What is the standard protocol for preparing Platelet-Rich Plasma (PRP)?

Standard Protocol for Platelet-Rich Plasma (PRP) Preparation

There is no single universally standardized PRP preparation protocol, but the most evidence-supported approach involves collecting anticoagulated blood (preferably with citrate), centrifuging at 170-270 g for 10 minutes, separating the platelet-rich layer, and optionally activating before clinical use. 1, 2

Critical Context: The Standardization Problem

Clinical preparations of PRP are poorly standardized, with content, purity, and biological properties varying widely—a fact unanimously agreed upon by expert consensus. 1 This lack of standardization directly impacts clinical efficacy and makes comparison across studies nearly impossible. 1

Step-by-Step Preparation Protocol

Blood Collection and Anticoagulation

• Collect autologous blood at the time of therapeutic application in a laboratory, operating theater, or clinic room. 2

• Use trisodium citrate as the preferred anticoagulant, as it is most widely used with minimal negative effects on PRP preparation. 2

• Acid citrate dextrose (ACD) and citrate phosphate dextrose (CPD), including ACD-A and CPD-A formulations, are effective alternatives. 2

• Absolutely avoid EDTA anticoagulant—it causes platelet swelling and activation, compromising the final product. 1, 2 This is one of the few definitive consensus statements from the International Society on Thrombosis and Haemostasis (ISTH). 1

Centrifugation Process

• Centrifuge anticoagulated blood at low force (170-200 g) for 10 minutes at room temperature. 2 Some protocols suggest up to 230-270 g for 10 minutes in a 15-mL conical tube for optimal PRP collection. 3

• This low-force, short-duration centrifugation provides a relatively pure preparation with good PRP yield while minimizing cellular contamination. 2

• Platelet concentration, yield, and recovery are critically dependent on the centrifugation protocol and collection methods utilized—this is another point of complete expert consensus. 1

Three Main Clinical Preparation Methods

Gravitational centrifugation techniques use varying g-force and centrifugation times that affect yield, concentration, purity, viability, and activation status. 2 This is the most common approach in clinical practice.

Standard cell separators operate on a full unit of blood using continuous-flow centrifuge bowl or disk separation technology. 2

Autologous selective filtration technology (plateletpheresis) is a specialized filtration method for platelet concentration. 2

Optional Concentration Step (For Platelet-Concentrated Plasma)

• For higher platelet concentrations, precipitate platelets by centrifuging PRP at >2300 g, remove 90% of supernatant plasma, and resuspend the platelets. 3

• This can achieve approximately 20-fold greater platelet concentration than whole blood. 3

PRP Activation

The need for pre-application activation remains uncertain in the literature (median expert score of 5 out of 9). 1

• When activation is used, thrombin is the standard activating agent. 2

• In some applications, collagen-rich tissues may activate PRP naturally, eliminating the need for pre-application activation. 1, 2

• The resulting clot provides a scaffold to capture secreted proteins and maintain their presence at the application site. 2

Timing and Stability

• Use PRP within 6 hours after preparation, though the exact stability window remains uncertain. 1, 2

Quality Control and Documentation Requirements

The ISTH emphasizes that any PRP preparation must include comprehensive documentation—this received the highest consensus scores (median 9 out of 9). 1

Essential Parameters to Document:

• Amount of autologous blood collected 1

• Baseline platelet number, volume, and concentration utilized 1

• Full description of PRP preparation methodology 1

• Purity of the final preparation (red blood cell and white blood cell contamination levels, plasma content) 1

• Platelet activation status prior to clinical use (to verify platelets haven't prematurely lost their granular content) 1

• Activation procedure, if used 1

Critical Pitfalls and How to Avoid Them

Commercial PRP preparation devices result in significant variation in product content—platelet concentration can vary widely between systems even when following manufacturer protocols. 2, 4 A 2019 systematic review found that platelet concentration was directly correlated with both volume of blood collected and device centrifugal force. 4

Temperature control during preparation shows strong correlation with PRP efficacy (r = 0.79 in a 2024 systematic review of 75 RCTs). 5 Maintain consistent temperature throughout the preparation process.

The presence of leukocytes may impact biological properties through immune and antibacterial effects, potentially influencing tissue healing outcomes. 2 Document whether your preparation is leukocyte-rich or leukocyte-poor.

Many trials fail to fully define the content, purity, and biological properties of platelet preparations—this received unanimous expert agreement as a major problem in the field. 1, 6

Classification System

The ISTH proposed a classification system based on five key variables: 2

• Leukocyte content
• Red blood cell content
• Activation method
• Platelet concentration
• Preparation technique

This classification helps standardize reporting and comparison across studies, though implementation remains inconsistent. 2

Safety Considerations

Autologous and allogeneic sterile PRP preparations are clinically safe when proper sterile technique is maintained during preparation and application. 1, 6