I had a recent clinical research meeting with our fellows and one of the topics brought up was PRP dosing. They thought that this was a simple matter of counting the platelets. However, once I explained how complex PRP dosing really was, their minds had exploded. Hence, I told them that I would blog on it.
PRP and Classification Systems
PRP stands for platelet-rich plasma. This means that the doctor takes a blood sample and uses a centrifuge to concentrate the platelets in your plasma. These platelets contain healing growth factors that can help damaged tissues.
There are several different types of PRP, but the two main types commonly used for injection include the “red” leukocyte rich variety and the “amber” leukocyte poor type. These are red and amber because the red blood cells come along for the ride with the white blood cells (leukocytes). Meaning leukocyte rich has lots of red blood cells as well and leukocyte poor has few.
While several classification systems for PRP have been proposed, the one that seems to be used the most often is the PLRA (1). This means that to classify PRP, we need to know the platelet count (P), the leukocyte content (L), the red blood cell content (R) and whether or not the PRP was activated (A). However, as I’ll show below, while this is a good start, quantifying or dosing PRP is actually MUCH more complicated.
Counting Platelets in PRP Can Be Unreliable at Best
Counting platelets in whole blood is easy and done every day. However, counting platelets in PRP is harder. Why? All of that centrifugation that’s needed to concentrate the platelets causes some to clump together in doublets and triplets. So when you look at PRP under the microscope, you tend to see a reasonable number of platelets stuck together. Cell counters will either count these double or triple platelets as one platelet or just ignore them because the counter believes these are larger red or white blood cells. Meaning that counting platelet numbers in PRP is usually inaccurate.
All Platelets are Not Created Equal
The first thing that blew the collective minds of my fellows was the fact that the growth factor content of platelets varies widely between patients. Remember that platelets have growth factors that live in alpha vesicles and then those vesicles degranulate over about a week. That means they release their growth factors out of the cell into the local environment. However, the number of growth factors packed into each platelet varies widely. For example, this GF content usually drops as the patient ages and with gender (2).
In addition, the timing and pattern of the GF release depends on what’s in the local microenvironment. Meaning unlike a pill dissolving in water, which GFs the platelet releases and when depends on what the platelet senses. For example, just by varying the levels of calcium and thrombin in the mix, one author was able to get widely different GFs to degranulate at different times (3). So to continue our pill analogy, platelets are “smart pills”.
Cellular Response to Platelet GFs Varies Widely with Age
We’ve been studying how different cells react to platelets for more than a decade. Remember that the GFs in platelets can act like espresso shots for the local cells. Hence, more GFs coming out of more platelets can get cells to grow more quickly (proliferate) or get cells with a specific job (like tenocytes) to repair a gap faster. However, these espresso shot effects of platelets tend to max out at lower doses with younger cells while older cells have a direct dose-response relationship all the way up to 20-40X (4). Meaning that if you double the platelet concentration (2X PRP) and compare how cells react to that compared to a 5X PRP, young cells won’t show much difference. However, do the same experiment with older cells and they will show much more proliferation with the 5X over the 2X.
Why does this happen? My thought is that it’s related to receptors. Remember that GFs from platelets must bind to receptors on the target cell to stimulate a response. The body is good at altering the density of those receptors on the cell based on what’s needed. Meaning, place cells in a GF rich environment like a young body and the cell will reduce the number of receptors on its surface. The opposite happens when the cell is placed in a GF poor environment (like an old body). The cells then ramp up the receptor density to take advantage of the few GFs floating around. So when we flood lots of GFs around receptor poor young cells, they can’t take advantage of the extra GFs as their few receptors fill up quickly. However, the greater number of receptors on old cells can take advantage of those higher GF concentrations.
Why Young Minds Were Blown
After giving this little talk in front of my fellows, I could tell their brains were now strewn all over the conference table. Meaning, they believed that the PLRA system was all that you needed to quantify and classify PRP. While it’s a great start, they could see that it wasn’t close to telling the whole story about PRP from a research standpoint. So what’s needed?
- A device that can accurately count platelet doublets and triplets in PRP as two and three platelets. With advances in AI, we will get there.
- A device that can determine the GF content of the platelets.
- Measurements of the microenvironment where platelets are being introduced.
- A recognition that younger people will respond differently to PRP dosing than older people. Perhaps a measurement of receptor density on cells?
The upshot? As you can see, we still have much to learn to more accurately dose PRP. While PLRA is a great start, it’s not close to quantifying and classifying everything about PRP. However, I have faith that the next generation of regenerative medicine researchers (as represented by those mind blown fellows) will solve these problems. Hopefully, by then, I’ll be sipping a rum drink on some beach somewhere!
(1) Mautner, Kenneth & Malanga, Gerard & Shiple, Brian & Ibrahim, Victor & Sampson, Steven & Bowen, Jay. (2015). A Call for a Standard Classification System for Future Biologic Research: The Rationale for New PRP Nomenclature. PM&R. 7. 10.1016/j.pmrj.2015.02.005.
(2) Evanson R, et al. Gender and Age Differences in Growth Factor Concentrations
From Platelet-Rich Plasma in Adults. MILITARY MEDICINE, 179, 7:799, 2014. Link to artcile.
(3) Martineau, I & Lacoste, E & Gagnon, G. (2004). Effects of calcium and thrombin on growth factor release from platelet concentrates: Kinetics and regulation of endothelial cell proliferation. Biomaterials. 25. 4489-502. 10.1016/j.biomaterials.2003.11.013.
(4) D. R. Berger, C. J. Centeno, and N. J. Steinmetz. Platelet lysates from aged donors promote human tenocyte proliferation and migration in a concentration-dependent manner. Bone & Joint Research 2019 8:1, 32-40. Link to artcile.