A Cost-Effective Method for Obtaining Standard Platelet-Rich Plasma

Introduction. Platelet-rich plasma (PRP) is used in several different areas of surgery to enhance natural healing and the regenerative process by delivering increased concentrations of autologous platelets. However, there are controversies in the literature regarding the potential benefits of PRP, due partly to the lack of optimized and standardized preparation protocols. The aim of this study was to develop a standardized PRP preparation protocol. Material and Methods. Whole blood was drawn from 18 healthy participants. Double centrifugation protocol was applied. The blood from each person was divided into 6 tubes according to second stage centrifugation force applied, varying from 300 g to 2000 g. Platelet counts and platelet concentration factors were determined and the data obtained were submitted to statistical analysis (repeated measures ANOVA, Bonferroni, P < 0.05). Results. When compared to whole blood, the mean platelet counts increased significantly in all centrifugation groups, and this increase is common with centrifugation force increase. While there was no significant difference between the 300 g and 500 g groups (P = 0.051), there were differences between the 500 g and 750 g groups (P < 0.001), and the 750 g and 1000 g groups (P < 0.001). The mean platelet counts were not different between upper g groups (1000g, 1500g, 2000g, P = 0.114). The platelet concentration factor varied from 1.92-fold to 3.76-fold. There were differences between the 500 g and 750 g groups (P < 0.001), and between the 750 g and 1000 g groups (P < 0.001). Conclusion. The present study indicates it may be possible to get a standard platelet concentration by adjusting centrifugation force individually according to each baseline value.

Platelet-rich plasma (PRP) is defined as plasma with a platelet level above peripheral blood concentration.^1,2 Platelets are the storage pools of growth factors including platelet-derived growth factor, transforming growth factor-ß, platelet-derived epidermal growth factor, vascular endothelial growth factor, insulin-like growth factor-1, fibroblastic growth factor, and epidermal growth factor.³ They also contain cytokines and many other proteins.^3-5 When platelets come into contact with exposed endothelium within wounds or damaged tissues, these factors are released and work in harmony with tissue-repair mechanisms such as chemotaxis, cell proliferation, angiogenesis, extracellular matrix deposition, and remodeling to promote appropriate wound healing.⁶ Hence, the idea was proposed that increasing platelet concentration in an injured tissue would result in increased levels of multiple bioactive factors and, subsequently, improve the natural healing process.⁷

  Platelet-rich plasma was first used as an effective agent for bone and tissue repair within the field of dentistry.^8,9 Its applicability then expanded to other fields, including plastic surgery,^1,6,10,11 orthopedic surgery,^3,7 cardiac surgery,¹² ophthalmology,¹³ and sports medicine.¹⁴ However, there are controversies in the literature regarding the potential benefits of this procedure. Although some authors have reported significant improvements in wound healing and bone formation using PRP,^11,14-16 others have not.^17-20 Such discrepancies are probably a result of study type. Although there are numerous basic science studies, animal studies, and small case reports evaluating the effects of PRP-related products, there are only a few randomized controlled clinical studies that provide a high level of medical evidence about the potential benefits of PRP. The number of participants in these studies is typically small, and the majority of these studies do not have enough statistical power to confirm the benefits of PRP.

  Conflicting results can also be explained by the use of different PRP products. There are substantial differences in the content of platelet concentrates produced by the various automated and manual protocols described in the literature.²¹ Variations in some key properties of PRP, such as the platelet concentration, can greatly influence the different biological effects.²² So, using different PRP preparations can impede the establishment of standards necessary to compare the data from different studies.^2,23,24

  The purpose of this study was to develop a preparation protocol for PRP standardization using common laboratory ware and equipment. This would contribute to the establishment of a reliable and practical method to maximize the reliability of future studies.

After the Local Ethics Committee granted approval, 18 healthy adult volunteers between 22 and 50 years of age were enrolled to the study. All participants gave informed consent. None of the donors were on any medication, including aspirin and other nonsteroidal anti-inflammatory drugs, for 10 days before the experiment.

  The participants’ blood was typically drawn from the antecubital region using a butterfly cannula. A 3-way valve was attached to the back end of the butterfly. One mL of 3,8w/v% sodium citrate solution was allowed to get in the 10 mL vacuum tube through the side valve. Then, 9 mL blood was drawn from the patient. An 18-gauge needle was used in an effort to reduce irritation and trauma to the platelets so they would remain in a relative inactive state. Samples were gently agitated to thoroughly mix the anticoagulant with the blood. A total of 55 mL of blood was collected from each participant, filling 6 tubes. Approximately 1 mL of whole blood was separated for baseline whole blood analysis.

  Since free calcium ions are required for blood clotting, sodium citrate, an effective anticoagulant, was used to bind free calcium ions. Since both sodium and citrate are found naturally in the body, it would be safe, in future clinical applications, to use PRP samples obtained by this study protocol.

  The centrifugation process was carried out with a standard laboratory centrifuge in 2 steps. First spin (soft spin) protocol was chosen as 250 g for 10 minutes based on the initial information that a centrifugation force of 230-270 g would be the most likely to produce the maximum platelet recovery from a whole blood sample in a 10-minute spin.²⁵ After the first spin, 3 layers appeared. This is due to differences in the density of the blood components: the deep layer consists of red blood cells, the middle layer contains platelets and leukocytes, and the top layer is made up of platelet-poor plasma (Figure 1A). The bottom part of the middle layer, also called a buffy coat, is rich in leukocytes. The middle layer and top layer were collected directly by gentle aspiration with a pipette and transferred to a new, sterile centrifuge tube. This product was generally contaminated by red blood cells from the pellet.

  The samples of each participant were divided into 6 groups, according to the centrifugation force of the second spin, to investigate the effects of centrifugation force on PRP elements. At the second spin, centrifugation force was 300 g for group 1; 500 g for group 2; 750 g for group 3; 1000 g for group 4; 1500 g for group 5; and 2000 g for group 6. After the second spin, the top two-thirds of the portions accepted as platelet-poor plasma were removed by gentle aspiration, amounting to approximately 3 mL/tube. Then, approximately 1.5 mL PRP was obtained (Figure 1B). From each prepared PRP, 300 µL samples were taken to determine platelet counts. Pellets containing platelets were resuspended in the residual plasma to analyze the platelet counts in PRP correctly, since platelets precipitate rapidly in this suspension.²⁶

  In addition to the platelet counts, 2 other parameters were evaluated for the PRP samples: platelet concentration factor and platelet recovery rate. Platelet recovery rate is the total amount of platelets in obtained PRP compared to that of the whole blood. Platelet concentration factor is the platelet count in PRP groups compared to the platelet count in whole blood.

  The significance of differences between the whole blood and the PRP platelet counts, as well as between the PRP platelet counts of groups, was determined by repeated measures ANOVA (analysis of variance), followed by a post hoc Bonferroni test when the ANOVA suggested a significant difference between groups (P < 0.05). The differences between the means of platelet concentration factors of groups were also determined using the same test.

The mean platelet counts in the whole blood were 2.60 x 10⁵ (0.6 x 10⁵)/µL and increased in the PRP groups, ranging from 4.98 x 10⁵ (0.3 x 10⁵)/µL to 9.76 x 10⁵ (0.3 x 10⁵)/µL. When compared to whole blood, the mean of platelet counts increased significantly in all centrifugation groups and this increase is common with centrifugation force increase (Figure 2). While there was no significant difference between the 300 g and 500 g groups (P = 0.051), there were differences between the 500 g and 750 g groups (P < 0.001), and the 750 g and 1000 g groups (P < 0.001). The means of platelet counts were not different between upper g groups (1000 g, 1500 g, 2000 g, P = 0.114).

  The platelet concentration factor increased as the centrifugal force of the second spin increased from 300 g to 2000 g. This increase was found as 1.92-fold, 2.16-fold, 2.80-fold, 3.48-fold, 3.67-fold, and 3.76-fold after a 10-minute second centrifugation at 300 g, 500 g, 750 g, 1000 g, 1500 g, and 2000 g, respectively (Figure 3). While there was no significant difference between the 300 g and 500 g groups (P = 0.053), there were differences between the 500 g and 750 g groups (P < 0.001), and between the 750 g and 1000 g groups (P < 0.001). The means of platelet concentration factors were not statistically different between upper g groups (100 g, 1500 g, and 2000 g, P = 0.076). The platelet recovery rate also increased as the centrifugation force increased. This rate varied from 32% to 63%.

  Using these results, it was investigated whether this protocol would work in obtaining standard PRP. Since these findings revealed 3 statistically different centrifugal force steps, 3 groups were created virtually according to their baseline platelet counts. Platelet counts obtained by 1000 g centrifugation force were accepted as the test results for the first group that had the lowest baseline platelet counts. For the second group that had middle baseline values, 750 g was chosen while 500 g centrifugation force results were taken for the third group that had the highest baseline platelet counts. In comparison, there was not a statistically significant difference between the mean platelet counts of the groups (P = 0.479, ANOVA). This result revealed that standard PRP could be obtained by changing centrifugation force according to baseline platelet counts (Table 1).

The present study revealed that a handmade standard PRP product could be prepared reliably without using a commercial kit. There are many ready-to-use commercially available disposable kits ranging in price from $175 to $1150 per kit,²⁷ whereas the cost of PRP using the authors’ method is considerably lower, at approximately $10 per person, and the equipment is available in any clinical setting. High costs and the need to have specific equipment to prepare PRP have critically reduced the use of autologous platelets. The authors propose more cost effective and more simplified PRP preparation methods would make PRP more readily available to a greater number of patients and treatment centers. This, in turn, would help accumulate clinical data and would facilitate the development and design of adequately powered clinical studies evaluating therapeutic efficiency of PRP. In addition, using standard PRP would greatly improve the comparability of studies.

  In the present study, platelet recovery rates and platelet concentration factor changed with centrifugation force, varying from 32% to 63% and from 1.92-fold to 3.76-fold, respectively. These findings are comparable with the results of the studies evaluating commercial PRP kits. Le et al²⁸ compared 3 commercially available kits—Curasan (Kleinostheim, Germany), Plateltex PRP system (Boland Cell, Bratislava, Slovakia), and RegenLab SA (Lausanne, Switzerland)—and a handmade protocol, obtaining a 1.55-fold to 3.43-fold increase in platelet concentration. The platelet recovery rate varied from 20% to 79% in their study.²⁸ Sundman et al²⁹ reported a 1.99-fold increase in platelet concentration using an Arthrex ACP Kit Series (Arthrex, Naples, FL) and a 4.69-fold increase using a GPS III System (Biomet, Warsaw, IN). Mazzucco et al³⁰ compared 4 different PRP protocols: Fibrinet Autologous Platelet System- BK120008 (Cascade Medical Enterprises, LLC, Wayne, NJ), RegenLab SA (Lausanne, Switzerland), Plateltex PRP System (Boland Cell, Bratislava, Slovakia), and a handmade procedure. They obtained 1.6-fold, 3.95-fold, 4.4-fold, and 4.4-fold increases in platelet concentration, respectively.30 Castillo et al³¹ compared the Cascade (MTF Sports Medicine, Edison, NJ), GPS III, and Magellan (Arteriocyte, Hopkinton, MA) systems, and reported that platelet concentration was a 1.62-fold, 2.07-fold, and 2.80-fold increase with a 67.6%, 22.6%, and 65.5% platelet recovery rate, respectively.

  As in all bioactive agents, there should be an optimal dose range of PRP. The idea that more platelets might be better seems incorrect, because a highly concentrated PRP could have an inhibitory effect on wound healing, as has been shown in a rat study of intestinal anastomosis.³² Its effect may change according to the application dose. It was reported that the biological stimulation of cell growth varies with PRP dose.³³ While application of the PRP enhances mesenchymal stem cell migration and proliferation, overexposure of cells to PRP yields many cells, but limited differentiation of those cells into appropriate cell lines.³⁴ Choi et al³⁵ also noted that the alveolar bone cell proliferation was stimulated by low, but suppressed by high, concentration of PRP.³⁵ Therefore, PRP dose is an important matter that has to be considered in the evaluation of its biological effects.

  Platelet-rich plasma dose is mostly described as an x-fold increase compared with baseline values and some investigators have suggested that platelet concentration factors should be between 2-fold and 8-fold in an effective application.^10,24,36 Since baseline platelet value varies widely from person to person, reports with an x-fold increase lead to difficulties in suggesting a fixed concentration required to demonstrate the benefits of PRP. As platelets are presumed to supply regenerative efficacy, it may be considered that a standard platelet concentration is required to obtain a standard biological effect.

  It was reported that the increase in the concentration of growth factors is proportional to the increase in platelet concentration in the PRP,^23,24,37 so platelet count was chosen as a standardization criterion in the present study. It may be proposed that one of the growth factors or other cytokines platelets secrete can be used as a target bioactive agent instead of platelet counts in PRP standardization. However, this proposal harbors some difficulties. First, platelets work in healing and regenerative processes by several growth factors and cytokines. It is difficult to decide which one of them best represents platelet function. Second, the concentrations of these factors are measured only after platelet activation. Nevertheless, some authors obtained better results using PRP without platelet activation and advocated not to activate platelets in PRP application.³⁸ On the other hand, platelets can be counted without interfering with the prepared PRP. Third, before the application, each prepared PRP should be checked in term of standardization in studies evaluating therapeutic efficiency of PRP. Measuring the concentration of any 1 of these bioactive components is costly and requires specific equipment, whereas the cost of platelet counting is considerably low and the equipment is available in any clinical setting.

  An important variable that can have a critical impact on PRP quality is the centrifugation protocols. The number of times centrifugation should be completed is controversial. Several studies suggest that double spin processes are preferable since platelet isolation is easier.^21,25,28 In addition to the number of centrifugations, centrifugation force is another important variable that can affect PRP bioactivity. The term centrifugation force needs clarification as some authors use it to refer to g force and others to mean revolutions per minute. The determining factor in centrifugation is the acceleration rate to be applied to the sample, rather than rotational speed such as RPM. This distinction is important because 2 rotors with different diameters running at the same rotational speed will subject samples to different acceleration rates. Therefore, protocols for centrifugation should specify the amount of acceleration. The acceleration is measured in multiples of “gg” (or x “g”), the standard acceleration due to gravity at the Earth’s surface. During circular motion, the amount of g force is equal to the product of the radius and the square of the angular velocity, and the acceleration relative to “g” is traditionally named “relative centrifugal force.”

  The present study showed the platelet concentration factor can be changed by the centrifugation force applied in PRP preparation. Published studies also support this finding^13,25,39; however, the means of platelet concentration factor and platelet counts were not different between the 300 g and 500 g groups and between the 1000 g, 1500 g, and 2000 g groups. These findings showed there were only 3 statistically significant centrifugation force steps: 500 g, 750 g, and 1000 g. It was considered that obtaining a definite platelet concentration might be possible by adjusting centrifugation force individually according to the personal baseline value. This idea was tested by using the current study’s results. In a comparison between the test results of 1000 g centrifugation force for the lowest baseline values group, 750 g centrifugation force for the middle baseline values group, and 500 g centrifugation force for the highest baseline values group, there was not any statistically significant difference. This result demonstrated standard PRP could be obtained by adjusting centrifugation force, according to baseline platelet counts.

  Eighteen volunteers participated in the current study. The number of samples studied was greater than similar studies; however, a larger sample size with a consistent graph outlining the results to serve as a normogram would add to the power of the study. All centrifugation processes, including pipetting tubes and transferring materials were performed by the same biologist. This might have assisted the standardization of the protocol studied. On the other hand, it could be accepted as a limitation of this study since different operators could get different platelet concentration factor values. Thus, it might be proposed that each laboratory should establish its own protocol for obtaining standard PRP.

  The present study revealed that 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. Therefore, by adjusting centrifugation force individually according to each baseline value, it may be possible to get a standard platelet concentration.

Affiliations: The authors are from the Izmir Education and Research Hospital, Plastic and Reconstructive Surgery, Ismir, Turkey.

Address correspondence to:
Yavuz Kececi, MD
Izmir Education and Research Hospital
Bozyaka, Karabaglar, Izmir
35350 Turkey
yavuz.kececi@gmail.com

Disclosure: This authors disclose no financial or other conflicts of interest.