Chapter 4 PRP and Autologous Conditioned Sera

Question 1

Which part of platelets contain growth factors/cytokines/vasoactive peptides/extra cellular matrix proteins?

Answer 1

Alpha granules

Question 2

List 5 factors that can be derived from platelets

Answer 2

1. Platelet derived growth factor (PDGF)
2. Vascular Endothelial Growth Factor (VEGF)
3. Transforming Growth Factor-β (TGF-β)
4. basic Fibroblast Growth Factor (bFGF)
5. Insulin-Like Growth Factor (IGF)
6. Hepatocyte Growth Factor (HGF)
7. Endothelial Growth Factor (EGF)
8. Connective Tissue Growth Factor (CTGF)

Question 3

What is the definition of PRP

Answer 3

Plasma that contains a platelet concentration greater than that of peripheral blood.

In reality, most authors agree that shoud be x3-5 concerntration fo normal blood.

Question 4

What are the two types of PRP called and what is the difference between them?

How does the isolation method differ between the two types of PRP?

Answer 4

Pure-PRP (P-PRP) and Leukocyte-PRP (L-PRP). Leukocyte PRP contains a proportion of WBCs (controversial whether this is beneficial or detrimental)

For production of P-PRP, once the sample has been centrifuged (softspin) only platelet-poor-plasma and part of buffy coat are harvested (ie no RBCs).

For L-PRP, whole buffy coat (and pletelet poor plasma) and some RBCs are harvested

Question 5

What are the two methods for PRP production. Briefly outline each method.

Answer 5

Centrifugation vs filtration.

Centrifugation:

• 10-110 ml blood collected w anticoagulant
• Softspin centrifugation
• Desired sample harvested (without/with some RBCs depending whether for P-PPP/L-PPP)
• Hardspin centrifugation
• Platelet poor plasma discarded, PRP collected

Pros: some plasma also part of delivered solution

Cons: High centrifugation may –> fragmentation/activation of platelets

Filtration:

• 60ml anticoagulated whole blood into resevoir bag (N.B. careful phlebotomy!)
• Capture solution (sterile water) mixed with blood –> platelet swelling.
• Gravity ro pass blood through filter. Filter catches RBCs, some WBCs, platelets.
• Sterile saline flushed in reverse direction to displace cells from filter –> leukocyte and platelet rich plasma

Pros = quick (6-10 min)

Cons = cells suspended in saline –> ?loss of benefits of plasma and associated proteins

Question 6

Name two instances in which PRP is contraindicated

Answer 6

Thombocytopaenia and coagulation defects

Question 7

Name 2 examples of commercially available PRP systems (according to Tobias)

Answer 7

1. Protec PRP, PulseVet
2. MediVet PRP, MediVet America
3. C-PET, Pall Corporation
4. SmartPReP2, Harvest Technologies
5. Angel, Arthrex Vet Systems

Angel, Arthrex looks best according to Tobias table (x4 increased concentration of platelets cf blood and v low level WBCs)

Question 8

What is autologous conditioned sera?

Answer 8

Incubated serum samples containing increased concentration of Interleukin-1 receptor antagonist (IL-1RA aka Interleukin-1 receptor antagonis proteion (IRAP))

Osteoarthritis is mediated by a number of cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β).37,46,90 IL-1β and TNF-α potentiate osteoarthritis by increasing the expression of matrix metalloproteinases that directly degrade the extracellular matrix of articular cartilage. In addition, IL-1β and TNF-αstimulate the production of downstream inflammatory agents such as nitrous oxide, cyclooxygenase-2, and prostaglandins.2,20,43 The result is transition of the joint from a homeostatic state toward a catabolic state of matrix degradation and chondrocyte apoptosis. Importantly, it has been shown that the synovial membrane of dogs affected by osteoarthritis expresses several cytokines linked to osteoarthritis, including IL-1β, IL-6, IL-10, and many others.59 Thus, it appears that IL-1β is involved in the pathogenesis of osteoarthritis in the dog as well as other domestic species.

Due to the importance of the IL-1β pathway in the pathogenesis of osteoarthritis, modulation of IL-1β protein expression, receptor binding, and downstream signaling events are compelling therapeutic targets. Interleukin-1 receptor antagonist (IL-1RA), also known as interleukin-1 receptor antagonist protein (IRAP), is an endogenous protein produced by a number of organs, including joints. IL-1RA competitively inhibits IL-1βactivity by directly binding the IL-1 receptor, preventing IL-1βligand/receptor interactions and downstream signaling events.

Question 9

What ratio of IL-1RA:IL-1 has been suggested to abolish the negative effects of IL-1β

Answer 9

130:1

Question 10

Briefly outline harvest technique of autologous conditioned sera.

Answer 10

• Collect 10-50ml whole blood (N.B. excellent venipuncture technique to avoid haemolysis)
• Injected into sterile chamber containing borosilicate beads (these –> leukocyte adherence + activation –> increased IL-1RA production)
• Sample incubated at 37º for 6-24 hours
• Centrifuged
• Serum harvested