What is the procedure for preparing platelet-rich plasma (PRP)?

Platelet-Rich Plasma (PRP) Preparation Procedure

The standard procedure for preparing platelet-rich plasma (PRP) involves collecting anticoagulated blood, centrifugation at specific speeds, separation of components, and optional activation before clinical application. 1

Blood Collection and Anticoagulation

• Collect autologous blood at the time of therapeutic application in a laboratory, operating theater, or clinic room 1
• Use appropriate anticoagulants:
  • Trisodium citrate is most widely used with few negative effects on PRP preparation 1
  • Acid citrate dextrose (ACD) and citrate phosphate dextrose (CPD), including those with adenine (ACD-A, CPD-A) are effective alternatives 1
  • AVOID EDTA as it causes platelet swelling and activation 1
• For research purposes, blood samples should be collected in EDTA-coated tubes for quality control measurements to maintain stable platelet counts for up to 24 hours 2

Centrifugation Process

Traditional Single-Step Method

• Centrifuge anticoagulated blood at low force (170-200 g) for 10 minutes at room temperature 1
• This provides a relatively pure preparation with good PRP yield 1

Optimized Two-Step Method

• First spin: 100 g for 10 minutes (promotes high platelet recovery) 3
• Second spin: 400 g for 10 minutes 3
• Withdraw 2/3 of remnant plasma to achieve high platelet recovery (70-80%) and concentration (5x) while maintaining platelet integrity and viability 3
• Process smaller blood volumes (3.5 mL) for better platelet recovery compared to larger volumes (8.5 mL) 3

Main Clinical Preparation Methods

Three main methods are available for sterile PRP product preparation 1:

1. Gravitational centrifugation techniques:

   • Most common and accessible method
   • Uses varying g-force and centrifugation times that affect yield, concentration, purity, viability, and activation status 1

2. Standard cell separators:

   • Operate on a full unit of blood
   • Use continuous-flow centrifuge bowl or disk separation technology
   • Combine hard and soft centrifugation steps
   • High g-force centrifugation isolates the buffy coat layer containing platelets and leukocytes 1

3. Autologous selective filtration technology (plateletpheresis):

   • Specialized filtration method for platelet concentration 1

PRP Activation

• PRP must be activated for platelets to release their α-granule contents 1
• The resulting clot provides a scaffold to capture secreted proteins and maintain their presence at the application site 1
• Activation methods include:
  • Thrombin
  • Re-calcification
  • Contact with collagen-rich tissues (in some applications) 1
• Consider whether activation should be performed prior to or at the time of clinical application 1

PRP Classification and Quality Control

• PRP preparations vary widely in content, purity, and biological properties, affecting clinical efficacy 1
• The International Society on Thrombosis and Haemostasis (ISTH) proposed a classification system based on:
  • Leukocyte content (≥1%)
  • Red blood cell content (≥10%)
  • Activation method
  • Platelet concentration
  • Preparation technique 1
• For quality control, samples should be taken in EDTA-coated tubes and analyzed using validated cell counters 2

Important Considerations and Pitfalls

• PRP should be used within 6 hours after preparation (though stability remains uncertain) 1
• Different centrifugation protocols significantly impact platelet yield, concentration, and viability 3
• Commercial PRP preparation devices result in significant variation in product content 1
• The presence of leukocytes may impact biological properties through immune and antibacterial effects and influence tissue healing 1
• Lack of standardization in preparation methods contributes to variable clinical outcomes 4
• Processing larger blood volumes may reduce platelet recovery efficiency 3

Emerging Techniques

• Newer ultrafiltration technologies incorporate platelet-poor plasma (PPP) processing to create protein-rich PRP (PR-PRP) matrices 5
• These techniques concentrate not only platelets but also functional plasma proteins like fibrinogen, albumin, and alpha-2-macroglobulin 5