What is platelet-rich plasma (PRP)?

Platelet-rich plasma (PRP) is a platelet- and growth factor-enriched solution prepared from a patient’s own blood. Injections of PRP are used to promote tissue repair and bone growth in a wide variety of medical conditions, including hair loss, osteoarthritis, and damage to tendons or ligaments. 

While typical platelet concentrations are around 200,000/µL, those in PRP should exceed 1,000,000/µL or 5× the basal level [1]. 

Platelet-rich plasma (PRP) is derived from the patient's own blood.

How can this >5× increase in platelet concentration be achieved in practice? And what other steps are important to maintain other potentially important properties of the PRP, such as platelet morphology, platelet activation, or growth factor concentration?

PRP preparation broadly involves three main steps:

1. Sample collection—generating plasma after collecting the patient’s blood;
2. Centrifugation—concentrating platelets and other wound-healing components to obtain the PRP;
3. Storage—maintaining the quality of the PRP prior to its clinical application.

In the closed method of preparing PRP, all of these steps are carried out in a predefined way within an entirely closed and aseptic all-in-one system. The open method, in contrast, offers the chance to optimize each step independently, although care must be taken to maintain sterility at all times.

Here we focus on the open method and summarize the latest evidence regarding optimal protocols for the collection, centrifugation, and storage of PRP.

PRP sample collection

PRP is derived from plasma, so the first step in preparing PRP is to generate plasma from the patient’s blood. This is performed by collecting blood into tubes containing an anticoagulant that prevents blood clotting. 

Different anticoagulant coatings on tubes can affect the quality of the PRP.

Overall, evidence from research studies suggests that ACD-A yields PRP with superior platelet concentration, activation, and morphology compared with EDTA or sodium citrate [2]. In contrast, growth factor concentrations do not differ depending on whether EDTA, ACD-A, or sodium citrate are used, although mesenchymal stem cells proliferate more readily in PRP prepared using ACD-A or sodium citrate [3]. 

These findings suggest that ACD-A is the anticoagulant of choice for preparing plasma for PRP prior to centrifugation.

Optimal centrifugation protocols for PRP

Using differential centrifugation, platelets can be separated and concentrated relative to the other components of the plasma. However, to optimize platelet and growth factor concentrations while preserving platelet integrity and viability, it is essential to use an appropriate centrifugation protocol.

Selection of centrifuge model

Which considerations should be made in selecting the model of centrifuge for concentrating platelets and growth factors in the PRP? 

In general, swing-out (horizontal) centrifuges are the superior choice for preparing PRP, as they allow for better separation of plasma components based on their density [4]. In addition, they reduce the level of cell trauma and damage relative to that from angled centrifuges, improving the quality of the PRP.

Recommended swing-out models that are commonly used to prepare PRP include the Hermle Z206A Compact Centrifuge (above), the Eppendorf 5810R High Capacity Centrifuge, and the Drucker Diagnostics HORIZON 24 Flex Centrifuge. 

Care should be taken to select the correct centrifuge for the type of collection tubes used, especially when using commercial PRP preparation kits. In such cases, the manufacturer’s instructions should be followed to select the appropriate centrifuge.

Temperature during centrifugation

PRP centrifugation is typically carried out at room temperature, with the American Association of Blood Banks Technical Manual recommending 20°C–24°C during centrifugation [5]. However, some evidence suggests that a lower temperature of 12°C–16°C may improve platelet recovery and viability [6].

Single vs. double centrifugation

In general, double centrifugation has been found to be more effective than single centrifugation at increasing platelet concentrations [7]. 

The choice of single or double centrifugation depends on the blood components required for the PRP treatment.
[Copyright: Karl-Ludwig Poggemann via NCI-Frederick. Licence: CC BY 2.0 ]

However, double centrifugation also tends to decrease leukocyte concentration, which may reduce levels of growth factors, such as HGF, IGF-1, and PDGF-AB, relative to single centrifugation [8]. In addition, single centrifugation is better for preparing PRP gel, as double centrifugation can lead to fibrin aggregation, causing ultrastructural alterations to the gel [9].

Therefore, the choice of single or double centrifugation will depend on the optimal levels of the different PRP components required to treat the patient.

Centrifugal force and duration

After comparing a range of single and double centrifugation protocols, Perez et al. (2014) found that centrifugation at 100 g for 10 min followed by 400 g for 10 min was optimal [10]. This protocol yielded a 5× increase in platelet concentration above the basal level and preserved platelet integrity. 

Similarly, Yin et al. (2017) compared a range of centrifugal gs and durations, finding that double centrifugation with 160 g for 10 min followed by 250 g for 15 min produced the greatest increases in platelet and growth factor concentrations [11].

In terms of single centrifugation protocols, Perez et al. (2014) reported that centrifugation at 70–100 g for 10 min was optimal, while >190 g decreased platelet concentration [10].

Overall, current research suggests that double centrifugation at a moderate but not excessive centrifugal force (100–160 g followed by 250–400 g) for 10–15 min yields the greatest increase in platelet concentrations.

Storage conditions for PRP

In terms of best practice for storing PRP, evidence suggests that storage at room temperature is adequate—however, care should be taken to preserve the sample if it is stored for more than a few hours.

Ideally, PRP should be used within 8 hours of centrifugation, as this maintains the leukocyte concentration and pH of the solution [12]. 

However, PRP can safely be stored for 24 hours at room temperature without affecting platelet counts or activation [12]. Therefore, a longer storage time may be possible if platelets are the key component required for clinical treatment.

Conclusions

Although PRP is widely used therapeutically to aid tissue repair and bone growth, understanding the best way to prepare it is not always obvious given the multitude of possible methods. With the help of the evidence and recommendations in this article, we hope that the preparation of PRP in your own clinic or lab can be optimized to improve the quality of treatments for your patients.

References

[1] Marx, R. E. (2004). Platelet-rich plasma: evidence to support its use. Journal of Oral and Maxillofacial Surgery, 62(4), 489-496. CrossRef Full Text

[2] Singh, S. (2018). Comparative (quantitative and qualitative) analysis of three different reagents for preparation of platelet-rich plasma for hair rejuvenation. Journal of Cutaneous and Aesthetic Surgery, 11(3), 127. CrossRef Full Text

[3] do Amaral, R. J. F. C., da Silva, N. P., Haddad, N. F., Lopes, L. S., Ferreira, F. D., Cappelletti, P. A., ... & Balduino, A. (2016). Platelet-rich plasma obtained with different anticoagulants and their effect on platelet numbers and mesenchymal stromal cells behavior in vitro. Stem Cells International, 2016, Article ID 7414036. CrossRef Full Text

[4] Arora, G., & Arora, S. (2021). Platelet‐rich plasma—Where do we stand today? A critical narrative review and analysis. Dermatologic Therapy, 34(1), e14343. CrossRef Full Text

[5] Cohn, C. S., Delaney, M., Johnson, S. T., & Katz, L. M. (Eds.) (2020). Technical manual, 20th edition. AABB. Buy it here or read the PRP preparation method 6-12 here.

[6] Dhurat, R., & Sukesh, M. S. (2014). Principles and methods of preparation of platelet-rich plasma: a review and author's perspective. Journal of Cutaneous and Aesthetic Surgery, 7(4), 189. CrossRef Full Text

[7] Pachito, D. V., Bagattini, Â. M., de Almeida, A. M., Mendrone-Júnior, A., & Riera, R. (2020). Technical procedures for preparation and administration of platelet-rich plasma and related products: a scoping review. Frontiers in Cell and Developmental Biology, 8, 1556. CrossRef Full Text

[8] Mazzocca, A. D., McCarthy, M. B. R., Chowaniec, D. M., Cote, M. P., Romeo, A. A., Bradley, J. P., ... & Beitzel, K. (2012). Platelet-rich plasma differs according to preparation method and human variability. JBJS, 94(4), 308-316. CrossRef Full Text

[9] Tamimi, F. M., Montalvo, S., Tresguerres, I., & Jerez, L. B. (2007). A comparative study of 2 methods for obtaining platelet-rich plasma. Journal of Oral and Maxillofacial Surgery, 65(6), 1084-1093. CrossRef Full Text

[10] Perez, A. G., Lana, J. F. S., Rodrigues, A. A., Luzo, A. C. M., Belangero, W. D., & Santana, M. H. A. (2014). Relevant aspects of centrifugation step in the preparation of platelet-rich plasma. International Scholarly Research Notices, 2014. CrossRef Full Text

[11] Yin, W., Xu, H., Sheng, J., Zhu, Z., Jin, D., Hsu, P., ... & Zhang, C. (2017). Optimization of pure platelet‑rich plasma preparation: A comparative study of pure platelet‑rich plasma obtained using different centrifugal conditions in a single‑donor model. Experimental and Therapeutic Medicine, 14(3), 2060-2070. CrossRef Full Text

[12] Abu Kasim, H., & Al-Hassan, F. (2016). Assessment of platelet concentrate prepared from fresh and overnight held whole blood. Asian Journal of Pharmaceutical and Clinical Research, 9, 16–20. CrossRef Full Text