What is the best centrifugation protocol for making PRP?

(Platelet-Rich Plasma) What is PRP therapy?

Platelet-Rich Plasma (PRP) is a solution rich in platelets and growth factors prepared from a patient’s own blood. Injections of PRP are used to promote tissue repair and bone growth in a variety of conditions, including hair loss, osteoarthritis, and tendon or ligament injuries.

Typical platelet concentrations are about 200,000/µL, but the platelet concentration in PRP should exceed 1,000,000/µL or 5 times the baseline normal level.

How is a 5-fold increase in platelet concentration achieved in practice?
What other steps are important to maintain other potentially important properties of PRP, such as platelet morphology, platelet activation, or growth factor concentrations?

PRP preparation generally involves 3 important steps:

1. Sample collection – first collect the patient’s blood and generate plasma;
2. Centrifugation – concentrate platelets and other wound healing components to obtain PRP;
3. Storage – the quality of PRP must be maintained before clinical application.

In the closed method of preparing PRP, all of these steps are carried out in a predefined manner in a completely closed and sterile integrated system.

In contrast, the open method provides the opportunity to optimize each step independently, although attention must always be paid to maintaining sterility.

Here, we focus on the open method and summarize the latest data on the best protocols for collecting, centrifuging and storing 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 step involves collecting the blood into a tube containing an anticoagulant to prevent the blood from clotting.

However, different anticoagulants can affect the composition and properties of plasma in various ways. So, among the various anticoagulants available – EDTA, ACD-A, sodium citrate, etc., which one is best for generating PRP?

Overall, the research evidence suggests that ACD-A produces PRP with better platelet concentration, viability, and morphology than EDTA or sodium citrate. Conversely, growth factor concentration did not differ based on the use of EDTA, ACD-A, or sodium citrate, although mesenchymal stem cells proliferated more readily in PRP prepared with ACD-A or sodium citrate.

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

Optimal Centrifugation Protocol for PRP
Using differential centrifugation, platelets can be separated and concentrated relative to other components of plasma. However, in order to optimize platelet and growth factor concentrations while maintaining platelet integrity and viability, an appropriate centrifugation protocol must be used.

Next, we summarize key recommendations for selecting the best PRP centrifuge, as well as the optimal temperature, centrifugal force, and spin protocol for generating PRP.

Centrifuge Model Selection
What factors should be considered when selecting a centrifuge model for concentrating platelets and growth factors in PRP?

Overall, a swing-out centrifuge is the best choice for preparing PRP because it allows for better separation of plasma components based on density. In addition, swing-out centrifuges can improve the quality of PRP by reducing the amount of cell damage and injury compared to tilting centrifuges.

Recommended swing-out models commonly used for preparing PRP include the MM10 Compact Centrifuge (pictured above)

Care should be taken to select a centrifuge that is appropriate for the type of collection tube being used, especially when using a commercial PRP preparation kit. In this case, the manufacturer’s instructions should be followed to select an appropriate centrifuge.

What is the temperature during centrifugation?

PRP centrifugation is typically performed at room temperature, and the American Association of Blood Banks Technical Manual recommends a centrifugation temperature of 20°C–24°C. However, some evidence suggests that cooler temperatures of 12°C–16°C may improve platelet recovery and viability.

Single vs. Double Centrifugation
In general, double centrifugation is more effective than single centrifugation in increasing platelet concentration.
However, double centrifugation also tends to reduce leukocyte concentration compared to single centrifugation, which may reduce the levels of growth factors such as HGF, IGF-1, and PDGF-AB. In addition, single centrifugation is more suitable for preparing PRP gels, as double centrifugation can cause fibrin aggregation, which can lead to changes in the gel ultrastructure.

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

A note on storage conditions for PRP
In terms of best practices for storing PRP, there is a lot of evidence that storage at room temperature is sufficient – however, if storage is required for more than a few hours, care should be taken to store the sample.

In general, PRP should be used within 8 hours of centrifugation as this maintains the leukocyte concentration and pH of the solution.

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

Summary
While PRP is widely used therapeutically to aid tissue repair and bone growth, understanding the best way to prepare PRP is not always obvious given the number of possible methods. With the evidence and recommendations in this article, we hope to optimize the way you prepare PRP in your own clinic or laboratory to improve the quality of care for your patients.