What is the best double spin protocol for Platelet-Rich Plasma (PRP)?

Optimal Double Spin Protocol for Platelet-Rich Plasma (PRP)

The optimal double spin protocol for PRP involves an initial centrifugation at 1000g for 5 minutes followed by a second centrifugation at 1500g for 15 minutes, which yields approximately 80% platelet recovery and a six-fold concentration increase. 1 This protocol has been shown to produce the highest concentration of growth factors such as PDGF-BB in activated fractions.

Key Components of an Effective Double Spin Protocol

Blood Collection

• Use plastic blood collection tubes containing citrate anticoagulant (0.109 M) 2
• Avoid EDTA anticoagulant as it should not be used for PRP preparation 2
• Draw blood directly into the tube rather than using a syringe first 2
• Process blood as soon as possible, preferably within 1 hour of collection 2

First Centrifugation Step

• Purpose: To separate red blood cells from platelet-rich plasma
• Optimal parameters: 1000g for 5 minutes 1
• Alternative method: Low-g centrifugation (170g for 10 minutes) may also be effective, though this remains uncertain 2
• Temperature: Maintain at controlled room temperature to avoid precipitation and cold activation 2

Collection of Platelet-Rich Plasma

• Carefully transfer the platelet-rich plasma (PRP) layer to a new tube
• Avoid disturbing the buffy coat layer to prevent contamination with white blood cells
• For closed systems, this can be done without opening the container 3

Second Centrifugation Step

• Purpose: To concentrate platelets into a pellet
• Optimal parameters: 1500g for 15 minutes 1
• This step creates a platelet-poor plasma supernatant and a platelet pellet
• Temperature: Maintain at controlled room temperature 2

Final Preparation

• Remove most of the platelet-poor plasma
• Resuspend the platelet pellet in the desired volume of remaining plasma
• Gently mix to ensure even distribution of platelets
• Use within 4 hours if kept at room temperature 2

Expected Outcomes

• Platelet concentration: Approximately 6× baseline concentration 1
• Platelet recovery: Approximately 80% of platelets from whole blood 1
• Growth factor yield: Highest concentration of PDGF-BB in activated fractions 1

Important Considerations

Standardization Issues

• Clinical preparations of PRP are poorly standardized, which may impact efficacy 2
• The content, purity, and biological properties of PRP vary widely 2
• Many studies fail to fully define the content and properties of platelet preparations 2

Activation Considerations

• It is uncertain whether PRP must be activated for platelets to release their granule contents before application 2
• In some applications, collagen-rich tissues may activate PRP, eliminating the need for pre-application activation 2

Quality Control

• Platelet concentration, yield, and recovery are dependent on the centrifugation protocol and collection methods 2
• For research purposes, platelet counts should be measured to ensure consistency
• Avoid premature platelet activation by gentle handling throughout the process

Pitfalls to Avoid

• Using EDTA as an anticoagulant, which is inappropriate for PRP preparation 2
• Storing blood at 4°C, which can cause cold activation of platelets 2
• Excessive delays between blood collection and processing, which can affect platelet viability
• Improper centrifugation speeds or times, which can result in suboptimal platelet recovery
• Rough handling during transfer steps, which can cause premature platelet activation
• Using expired or improperly stored PRP (should be used within 4 hours if kept at room temperature) 2

While there are multiple protocols available, the evidence supports that this specific double-spin protocol (1000g for 5 minutes followed by 1500g for 15 minutes) provides optimal platelet concentration and growth factor yield for clinical applications.