPP over time and would reduce the platelet count of the PRP preparation.[3]

9. For counting the platelets in the final PRP concentrate, it must be re-suspended for at least 5-10 min to allow for equal distribution of platelets before counting.[31]

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Figure 2

Venipuncture using BD Eclipse™ blood collection needle

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To obtain a yield of platelet in PRP more than 10 lakhs/mL, all variables like rpm, time and temperature should be standardized and also consistency of platelet concentrate has to be maintained over the period After several attempts with various permutations & combinations, the authors found that at 900g x 5 mins for 1st centrifugation and 1000g x 10 mins for 2nd centrifugation at 16° C in a refrigerated centrifuge (RemiCM8plus; Remiworld, India), the PRP yield was found to be optimum. Authors have consistently recorded platelet count exceeding 10 lakhs/ml using these parameters [Figure 3].

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Figure 3

Consistency of PRP yield

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CONCLUSION

The utility of PRP is increasing in the field of dermatology and aesthetics. Though commercially marketed PRP systems offer ready-to-apply platelet-rich suspensions in a sterile manner, high cost, limited volume of drawn blood, differences in centrifugation guidelines, wide variation in platelet concentration, makes it difficult for the clinician to choose amongst the readily available PRP kits.

There are many protocols for preparation of PRP each having its own standardized parameters and claimed results, as mentioned in the review. It is advisable to standardize individual preparation protocols, which are cost-effective and easy to adapt in clinical settings.

Understanding of basic principles of centrifugation is of paramount importance in preparation of PRP. Repeated methodology trials by ‘trial and error approach’, strict vigilance at maintaining sterility and regular cross-checking of the platelet values are a must to obtain consistent results.