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

Optimal Double Spin Protocol for Liquid PRP Preparation

The optimal double spin protocol for liquid PRP preparation involves a first spin at 100-300g for 5-10 minutes followed by a second spin at 400-700g for 10-17 minutes, using citrate anticoagulated blood, with processing completed within one hour of collection to maximize platelet concentration and viability. 1

Anticoagulant Selection

• Use plastic blood collection tubes containing citrate anticoagulant (0.109 M) 2
• AVOID using EDTA anticoagulant as it causes platelet swelling and activation 3, 2
• Acid citrate dextrose (ACD) and citrate phosphate dextrose (CPD) are also effective alternatives 3
• Ethylene di-amine tetra-acetic acid disodium has been shown to inhibit platelet aggregation more efficiently than acid citrate dextrose solution, resulting in higher non-aggregated platelet yield 4

First Centrifugation (Soft Spin)

• Parameters: 100-300g for 5-10 minutes 1
• Purpose: Separates red blood cells from plasma containing platelets and leukocytes
• Alternative parameters that have shown efficacy:
  • 170-200g for 10 minutes at room temperature 3
  • 230-270g for 10 minutes (for PRP without white blood cells) 4
  • 70g for 10 minutes (for white blood cell-containing PRP) 4

Collection After First Spin

• Collect the plasma and buffy coat layer (containing platelets)
• Be careful not to disturb the red blood cell layer
• Transfer to a fresh tube for the second centrifugation

Second Centrifugation (Hard Spin)

• Parameters: 400-700g for 10-17 minutes 1
• Purpose: Concentrates platelets into a pellet at the bottom of the tube
• Alternative parameters with demonstrated efficacy:
  • 1500g for 15 minutes at room temperature 2

  • 2300g (for highly concentrated PCP preparation) 4

  • 1550 rpm for 10 minutes 5

Final Processing

1. After second centrifugation, the sample separates into:
   • Upper layer: Platelet-poor plasma (PPP)
   • Bottom: Platelet pellet
2. Remove approximately 2/3 to 3/4 of the PPP
3. Resuspend the platelet pellet in the remaining plasma to create liquid PRP
4. Target concentration: 1-1.5 million platelets/μL 1

Critical Considerations

• Process blood within 1 hour of collection to ensure optimal platelet viability 2
• Maintain blood at controlled room temperature throughout processing 2
• Handle tubes gently to prevent premature platelet activation
• The entire preparation process should take approximately 35-40 minutes 5
• PRP should be used within 4-6 hours if kept at room temperature 2

Common Pitfalls to Avoid

1. Using EDTA as anticoagulant (causes platelet activation)
2. Storing blood at 4°C (causes cold activation of platelets)
3. Excessive delays between collection and processing
4. Improper centrifugation speeds or times
5. Rough handling during transfer steps
6. Failure to properly resuspend platelet pellet

Activation Considerations

• Activation of PRP before application is not always necessary 1
• In some applications, collagen-rich tissues may activate PRP naturally 3
• If activation is desired, thrombin can be used 4

This protocol can yield a platelet concentration approximately 3-5 times higher than baseline 5, 6, which is within the therapeutic range for most clinical applications. The exact parameters may need slight adjustments based on the specific centrifuge model used, as the radius of the centrifuge affects the final g force 3.