How do doctors increase platelet concentration when preparing tubes for PRP?

Question

I am reading from multiple doctors responding on RealSelf that most PRP is low quality because systems only use 2x concentration of PRP. They say it is most effective when platelets are 6x or 1.5 million per ml. How does a doctor increase their PRP effectiveness from 2x to 6x? Is it dependent on time in the centrifuge?

Jeff Rapaport, MD · Answer

The  available concentration of PRP in the tube Is based upon the platelet gradient.  Larger tubes or containers ( 30 Cc or larger) combined with  a high efficiency of capture of platelets allows for more platelets in the tube and therefore more volume of a higher concentration PRP.   This is inherent in the dynamics of the tube Or container. Therefore, the system 
used makes a big difference. ( I like Progen.)  In my opinion, it's the total number of platelets that one injects that is the most important factor.  This can be determined by the volume of the injection times the concentration of what is injected.  It is assumed, but never been proven outside the lab, that the desired range of concentration is 3X to 6X.  Therefore, the largest volume that one can inject at this concentration is Most effective. Find a PRP expert not just an injector.

Jeff Rapaport MD FAAD 
Realself Hall of Fame

Patrick Yam, MD · Answer

Thanks for the great question. Preparing PRP well is a bit of a delicate process and there are a variety of PRP systems on the market. Common ones in use are designed to be quick and yield a high volume of PRP, but not necessarily a high concentration of platelets. In general, PRP can be prepared through a single- or double-spin process. Single spin is limited in terms of the concentration which can be produced, maximum usually around 2-2.3x, but usually lower, on average 1.25x. This is from a recent (Magalon et al, 2020) comprehensive review of 34 different PRP systems which had been characterized in the scientific literature. Normal starting platelet concentration varies, anywhere from 150-440 depending on the lab, but for the sake of discussion, let's say average platelet count is around 220,000/µL. 1.25x = 275k, far from 1 million/µL, the classic definition of PRP (Marx, 2001). 
To achieve 1.5 million platelets per microliter from a starting concentration of 220 would require 6.8x. This could only be reached with a double-spin system. Basically, you take the blood through a first, soft spin, and then collect the plasma containing platelets (PCP) and go into a second spin where you concentrate even further. After this second step, most of the platelets have collected into a pellet at the bottom of the tube with platelet-poor plasma (PPP) above. A certain amount of PPP is discarded and the platelets are resuspended into the remaining plasma volume to make a higher concentration. As you can imagine, if you discard more PPP, there will be a higher concentration of platelets in the final PRP. 
If you made it this far, you might start to realize that concentration is not the only important factor. The actual number of platelets delivered (platelet dose) is also important. For example, for hair treatments the target is 3-6 billion platelets delivered to back to the patient. At a concentration of 1 million/µL, there are 1 billion platelets in 1mL of PRP. So this is equal to approximately 3-6mL at 1 million/µL. A typical volume of injection would be around 6mL, so the concentration should be 0.5-1 million/µL.The best way to be sure of the concentration is to check the PRP with a hematology analyzer before injection. 
As far as the centrifuge goes, it's not just time, but also force that has to be considered. If you spin too fast or too long, you can get a very pure sample, with low contamination with red cells and white cells (leukocytes), but you also lose a lot of platelets. The purest PRP sample in Dr. Magalon's report above did not concentrate platelets, but the opposite and ended up with a lower platelet concentration than the starting blood sample.
Thanks for reading and I hope this helps.

How do doctors increase platelet concentration when preparing tubes for PRP?

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