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Platelet Rich Plasma

forum2Platelet Rich Plasma

An expert panel aims to demystify the often perplexing topic of platelet-rich plasma therapy systems, and offers their recommendations for obtaining optimal results

 

Tropocells

Dr Dennis Wolf: The Tropocells kit comes with a 15ml blood collection tube that draws 10ml blood. The tube contains a precision measured anticoagulant and a separating gel. The anticoagulant is pH balanced making it more comfortable for the patients.

A butterfly blood taking set is used to aspirate and perform the venipuncture.

Once we have drawn the blood into the tube, gently agitate it to mix the anticoagulant with the blood and then insert it into the centrifuge. A RCF of 1500g is required for 10 minutes.

After centrifuging, the sample will have separated into three distinct layers—red blood cells with granulocytes at the bottom; a gel layer and the plasma. At this stage, the platelets are sitting on the gel leaving an increasingly platelet poor plasma higher in the tube.

Some of the platelet-poor plasma can be aspirated from the top of the tube to concentrate the platelets. After which, a gentle agitiation is created to resuspend the platelets that are on the gel.

Once resuspended, we then insert a filter funnel into the top of the tube. The plasma is pushed up through the membrane at the bottom of the filter funnel and into the internal part of the funnel.

We end up with platelet-rich plasma in the filter funnel which we can then extract through the cap of the filter funnel with a syringe. We can use the same 1ml syringes to extract the solution that we will use to inject into the patient.

I prefer to provide items such as syringes and needles from my clinic stock.  This PRP system provides only the parts unique to the system.

 

Dracula Therapy 

Dr Daniel Sister: The Dracula kit is different from many others—it still extracts platelet-rich plasma, but instead works like a cafetiére. Some systems have no separation between the red cells and the plasma, while others have a buffer that separates them. With a cafetiére, you push the plunger and the coffee goes through. Similarly, we push the plunger and the plasma goes through while the red cells are separated, so we end up with pure PRP and PPP. It’s very important to use the whole plasma, not just the PRP.

When drawing the blood, it should be taken as slowly as possible to ensure we don’t damage the platelets. There is no point in rushing to take the blood and when we harvest the plasma, it’s better to have system that takes it slowly than one which would create turbulence and destroy the platelets and growth factors.

When the solution is centrifuged at a specific, low speed for eight minutes, we have a separation of the plasma and the red cells. By pressing on the plunger, the red cells are pushed down and we have our plasma—20cc in one manipulation.

After that, we use a two-way connector to extract into a 1ml syringe if it’s easier to use or to mix it with hyaluronic acid. We get the plasma without any chemical separation and no need for human or bovine thrombin. I want the PRP to be totally autologous unless I want to mix it with hyaluronic acid.

 

Angel Lift System

Dr Kambiz Golchin: Rather than dealing with tubes, I like to use a fully-closed, automated system that does the job very simply. With the Angel system, we extract around 52ml of blood and push the plunger in, which then and it’s going to goes into a reservoir bag. The system then makes the PRP automatically, depending on the settings that we give it. We can choose the amount of red or white blood cells, and adjust the volume so we can generate less or more, depending on need.

The 2012 Cochrane Review stated that there is no clear evidence that PRP works. So why are we using it? Contrary to the Review, we know that there is evidence that it works. A systematic review in the Journal of Plastic & Aesthetic Surgery shows clearly that PRP works in fat grafts. There is some evidence in wound care for diabetic foot ulcers.

However, these are different indications and they need different formulas. So we can’t use the same PRP for all treatments and get great results; it just won’t work. PRP is not a filler, so we’re not going to get a filling effect from it.

As there are different indications, we have to be specific with formulations. Aesthetic indications require different settings, for example white cells do something different, as do platelets.

 

Selphyl 

Dr Hema Sundaram: The Selphyl system produces Platelet Rich Fibrin Matrix (PRFM), a unique type of PRP that has a fibrin scaffold from which there is gradual, controlled release of growth factors and cytokines. One of the important features is that the PRP is free of red blood cells—it’s pure yellow. If you’re looking at bringing PRP into your practice, look at two or three different systems and figure out what’s going to work for you and your patients. I just published a new peer-reviewed scientific paper. The Potential of Topical and Injectable Growth Factors and Cytokines for Skin Rejuvenation in the special issue of the journal, Facial Plastic Surgery that is in conjunction with the American Academy of Facial Plastic and Reconstructive Surgery  (AAFPRS) International Symposium. It provides an evidence-based update on PRP, with a comprehensive review of the available data and explanation of the different formulations.

With the Selphyl system, I’ve developed a two-layer technique to optimize results and minimize recovery time. I use a 27 gauge blunt cannula to inject PRFM into the superficial subdermal plane, for a nice, even distribution. This is non-traumatic to the skin. Patients who have a tendency to get swelling after HA filler injections often ask for PRFM. By making the procedure as non-traumatic as possible, as a general rule, my patients can walk out of my clinic with no bruises at all.

I then use a sharp 30 or 32 gauge needle for precise intradermal injection of PRFM. This dual plane injection technique achieves superficial volumising, with a subsequent skin boosting effect. It’s notable that the Selphyl system allows a full 10-minute window period between the formation of the PRFM and time of injection.

Injection of PRFM into the dermis can dramatically improve light reflectance of the skin, which is very helpful for dark circles under the eyes and to restore skin radiance. It isn’t just about volumising and generating collagen; to alter skin reflectance is a significant benefit.

 

Does PRP work?

Dr Fraser Duncan: PRP is not one single substance and the clinical evidence for its use is controversial. There are four main questions for anyone considering using PRP. The first is how pure is it and is it sterile? How much does it cost to set up and use? How easy is it to use? And does it work?

Dr Daniel Sister: In 1950, two doctors were awarded the Nobel Prize when they discovered a growth factor in plasma. In 1970, Marx proved that he helped to treat receding gums and peripheral bone structure and since then, more than 6,000 studies have been published. So there is hard evidence that PRP works. From my own experience, we are currently reviewing over 700 treatments I’ve performed and we have over 70% satisfaction rates.

When people say PRP is not a filler, I agree. It’s not a filler but because it helps to generate type 1 collagen, regenerate more vascularisation and helps all the cells to function, there is a filling effect—not as much as if you were to use a filler, but there is a filling effect. PRP is totally natural and autologous, so you can mix it with any other treatment. You can mix it with fractional laser, hyaluronic acid or with any other treatment to have a stronger or longer-lasting filling effect with fewer side-effects. It is definitely the most versatile and efficient treatment.

Dr Kambiz Golchin: I truly believe in PRP—I quoted the Cochrane Collaboration which looks at all the evidence that has been published. The National Institute for Health and Care Excellence have a report on the subject as well. The reason for all the confusion is because of the differences in PRPs.

What is the definition of PRP? There are differences in the definition—is it just superphysiological or does it have to be at least two-fold? There is no consensus on what the correct definition is. I agree that growth factors are key to the effect of PRP but growth factors are not directly related to platelet count.

Dr Alain Gondinet: We need to go back to the basics and to FDA approval. According to the FDA’s definition, when we use a medical device kit, we need to have a minimum of 250,000 platelets per microlitre and a minimum of 50% of living cells. The FDA also states that we have to use the PRP within four hours. We have to take the blood through the centrifuge and inject within a maximum of four hours—we are not haematologists or a blood bank.

The definition exists but there is currently no indication for PRP approved by the FDA. We wait for studies of PRP acting on the filling of nasio-labial folds, but it will come. We can’t be pessimists.

At RegenLab, we have a lot of studies in progress with the FDA and currently carrying out the 510(k); the file for the FDA approval. We are also starting to do the ATS-2 for thrombin. It’s not a filler—it’s “filler-like”. In regards to thrombin, we create a gel, but we don’t want to fill with the gel. We already have a filler, such as HA. But we do want to regenerate.

Dr Fraser Duncan: We have many different PRP systems but no specific evidential base for its use in aesthetics. There are almost as many peer-reviewed evidential papers that show PRP does not work as there are to say PRP does work

Dr Hema Sundaram: I couldn’t agree with that more. From an evidence-based perspective, there are evidence gaps with all PRP technologies. It’s very important to understand evidence levels and anything in vitro is evidence level five. We have to then connect the dots through to clinical outcomes. It’s an incredibly intriguing field that we all should be looking at getting into. But we shouldn’t fall in the trap of mistaking promotion and marketing for science.

 

Plasma purity

Dr Fraser Duncan: I’m a huge a fan of PRP and am using it extensively. However, I’m slightly concerned that we may potentially be extending its use into aesthetics too quickly. We could be killing that golden solution that may lay a beautifully golden egg for us.

Posing a question to the panel; how pure is the plasma fraction that these systems are producing in terms of erythrocytes, leucocytes and any substance which is likely to be allergenic?

Dr Alain Gondinet: With the ReGen system we don’t add anything to the PRP; we only have the blood. We don’t add calcium. We add the calcium when we carry out alternative wound care with topical application when we want to produce coagulum for stomalogy or when we want to produce membranes for orthopaedia. But for aesthetic applications, it’s better to avoid using calcium, particularly calcium chloride. It’s better to use calcium gluconate. We also have between 80–90% of living cells.

Dr Hema Sundaram: In my scientific paper, I’ve reviewed data from studies showing that there is significant variability between different batches of PRP produced by the same automated system, and even between two blood draws from the same patient. Purity may be defined as the percentage of the PRP that comprises platelets. With the Selphyl system, there is 74% purity—the platelet component is 74%. There are no erythrocytes because we don’t want haemosiderin, and there are no known external allergens.

In my opinion, there isn’t enough evidence to say what kind of calcium is advantageous or not. We need to wait until we have peer-reviewed publications, not promotional studies from companies, so the jury is still out.

Dr Kambiz Golchin: As the Angel is a fully-automated system, there are optical sensors which can detect the platelets, red cells and the plasma. This gives very accurate and consistent results.

Dr Fraser Duncan: In clinical practice, we give both calcium chloride and calcium gluconate in many patients for the same reason; to potentially increase their blood pressure in high infusion of blood. Both are completely safe in clinical practice.

Dr Xavier Abad: PRGF-Endoret protocol was tested by Harvard Medical School when we obtained FDA approval. Endoret doesn’t have red or white cells. Platelets have been activated before the treatment, so that’s the reason that we are using calcium. I don’t agree that the calcium chloride is more harmful than calcium gluconate—there are specific warnings about the use of calcium gluconate for some applications but no warnings about calcium chloride.

Dr Daniel Sister: The Dracula system is very simple. There is no chemical buffer so the solution is totally pure; all plasma. There are no red cells. Growth factors and around 37 cytokines in plasma work for bones, skin, hair regrowth and cartilage. The body is clever enough to take what it needs from the plasma to do what it wants.

Dr Fraser Duncan: There is some clinical evidence in the literature, that leukocytes in this situation may be catabolic. It may actually negate the anabolic effect from cytokines and other growth factors.

Dr Daniel Sister: I agree but with the Dracula system, when we push the plunger, we get the plasma and platelets, but no red cells, no leucocytes and no chemical buffer in the middle.

Dr Dennis Wolf: Tropocells gives us a yield of higher than 80% of functional viable platelets. I can also create a four to five-fold increase in platelet concentration. The gel locks all red blood cells and granulocytes beneath the gel layer so you don’t have any contamination—no release of metalloproteinases once injected as well, which obviously has a beneficial effect.

The filter also contributes to purity. So there is no risk of drawing up gel when you use a needle to extract the plasma.

This PRP system is used worldwide and in the UK for orthopaedics with published papers. It is also used for wound healing.  In this case, the PRP is activated with calcium chloride before topical application. There is published evidence on the benefits for wound healing.

Dr Hema Sundaram: I have discussed this in my scientific paper. There Is no consensus regarding leukocytes. Some researchers are concerned about neutrophils becuase they upregulate the activity of matrix metalloproteinases, which are collagenases.

 

Contamination

Dr Fraser Duncan: The one thing that would worry me is that apart from the potential for infection of taking blood—which is universal using aseptic technique—are your systems closed, semi-closed or open? Is infection a potential risk in a clinic situation rather than a hospital situation?

Dr Alain Gondinet: With the ReGen system, everything is closed so you have no risk of oxygen contamination or oxidation of the plasma. But, of course, if the tube is opened, there may be a risk.

Dr Hema Sundaram: The Selphyl system is closed and sterile in its entirety. The needle that is ensheathed with it is also part of the closed system and is sterile.

Dr Kambiz Golchin: When you talk about sterility in microbiological terms, there is a term called the sterility assurance level (SAL)—you can’t get absolute zero. For test tubes, the SAL is 1–1000. The Angel system is 1–1,000,000 which means that the system is actually cleared to be used in an operating room; for orthopaedics and for stem cells. You can use the system knowing that as it is a fully closed system, there is no multiple penetration into test tubes with different needles. The collection is just made into a sterile syringe and you can use it straight away.

Dr Xavier Abad: With Endoret technology, we perform the blood extraction in the closed system, then open the system for the fractioning. We tested this protocol with 150 patients at Harvard Medical School and more than 700,000 patients over 15 years. We also published two scientific papers when we have bacteriostatic potential of the plasma with the platelets, so we avoid any kind of contamination.

Dr Daniel Sister: When we move the blood from the centrifuge to the syringe with the Dracula system, it’s a closed process with no contamination.

Dr Dennis Wolf: Tropocells is a semi open system.  Blood is taken directly into the tube maintaining a closed environment and the tube is opened only for PPP removal and resuspension of platelets. The filter sleeve facilitates PRP complete harvest and keeps PRP in a closed environment. Importantly, each component of the system is packed in its own sterile pouch, maintaining sterility until used. Tropocells also has a closed system.

 

Calcium and thrombin

Audience member: How are thrombin and calcium used in forming PRP and PRFM?

Dr Fraser Duncan: In the US, there are more than 20 systems available and far fewer than that in UK. Globally, there are many systems which purport to give exactly the same substance, but they don’t. They do not produce the same PRP substance. The matrix in the Selphyl and the Endoret systems is activated using calcium. This forms the matrix—the suggestion is that within this matrix, the platelets are trapped so it’s an artificial clot which is injected and which sets. There’s also the potential of a thrombin clot which offers another substance. So PRP is not a uniform substance and can have as many variations as there are manufacturers of systems generating it.

Dr Alain Gondinet: However, it’s not as simple as that. In the blood we have fibrinogen. Fibrin needs the thrombin as a coagulating enzyme and the calcium is a coactivator of the thrombin. So, with the autologous thrombin, you activate the fibrinogen into fibrin and the fibrin will embed the cells and then you have the matrix.

Audience member: Where does the thrombin come from?

Dr Alain Gondinet: The thrombin comes from a tube without anticoagulant. When you centrifuge the blood and separate the red cells, you end up with a fibrin clot. When you destroy the clot, you have thrombin. You then mix the thrombin with the anticoagulant PRP, and obtain a gel. The thrombin is natural; autologous. There is therefore no risk of coagulation when we inject it.

Dr Xavier Abad: The release of growth factors when using thrombin is different to the release of growth factors when using calcium.

Dr Alain Gondinet: Absolutely. This is why PRFM is only fibrin and a small number of platelets and growth factors—the platelets are embedded and are not able to release the growth factors. So you have to centrifuge the anticoagulated blood first to release the growth factors and after that, you can activate with the calcium.

Audience member: Is calcium required or not?

Dr Kambiz Golchin: The reason we need the calcium is because we anticoagulate the blood so we don’t get a clot instantly. The calcium reverses the anticoagulation. So, yes, we do need calcium but it is not absolutely essential for different indications. We do not need to activate the platelets every time—thrombin and calcium aside, there are other things that activate platelets, such as damaged collagen. For example, when we’re mixing it with fat, we don’t need to actually activate it.

Dr Hema Sundaram: Calcium chloride is needed to make platelet-rich fibrin matrix which gives the controlled, sustained release of growth factors and cytokines. There are peer-reviewed scientific data on the Selphyl system showing clinical efficacy for wound healing and skin rejuvenation, such as papers by Sclafani and colleagues in respected plastic surgery journals. But we still need to keep studying the safety and efficacy of various PRP systems.

Dr Dennis Wolf: No additional chemicals are added to Tropocells and for aesthetic purposes, no activator.  Activation occurs naturally in the skin when used for skin rejuvenation. Platelets are viable for up to four hours after preparation.

Audience member: Do you use in vitro diagnostic tubes?

Dr Alain Gondinet: No. They are cheap but they are not produced for therapeutic injections. They are just produced for diagnostics; that’s it. If you do PRP with IVD, you will not be covered by your insurance.

Dr Kambiz Golchin: That’s absolutely correct. This is why the Angel system does not use tubes. It’s a disposal closed kit, so it’s as pure as you’re going to get.

Dr Xavier Abad: The reason is because of the presence of endotoxin. You have to have a very low level of endotoxin in these tubes.

Dr Alain Gondinet: You need to have pyrogen-free tubes by law.

Dr Dennis Wolf: The tubes are FDA approved for therapeutic injections, are non-coated and comply with regulatory law.

 

Improving results

Audience member: How would you look at your results in terms of improvement and client satisfaction?

Dr Alain Gondinet: Biology is biology. This means everybody will react differently because it depends on age, the renewal of the cells and the number of stem cells. Sometimes we have very nice results and sometimes we have less good results. Sometimes we have to repeat the session because when we inject living cells, they do what they want.

Dr Kambiz Golchin: Depending on how you look at your results, the results do look a bit “soft” if you’re just looking at PRP in aesthetic use on its own. But you do get a glow and an improvement in texture. The value of PRP is in combination treatments; the results are so much better.

Dr Xavier Abad: It’s very important not to give wrong expectations to the patients. This is not a standard filler. We know that we improve the quality of the skin and we have measured that. We have more hydration and a better pH. There are different parameters that we can measure but it’s not for changing the expression. It’s not Botox and it’s not a hyaluronic acid.

Dr Daniel Sister: Of the 700 treatments we’re currently reviewing for the London School of Medicine, the total number of patients is around 300. So some came for one treatment, some for two and others for more than two. Obviously, if they have come back for another treatment, it’s because they’ve seen a difference.

I have over 80% of our patients return without booking them another treatment straight away. I never tell patients they have to come back for a second or third treatment. I say, “If you like the results, come back and see me”.

Dr Dennis Wolf: In my aesthetic practice, I use PRP both on its own and as an adjunct. I use it in conjunction with laser, IPL, hyaluronic acids and toxins.

Dr Hema Sundaram: I agree with the use of PRP as an adjunct with synergistic potential. Again, we have to distinguish data from marketing. There is variability in patient response to PRP. Those who respond well are very happy.

References

1. Castillo TN, Pouliot MA, Kim HJ, Dragoo JL. Comparison of growth factor and platelet concentration from commercial platelet-rich plasma separation systems. Am J Sports Med. 2011;39(2):266–271

2. Sclafani AP, Saman M. Platelet-rich fibrin matrix for facial plastic surgery. Facial Plast Surg Clin North Am. 2012;20(2):177–186.

3. Sclafani AP. Platelet-rich fibrin matrix for improvement of deep nasolabial folds. J Cosmet Dermatol. 2010;9(1):66–71.

4. Fabi S, Sundaram H. The Potential of Topical and Injectable Growth Factors and Cytokines for Skin Rejuvenation. Facial Plast Surg. 2014;30:156–170. (AAFPRS International Symposium Special Issue).

5. Sundaram H, Carruthers J. Volumetric approach to the lower eyelid and midface. Clin Plast Surg. 2014; in press.

Author: bodylanguage

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