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The effect of different speeds of centrifugation on platelet-rich plasma preparation

Anbar, Tag S.a; El-Ammawy, Tarek E.a; El-Metwally, Yasser M.b; Abdel-Rahman, Amal T.a; Mohammed, Sahar S.a

Journal of the Egyptian Women's Dermatologic Society: September 2015 - Volume 12 - Issue 3 - p 150–155
doi: 10.1097/01.EWX.0000469302.93469.65
Original articles
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Background Platelet-rich plasma (PRP) therapy is an easy, safe, autologous, and cheap biologic treatment commonly used in various medical fields, including dermatologic therapy and plastic surgery, with good results. There are different methods of PRP preparation using either laboratory or commercial products. However, there is no standardized method for PRP preparation.

Objective To evaluate the effect of different speeds of centrifugation on PRP preparation.

Patients and methods A volume of 10 ml of whole venous blood was collected from 20 healthy volunteers (10 female and 10 male), aged 20–40 years. These samples were divided into two tubes and left to precipitate (sample 1) and the platelet concentration was measured in the supernatant. The sample was then resuspended and the first tube was centrifuged at low speeds of 200, 300, and 500 rpm, and the second tube was centrifuged at 700, 900, 1100, and 3000 rpm (samples 2–8, respectively) and the platelet concentration was measured and compared in the obtained PRP.

Platelet concentration was measured in the two PRP samples obtained from two more volunteers (both male) using the commercial Regen kit and compared with those obtained by means of the laboratory method from samples obtained from the same volunteers.

Results The platelet concentration was the highest in the PRP obtained in samples 1 and 2, followed by that obtained in samples subjected to lower speeds of centrifugation (300 and 500 rpm), and it was significantly higher compared with that obtained using a ready-to-use kit. Moreover, the white blood cell concentration was higher in the PRP obtained using laboratory method compared with that obtained using the kit method.

Conclusion Laboratory preparation of PRP, especially that obtained without centrifugation, or using lower centrifugation speed, yields higher platelet concentration. Centrifugation at low speed is preferable as it saves time, avoids sample contamination, and preserves platelets.

Departments of aDermatology, Andrology and STDs

bClinical Pathology, Minia University, Minia, Egypt

Correspondence to Tag S. Anbar, MD, Department of Dermatology, Andrology and STDs, Minia University, Minia 61111, Egypt Tel: +20 122 228 6333; e-mail: taganbar@yahoo.com

Received January 1, 2015

Accepted January 23, 2015

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Introduction

Platelet-rich plasma (PRP) comprises a high concentration of platelets in a small volume of plasma. PRP contains various growth factors (GFs) and cytokines that play a critical role in all aspects of the wound healing process 1.

The process of PRP extraction quantifies almost a 400% increase in the baseline platelet count 2. The resulting plasma is composed of 94% platelets and the remaining 6% is made up of red blood cells and white blood cells (WBCs) 3.

The activation of platelets releases several GFs such as basic fibroblast GF, platelet-derived GF, insulin-like GF, epidermal GF, vascular endothelial GF, transforming GF-β, platelet factor 4, and interleukin-1. Platelets also secrete other proteins, such as osteocalcin, osteonectin, fibrinogen, vitronectin, fibronectin, and thrombospondin-1, which play a critical role in the regulation of the cell–cell interactions and the cellular organization 4,5.

Autologous PRP has been safely used in many disciplines, including orthopedics, maxillofacial surgery 5, and cardiothoracic, plastic ,and reconstructive surgeries 6. In the field of dermatology, it can be used in the treatment of nonhealing wounds, such as trophic and vascular ulcers, decubitus wounds, fistulae, burns, and dermoepidermal dystrophies. It has also been found to be beneficial in acne scarring, fat transplantation, promotion of hair survival and growth, and as a useful adjuvant in the management of androgenetic alopecia 7–9.

There are different methods used to prepare PRP either using commercially available kits or using a laboratory method such as that used by Gonshor 10. However, there is no standardized method for PRP preparation. Therefore, the aim of the present study was to evaluate the effect of different speeds of centrifugation, as an important step, on PRP preparation.

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Patients and methods

The study has been approved by the Faculty of Medicine Council, Minia University. Each participant gave written consent form to be included in the trial.

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Platelet-rich plasma preparation

A volume of 10 ml of whole venous blood was collected from each of the 20 healthy volunteers (10 female and 10 male) included in the study.

Each sample was divided into two disposable sterile vacutainer tubes after adding 0.5 ml (1 : 10) acid citrate dextrose (ACD) to each and was left for 1–2 h at room temperature to precipitate. Thereafter, the resulting supernatant was aspirated carefully with a non-needled insulin syringe into another clean test tube and was counted using an automated cell counter device (BC-3600; Mindray Biomedical Electronics, Shenzhen, China). The average platelet number of the two tubes was then recorded. After counting, the supernatant from each tube was resuspended again into the primary one. The tubes were mixed well again and centrifuged. The first tube was centrifuged at lower speeds (200, 300, and 500 rpm) and the second tube was centrifuged at higher speeds (700, 900, 1100, and 3000 rpm) for 10 min at each speed, using an Eppendorf centrifuge. Following each speed the platelets were counted in the supernatant (the cloudy phase) and then were resuspended again into the primary tubes and were mixed well before recentrifugation at the next speed. At the end we compared platelet counts at different speeds of centrifugation to find the most ideal one.

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Platelet-rich plasma preparation using Regen kit

Eight milliliters of venous whole blood was collected from two volunteers into Regen BCT tubes containing Cell Selector Gel (Regen Lab SA, Le Mont-Sur-Lausanne, Switzerland). The tube was turned upside-down several times, and then it was centrifuged at 1500g (3000 rpm) for 5 min. The resulting supernatant was aspirated with a non-needled insulin syringe into another sterile tube to be counted for platelets using an automated cell counter. The Regen platelet count was then compared with that obtained from another 8 ml blood of the same volunteer into a disposable vacutainer tube that was left to precipitate for 1–2 h to obtain the laboratory platelet count without centrifugation.

Both Regen-prepared and laboratory-prepared PRP platelet counts were confirmed by manual counting of the platelets in all samples. PRP was diluted with ammonium oxalate solution to rupture other cell types except for the platelets in a ratio of 1 : 10, and then the platelets were counted in the specific chamber (hemocytometer) and the resulting number was multiplied by 10 3 (Fig. 1).

Figure 1

Figure 1

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Statistical analysis

Data were analyzed using statistical computer program statistical package for the social sciences (SPSS, version 13.0; SPSS Inc., Chicago, Illinois, USA) software. Quantitative data were presented as mean±SD. Comparison of quantitative data between two variables was made using Student’s t-test. One-way ANOVA test was used for comparison between more than two quantitative variables. Data were presented in tables and graphs. P values of 0.05 or less were considered statistically significant.

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Results

Platelet-rich plasma preparation using different centrifugation speeds

The highest platelet count was found in PRP obtained from sample 1, and it was inversely proportional to increasing centrifugation speed (Table 1 and Fig. 2).

Table 1

Table 1

Figure 2

Figure 2

In the present study, the baseline whole-blood platelet counts ranged from 150 to 329 mm3 (mean±SD 220.4±620.55 mm3). These counts were elevated by 1.36–3.52 folds in sample 1. The difference between the platelet count from the baseline sample and the mean platelet count in PRP prepared using different speeds of centrifugation was statistically significant (P<0.001). The mean platelet concentration factor is defined as the platelet count in the PRP divided by the platelet count in the baseline blood sample. It was the highest in sample 1 (2.5±0.6). When compared with sample 1, the platelet concentration factor was slightly lower in sample 2 (2.4±0.6) and in sample 3 (2.2±0.5), with no statistically significant difference (P>0.05), whereas the difference was significant in sample 4 (1.9±0.5, P<0.01). Meanwhile, a centrifugation performed at higher speeds (700–3000 rpm) decreased the platelet concentration factor compared with lower speeds of centrifugation (Table 2 and Fig. 2).

Table 2

Table 2

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Platelet-rich plasma preparation using Regen kit

PRP obtained using the ready-to-use Regen kit was compared with that obtained by leaving the sample to precipitate and by the lowest speed of centrifugation (200 rpm). We found that the platelet concentration was increased by an average of 3.1 folds in the first sample and by an average of 2.7 folds in the second sample in the laboratory method compared with (<1 fold) that in the kit method. The count was confirmed using a hemocytometer by means of manual counting of the platelets. Moreover, WBC concentration varied greatly, being higher in the PRP obtained with the laboratory method compared with that obtained using the kit method.

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Discussion

Currently, PRP is consistently defined only by the absolute quantity of platelets and not by the other components. However, normal platelet counts in blood range from ∼150 000 to 350 000/μl 11. PRP is often defined as at least 1 000 000 platelet/μl suspended in plasma 12. To ensure that the platelets are suspended and do not form a clot, PRP must be prepared from anticoagulated blood 13.

Anticoagulants influence the quality of PRP, which in turn is directly associated with its biological effects. On reviewing the literature, two types of anticoagulants were found to be used: the compound anticoagulants, including ACD and citrate–theophylline–adenosine–dipyridamole (CTAD), and the noncompound type, including heparin and sodium citrate. Compared with heparin and sodium citrate, the compound anticoagulants ACD and CTAD maintained platelet integrity for a longer time, reduced platelet spontaneous activation, increased the amount of GFs released from PRP, and consequently improved the efficacy of PRP in stimulating cell proliferation, facilitating tissue regeneration and/or wound healing 14. It is worthy to note that ACD is the anticoagulant that is being used by blood banks to store viable platelets for platelet transfusions, because ACD can maintain platelet viability for up to 6 h 15; that is why we used it as an anticoagulant.

In our study design, ideally, we should have withdrawn 35 ml of venous blood from each volunteer, to divide them into seven tubes and apply a single centrifugation speed to each one at a time, but this was not convenient and not accepted by the volunteers. We modified the design and thus withdrew 10 ml of venous blood from each patient and divided them into two tubes; one was centrifuged at low speeds (200, 300, and 500 rpm) and the other on high speeds (700, 900, 1100, and 3000 rpm) for 10 min each. It was found that platelet counts were inversely proportional to the increasing centrifugation velocity. It was higher in PRP obtained by means of centrifugation at lower velocities, followed by that obtained by centrifugation at higher velocities (200, 300, 500, 700, 900, 1100, and 3000 rpm), respectively. This decline in the platelet count can be due to either the repeated centrifugation of the samples and exhaustion of the platelets or the centrifugation speed itself. The first suggestion can be ruled out by the fact that when comparing the results of samples 2 and 5, which were centrifuged once, the platelet count was significantly lower in sample 5 than in sample 2. Similarly, if we compared sample 3 with sample 6 (centrifuged twice) and sample 4 with sample 7 (centrifuged 3 times) we obtain the same results. These findings are in favor of our proposal that the decline in the platelet number was mainly due to the increased centrifugation speed.

Most of the earlier studies gave little or no information on the range of platelet concentration that defines the PRP. They only provided the information that platelets usually increase 2–4 times from their original concentration. For example, Hsu et al.16 reported a 434% increase in platelet concentrations of PRP prepared from 20 ml venous blood by means of double-spin centrifugation (2400 rpm for 10 min and 3500 rpm for 15 min). This higher increase in both centrifugation speed and platelet concentrations may be due to the use of different types of centrifuges and a larger volume of blood compared with that used in our study. In contrast, Trink et al.17 reported a 3.5 times increase in platelet concentration of PRP prepared from 36 ml venous blood centrifuged at 70g for 8 min, which is consistent with that obtained in our study. Moreover, Bausset et al.18 observed that the lower centrifugation speeds were also better for the resting platelet morphology preservation (discoid form with low number of pseudopodia), whereas higher speeds showed morphological activation indicators (high number of pseudopodia and a trend toward centralization of granules) at 250 and 400g. Finally, they noticed that centrifugation at less than 400g maintained the procoagulant activity of platelets with higher time to clot formation. Taken together, with our results, we can say that centrifugation at lower speeds gives PRP of better quantity and quality compared with that obtained with centrifugation at higher speeds.

The several products available in the market to obtain autologous PRP can differ from each other in the preparation procedure and results. Different systems have different yields in terms of concentrated viable platelets, accounting for many of the criticisms in terms of the efficacy of PRP 19. However, most, if not all, of these products are collectively called PRP, which does not allow distinction between the different systems and protocols 20. In this study, we compared the blood obtained from two volunteers to prepare PRP using two different methods: the first method was carried out using the ready-to-use Regen kit and in the second method PRP was obtained by leaving the samples to precipitate alone and then centrifugation at 200 rpm. We found that platelet concentration was increased by an average of 3.1 and 2.7 in the two samples using the laboratory method compared with less than one fold increase in the kit method. The platelet count was confirmed by means of both the automated and manual methods. These results may be explained by the possible deleterious effect of higher centrifugation speed (3000 rpm) used to prepare PRP using the Regen kit on platelets.

There are no sufficient data available to demonstrate functional differences between leukocyte-rich and leukocyte-poor PRP. Some authors recommended that the leukocytes be discarded from the preparation to prevent the inflammatory processes 21, whereas some other authors reported that there is no good scientific reason to discard them 22. If we agree with the proposition of the deleterious effect of WBCs on the platelets as mentioned above, the lower speeds of centrifugation with its high WBCs content might represent a potential limitation of such protocol.

At least 16 commercial platelet separation systems are available today, many of which may vary significantly in the relative amounts of platelets, leukocytes, erythrocytes, and anabolic and catabolic GFs; thus, it is difficult to generalize results from clinical trials using a given PRP manufacturer 13. There is no evidence of standardization of PRP preparation and use. Different methods of preparation either using PRP from commercially available kits or using PRP from a laboratory method may produce different platelet concentrations, and each preparation may produce different products with unknown effects 18. This supports the view of Dohan Ehrenfest et al.23, who reported that without a consensus, this therapeutic field remains opaque.

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Conclusion

We propose a simple, fast, and cheap PRP preparation method based on leaving blood sample for 1–2 h to precipitate on its own. To save time and to avoid the damaging effect of leaving the sample for long time, such as sample contamination and platelet clumping, the sample can also be centrifuged at 200 rpm for 15 min with the same outcome. Finally, commercial PRP kits are not a must for PRP preparation.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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Keywords:

alone precipitation; centrifugation speeds; platelet-rich plasma

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