platelets will activate with mechanical trauma and after coming in contact with extra-cellular fluids

In my opinion PRP should be activated prior joint injection. I have some experience treating horses with OA and for me, the clincal results are better when PRP is activated.

I unreservedly agree with Preeti Singh. Platelets are very sensitive and can be partially activated during the preparation of PRP. Contact with thrombogenic surface after intraarticular injection is another very powerful stimulus. And, if the blood is in the joint cavity, is there thrombin too and then platelets have not a chance to remain inactive.

Dear Jaromir, I agree with your biological appreciation. However, this is my clinical appreciation about the use of PRP for the treatment of joint disease in horses. I think that PLT release reaction should be stimulated with activating substances to produce a massive release of growth factors. In my opinion a massive concetration of growth factors is the main biological trigger of the joint anabolism. Thus, if you put a non activated PRP in a joint the release of growth factors will be more slow and possibly the joint response will be different.

That's probably true dear Jorge. Definitely, the important are the platelet proteins, no platelets. And these proteins must be released from platelets. I do not know if and how quickly, definitely as much as possible.

How do you prepare PRP for veterinary use. We have a centrifuge Harvest and now trying filters Paul Medical FPC.

Most preparations such as ACP don´t need activation with calcium because the cytokines in the alpha granules of plateltes are released through the cetntrifugation process. Ca Citrat is used for anticoagulation to extend the use after centrifugation. If you apply PRP within 10 minutes after preparation you don´t need Ca Citrat at all.

Do not need activation in a degenerative knee.  The exposed collagen fibrils are a strong stimulus to alpha granule release.

We prefer to use sodium citrate instead of EDTA as anticoagulant because there is evidence that  EDTA may injury the platelet membrane. Then, calcium chloride is used to chelate de citrate, and allow the coagulation process, that will initiate platelet activation. Platelet activation can be further enhanced with the addition of thrombin, preferably autologous, to PRP. It will promote the formation of platelet gel, and a rapid release of growth factors. Otherwise, when PRP is injected, it will be exposed to thrombin in the tissue, and will activate anyway, but slower. Therefore, in cases where PRP will be injected, I prefer not to activate with thrombin. In cases where PRP will be added to the surgical site, as a gel, for example, I prefer to activate it.

I must admit that I agree with William Murrell that the platelets will activate very happily without the need to add CaCl. Citrate chelates the calcium relatively weakly and reversibly compared to EDTA for example and CaCl is added to swamp the citrate. In the tissues the citrate is metabolised/diluted quickly and the extracellular fluid will provide the Calcium necessary. 

There is an interesting debate though about whether activating the platelets quickly or slowly is better. Activating platelets with thrombin causes them to degranulate quickly so that they have lost 90% of their payload within 60 mins. However they continue to live for 8-10 days continuing to produce the factors in a low concentration.

Is it really that important to force the activation to have a quick release of factors?

I just read the BJSM article on PRP.  Most of the preparations used in the RCTs on Knee OA used activated PRP.

Robert is highlighting the debate. I certainly agree that most of the kits activate the pro before injection,but is it really necessary? Within the joint compartment there is plenty of collagen to activate the platelets, and calcium chloride merely neutralizes the effects of the citrate, which is quickly diluted in the tissues anyway.

I have not done a comparative study but pro works well for me without, and there is a logical argument that it could be better to activate them at the site of injection. The important thing is to have good quality platelets that will continue to live for ten days secreting small but significant platelet factors that are short lived once secreted.

Collecting peripheral blood in heparin does it have any detrimental effect on PRP gel preparation/ PRP activation?

Maybe for gel preparation, no for activation, yes for post injection bruising, would recommend only citrate volume appropriate.

I think there are several problems with using heparin with little or no advantage

Because citrate uprates glycloysis in the platelets there is over several hours an increased rate of spontaneous activation which is reduced if ACD-A anticoagulant is used. I have a separate question here on whether there is any significant difference in the spontaneous activation rates between pure citrate and ACD-A for periods less than 60mins. Nobody has yet offered any concrete evidence either way but there is the suggestion that on a theoretical basis ACD-A could be preferred. 

Another question (also asked here) is how much citrate is necessary to achieve anticoagulation. The present protocols are based on historical blood bank requirements where the blood should remain anticogulated for 3-4 weeks. In reality the amount of citrate can be significantly reduced to 0.5ml 3.8% citrate in 8.5ml blood without any coagulation at several days, thus potentially decreasing the increased activation related to uprated glycolysis.

PRP is activated by collagen, not just calcium. If you are injecting PRP into soft tissue there is no need for activators.  

After some experimentation on my own knees I am beginning to come to the conclusion that calcium to reverse the anticoagulant may improve the process when PRP is injected into closed spaces like knee joints.

This may be because part of the activation process is not just the collagen but is also related to calcium diffusing into the local area. Since I am injecting pretty large volumes of PRP (8ml) it takes time for this to happen, whereas adding calcium allows the production of a fibrin clot within a few inutes. It certainly feels different!

I doubt that the calcium is important in injections around tendinoses and into the skin for aesthetic treatments since there is both a lot of collagen and the calcium will diffuse into the area quickly. Possibly the need for an immediate fibrin clot is less because the platelets wont move straight away since they are in the dermis

As mentioned before and also based in the opinion of Dr Blatchley, PRP should be activated prior uts usage. We believe the masive granular release is paramount to redirect the inflammatory/regulatory effects unchained by this substance.