Topics Discussed Include the Following…

*The Most Common Criticism of PRP-Related Research
*Proposed methods of classifying PRP
*Should you or should you not activate the PRP with Calcium?
*What exactly happens when you inject PRP?
*Exercise pre PRP
*Why activate sometimes and sometimes not?

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Relevant Research, Transcript, Relevant Links

Relevant Research

Hamilton, Bruce, Johannes L. Tol, Wade Knez, and Hakim Chalabi. “Exercise and the Platelet Activator Calcium Chloride Both Influence the Growth Factor Content of Platelet-Rich Plasma (PRP): Overlooked Biochemical Factors That Could Influence PRP Treatment.” British Journal of Sports Medicine 49, no. 14 (July 1, 2015): 957–60.

Sheean, Andrew J., Adam W. Anz, and James P. Bradley. “Platelet-Rich Plasma: Fundamentals and Clinical Applications.” Arthroscopy: The Journal of Arthroscopic & Related Surgery 37, no. 9 (September 2021): 2732–34.

Toyoda, Toshihisa, Kazushige Isobe, Tetsuhiro Tsujino, Yasuo Koyata, Fumitaka Ohyagi, Taisuke Watanabe, Masayuki Nakamura, et al. “Direct Activation of Platelets by Addition of CaCl2 Leads Coagulation of Platelet-Rich Plasma.” International Journal of Implant Dentistry 4 (August 1, 2018): 23.

Ulasli, Alper Murat, Gokhan Tuna Ozturk, Bagdagul Cakir, Gulsemin Erturk Celik, and Fatih Bakir. “The Effect of the Anticoagulant on the Cellular Composition and Growth Factor Content of Platelet-Rich Plasma.” Cell and Tissue Banking, August 28, 2021.


Thank you guys for being here. I know there are many smart, very smart people—not just smart, but well-informed people regarding tonight’s subject. And so I promise you, I don’t claim to be the know-it-all, and I don’t claim to be able to give you a definite black and white answer. For example, we know two plus two equals four in this universe. I can’t give you an answer that definite concerning tonight’s subject (activation of PRP), but hopefully I can show you some of the ideas that have guided my thinking over the past decade of doing and teaching procedures with PRP. And hopefully, you can integrate those ideas into your practice, watch what happens, and then teach me something better.

If you have comments, I will definitely keep an open mic at the end of my presentation, which should take only about 15 minutes, showing you my favorite ideas in the literature, and then I hope you’ll jump in and tell me what you’re thinking.

All of the research with PRP is definitely growing logarithmically.

You can see here’s the research articles by year with platelet-rich plasma. Of course, we’re not through with 2021 yet. And all of the articles, without exception, where there’s a med analysis and all of the primary research with clinical trials, they all end in the same way in that we don’t have a good apples-to-apples comparison, a good definition of what platelet-rich plasma is.

The Most Common Criticism of PRP-Related Research

Every week when I get ready for this little webinar, I have dozens of new articles to look through to try to see what could be most helpful to our group, and all the articles have that criticism, which is justified. And you can see there’s talk, there’s always talk on the basic science level about how we make the platelet-rich plasma.

You can see there are people talking about making it with different devices.

So one of my favorite things I’ll show you right off the bat is this little infographic. Actually, I can give you the link before we shut down the webinar tonight, but this is a little infographic that was done by some guys who reviewed the research concerning orthopedics.

I don’t know if you’ve tried this yet. But next time you’re in a room full of dentists, ask them about platelet-rich plasma and everyone in the room will know what it is. And most of them will either have a centrifuge or one of their partners has a centrifuge, and they make use of it in their practice.

All the orthopedic surgeons will know what it is. But even a decade after introducing the O-Shot® procedure and the Priapus Shot® procedure, it’s still difficult to find urologists and gynecologists who understand exactly what the tool (PRP) is, much less the variables involved in preparing and using it.

Proposed methods of classifying PRP

So a lot of what I’m looking at each week in the way of new research, I’m pulling from the dentistry literature and the orthopedic literature. And so these guys have proposed ways to classify PRP.

  1. Is it leukocyte-poor or leukocyte-rich?
  2. What’s the concentration?
  3. How did you activate it?
  4. Endogenous versus exogenous?

And then, there you go. So their proposed PAW classification.

Now, I’ve always leaned towards activating it. Part of that is I was first introduced to the Selphyl kit, which as far as I know is still the only kit that actually comes with calcium chloride to activate the plasma before you… Well, to activate it in the syringe before you inject it.

Now, there’s a couple of things to note here that are not scientific that have to do more with just the much needed but also needed to be understood restrictions by the FDA. So for example, a kit other than Sephyl® was brought to my office by the salesperson who swore to me all about how the kit was self-contained.

And I suggested that I would like to activate the PRP with calcium chloride. He agreed that there was research to back up that idea and promptly went to his vehicle and came back with a calcium chloride vile, but he was not able to talk about that. He was not able to speak about the calcium chloride until I brought it up because it was off label for his centrifuge.

So just understand that when you’re speaking with a salesperson, most of these people are wonderfully ethical people who understand their kit, but they also are bound by the law to be careful what they can even say, and rightly so. So that limits the way the science is discussed by the people who are often educating the clinician salesperson for a particular kit.

Should you or should you not activate the PRP with Calcium?

So knowing that, let’s look at some of the science that’s been influential for me. Let’s see, I’ll just put this DOI link right now in the chatbox, so you can pull it up and have it before the meeting shuts down, so there you go. If you click on that, it’s in the chatbox. You’ll have this article. It will also be posted on the membership site later. But I liked this article for a couple of reasons.

One, is if you just suffer through the introduction, much of it you’ll already know, but having it put together, I think it’s the best display of the question, which is, “should you activate or not?”

And this particular study, they brought into the picture, how is exercise affecting it (PRP)? But the way they pose the study is worth just going through word for word, at least the part I’ve highlighted here.

So in this introduction, they say, “A recent…” Just bear with me because I want you to hear this word for word. “A recent med analysis on the efficacy of the widely-used autologous platelet-rich plasma, 14 musculoskeletal indications showed conflicting evidence.”

Okay. So this is really ubiquitous. All of the med analysis show conflicting evidence. And so the point they’re making though is, they think predominantly because of the shortcomings in the standardization of the study protocols and the confounding factors affecting the platelet application. So there’s so many variables. If you go back to this, if we’re all studying, say, how to inject the knee and you have leukocyte-poor versus leukocyte-rich, at least three different common ways of activating it, and then you have different concentrations of blood. Then even in the same patient, you can see that there’s multiple ways you could create your plasma and get different results.

So and extracellular elements, optimal concentrations of platelets, leukocytes, release growth factors, dose, timing, and activation: all of those are variables every time we do a procedure.

What exactly happens when you inject PRP?

So platelet-derived growth factors, PGF, are stored in alpha granules found within platelets. So even the people in our group, I think this should be in your bone marrow if you’re injecting PRP because this is about to tell you very clearly what it is you’re doing. Are stored in alpha granules found in platelets and are released in a selective manner upon activation. So it’s got to be something activated, whether it’s after you inject it or before is up for debate.

But until it’s activated, you’re not doing anything.

It’s why your plasma can sit at room temperature, in theory, in a container for three or four hours before you actually use it because it’s not activated.

Platelets, let’s see. Platelet activation is dependent on specific platelet membrane, glycoproteins binding to ligands, kinase activation and cytoplasmic calcium influx from both the dense tubular system and the extracellular milieu and may be initiated in vivo, here we go, by a range of factors including thrombin, calcium, collagen and shear stress.

Example of a variation in technique

Now, last week, or a couple of weeks ago, we reviewed a double-blind placebo control study done out of the University of Aristotle in Greece, where they injected the Corpus cavernosum with PRP. And in that study, their idea was to very slowly, over four or five minutes, per Corpus cavernosum, inject the PRP, obviously trying to prevent any activation at all until it was within the tissue. So that is one way of thinking versus activating it, which was my postulate before you inject it.

Who knows what’s the right thing? But I’m getting to my logic.

So what really happens?

Each platelet contains about 80 alpha granules, which, in addition to growth factors, contains adhesive proteins, the chemokines, fibrinolytic proteins and procoagulant molecules. In vitro calcium and thrombin are routinely utilized to induce growth factor release from European clinical practice. Pre activation is widely used; Regen makes a kit that includes thrombin, a way to make thrombin.

And they also make a kit that comes with an HA, non-cross-linked hyaluronic acid, which can activate PRP. Regen makes the kit that includes HA, we can’t get those yet in the US, as far as I know, I think they’re coming soon.

Selphyl® makes a kit that as I said, comes with a small aliquot of calcium chloride to add pre-injection.

However, evidence and consensus on the therapeutic requirement for pre-injection activation is lacking. That’s true, there’s no good consensus. So I’ll show you what my thinking is, and you can come up with your own consensus.

Exercise pre PRP

First thing of this study was to evaluate the effect of exercise on platelet rich plasma. You can see the list, basically all the stuff.

The second aim was to study the effect of the activating agent, calcium chloride on the growth factor, concentration-relation to different exercise states.

So they took these guys. If you look at the methods up here, they took the guys at 10 healthy men, and they had them do an hour of sub-maximal exercise. They drew the blood pre, post, and 18 hours afterwards, and then prepared the PRP and looked at it in vitro.

In this study, the exercise actually decreased the VEGF, which we’re very interested in, right? We want new blood flow, neovascularization, which the VEGF helps with. And that seemed to go down with exercise, which is conflicting with other studies. But then the calcium chloride activated the growth factors, but there was a different spectrum… And you can see up here, “activated with calcium chloride results, significant increase of PDGF and IGF-1.

There wasn’t a big change in the VEGF with the calcium chloride. So what does all that mean?

I think the next one will help put it together. So in this study, which came out of the dental literature… So just keep that in mind,

the calcium chloride did increase some of the growth factors and it changed the spectrum, there are other studies that show that same idea.

For example, when you activate with HA you get an increase in the VEGF.

What they’re leading up to is there could come a time to a part of the protocol for each procedure, whether it’s the knee versus a P-shot® versus the face.

We could be more specific about which growth factors we want and therefore change how we’re activating.

Based upon that idea, what I’m doing thus far is more simple than that, and it’s explained by this. The first idea that I get from this is that there is an increase in the amount of growth factors, reliably, the amount of growth factors that are released by adding the calcium chloride. At least in vitro, you’re going to reliably get an increase.

The question is, is that going to be more or more effective if that’s done in your syringe pre-injection versus letting it happen after you inject it?

What I just showed you is what the research talks about. What I’m about to tell you is my opinion.

My opinion is that the calcium chloride is literally a few drops and it assures me that those platelets have been activated rather than hoping they are activated to that full extent by my injection, through the sheer forces and the exposure to the collagen through the needle, where to me that feels less reliable.

Okay. Now the next part though, I think tells you another reason why I like activating with calcium chloride, and then I’ll tell you why. Even though I like it, I don’t always do it. I’m not going to make you suffer, you can read all this yourself, but I want to point out a couple of things. And again, this is from the dental research and the dentists were doing this at least 10 years before it was ever really used much cosmetically.

There were a few pioneers who were doing it, some of them in our group, but most were not thinking about it cosmetically until 10 years after the dentist thought about it. So let’s just, a little bit of the background because it helps us understand. Unlike sir Claude, who played a rich fibrin, because I get this question a lot,..

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“should we use PRF versus PRP?”

Well, the dentists use PRF quite a bit because it makes a gel and you can pack a wound with it for better handling efficiency and minimizing loss of growth factors to diffusion. PRP and some other derivatives in liquid form are usually clotted by addition of exogenous coagulation factors, such as thrombin and or calcium chloride. In other words, if you’re not going to make PRF if you want it to stay where you put it, and according to these authors, maybe you should think about either thrombin or calcium chloride.

The reason I don’t usually go for the PRF is I feel like there’s less research to back it up. And the handling of it feels less consistent to me because what we’re doing is through a needle, not packing a wound. And so venipuncture is performed with anticoagulants.

This is back to some of the science and the sodium citrate or acid citrate dextrose, ACD solution, it coagulates the calcium, that’s why it doesn’t form a clot. So adding the calcium back is undoing the mechanism of the anticoagulant in the two, in which you drew the blood.

So that’s worth thinking about, it’s logical but it’s maybe something I hadn’t really thought about specifically until I read this. So behind the clot formation, there’s an intrinsic coagulation pathway, and this takes you back to your basic science. You guys remember factor seven and all that, and calcium helps do all that.

So on a screen, there’s a chart down here. I think that shows us better, but let’s skip down here in contrast to calcium. When an alternative coagulation factor say thrombin is added to the situated PRP, the resulting fiber and fodder fibers are thin and often fuse together. In this study, we attempted to associate platelets from the coagulation pathway and evaluate the possible direct action of calcium.

What these guys did is then this was in vitro. They looked at what sort of structure was being built using electron microscopy. Basically when you put in what their conclusion was, if this were… If we were building a house, the calcium chloride provides more strong steel rebar to support the structure of the platelet-rich fibrin matrix than does thrombin.

This is the diagram I was looking for. So here’s the thrombin cascade that we’re talking about, where the glass surface activates it. And here’s calcium chloride which just does it for you.

Why activate sometimes and sometimes not?

So why do I use it sometimes and not others? If I’m doing an O-Shot® where I’m trying to create a firm, basically a liquid lift or liquid sling that changes the orientation of the urethra and helps with incontinence or creates healthier tissue, and I want it to stay in that place. I like adding the calcium chloride, so I get a stronger structure or a stronger platelet, rich fibrin matrix that happens more quickly. I get a clot and it stays where I want it to be more reliably, I think. And I think these two studies or these two descriptions, maybe they’re better just talked about as descriptors of the behavior of platelets. Back up that idea that you get a more reliable structure.

So how could that change how those procedures are done? If you look at the study, I mentioned earlier, their choice was to use a constrictor band and slowly inject over 4 minutes per Corpus cavernosum without activating —they were attempting to not activate the platelets; but then keep a constrictor band in place for 20 minutes.

During which time you’d get some mild hypoxia, which would cause some activation. In theory, you would get some activation and staying of the growth factors within the penis.

The downside to that as we discussed is that you’re avoiding the half of the Corpus cavernosum that lies deep to the base of the penis, looking from the outside or the pubic symphysis pubis there’s… Goes back along the pubic rami like with the female, so all that’s not treated. And we know by treating the face that by injecting the plasma, you can see it doesn’t go flat.

It stays expanded because you’re forming a matrix in there, which perhaps would change the way some of the urologists and gynecologists think about this PRP if they could see the behavior in the face. You don’t use a constrictor band to keep it in the face when you inject it.

So my preference is to activate it: One, because I know I’m reliably getting all the platelets to release their alpha granules; two, because I feel like I’m more reliably getting a fibrin matrix; and I prefer the PRP over a PRF because again, I think it’s more reliable and I can handle it more accurately through a needle.

I like to always use the calcium and the O-Shot® and the P-shot® because I’m trying to get the material to stay in a specific place.

I don’t always use CaCl (although many of our providers do) with hair and with the face because adding the calcium, whether it’s chloride or gluconate, and the gluconate does hurt less and works as well.

But adding the calcium can increase the pain in the face and the scalp, Also, in the face and scalp I want it to spread. I want it to spread more throughout the scalp and throughout the face where more ubiquitous covering of that tissue.

Wherewith the P-shot® and the O-Shot®, I’m trying to keep it more local.

When treating the breast, when treating loss of sensation in the nipple, I also like to use calcium for the same reason: so it stays right under the areola; but if I’m just injecting the fatty tissue of the breast, I don’t use calcium because I want it to spread.

Now, all that could be wrong, but that’s the way I think about it. If you look at the research and the references, then I think I’m that backed up by that. But I started this by telling you, “I can’t give you a definite right.”

And as was pointed out, you can find lots of research about PRF. And there are many people that are going to jump up and down and say that everything I just said was wrong, which is okay. What really needs to happen is we do research where we do, a hundred P-shots®, one way and 100 P-shots® in another way. And that’s my thinking, so let’s see…

What else do you guys have questions or comments? I’m happy to unmute anybody’s microphone if you want to talk about it…(silence) You just click the little button, I’ll unmute you and you can tell me where I’m wrong or where I need to be educated.

The problem with teaching something for 10 years is you start to believe everything you’re saying, which obviously… Everything I’m saying will eventually proven to be, there’s a better way, but this is my best understanding at the moment.

The other thing I’ve noticed is that when someone tells me they’re consistently not getting good results, not so much with hair, I don’t hear it or with the face, but especially with the O-Shot®, oftentimes all I have to do is have them start activating the PRP and they’ll start seeing better results.

Let’s see, I’m looking at some of the questions…(silence) When it comes to who I’ll treat and who I won’t treat it. The guiding principle I use is, could this person have surgery? If they would not be a candidate for surgery, let’s say high dose corticosteroids, or even if really heavy smoker, you can make an argument there. They’re not going to respond as well. If they have hepatitis C or HIV, absolutely, I would treat them, if they have just like… I would do surgery on them and have many times in the ER. If they’re on anticoagulants, I’ll still treat them… You can still have an IM injection if you’re on anticoagulants, but I’ll just warn them, they may have more bruising. And let’s see, but if they’re on other anti-inflammatory agents or smokers. Just like for the same reason, you have more poor wound healing if you have facial plastic surgery, our procedures don’t work as well.

For a while, I wouldn’t do them at all. Now I will do them, but always with a disclaimer that their results may not be as noticeable. And I think with that, I’ll shut it down, I’m always honored that you guys have an interest in what we’re noticing. Again, I’m bringing to you… Not just, as I mentioned, not just my ideas, but what I’ve gathered from our 4,000 plus members. And indirectly, like I said, I’ve often had probably a dozen times, had someone who said, “Hmm, my O-shots® are just not working.” And when I found out they weren’t using calcium and they started adding that in, they became more consistent.

The other thing is, the question comes up as it’s still minimal manipulation if you add the calcium chloride. And yeah, we actually had one of the head people who, the FDA on a lecture that was on a different webinar, and that question was asked. They do consider adding calcium chloride it’ll still be minimal manipulation because it’s still autologous. It’s still homologous because, in a person’s body, the platelets are activated in a similar way. So you’re not doing something the body wouldn’t normally do with the platelets. Okay. I think with that, we’ll shut it down. Thank you, guys. I hope you have a good night and I will put links to all this in the email that goes out and on membership sites. Bye-bye.

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Charles Runels, MD






Cellular Medicine Association


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