Investigating Platelet Rich Plasma

By Dr Sarah Tonks / 01 Mar 2014

Dr Sarah Tonks discusses what you need to know when selecting a PRP method for your practice

The use of Platelet Rich Plasma (PRP) in aesthetics is relatively recent but has gained considerable publicity due to its adoption by celebrities such as Kim Kardashian and Bar Refaeli, who were both depicted with their faces smeared with whole blood rather than PRP, resulting in a dramatic effect. However, PRP has been used clinically in humans since the 1970s in opthalmic surgery, orthopaedics and sports medicine.

Much of the interest around PRP has come from the idea that this is an autologous substance and therefore considered to be ‘safer’ than traditional dermal fillers and botulinum toxins. WhatClinic.com reported an increase in enquiries regarding the procedure of 807% over the previous year in 2013. Although PRP is now offered by many clinics and there are numerous systems on the market, some uncertainty remains over its efficacy and proposed mode of action. Some have expressed doubts as to whether this truly represents evidence- based medicine due to the lack of robust clinical trials and outcomes. Indeed a recent Cochrane review in 2012 stated that at the moment there was insufficient evidence to support the use of PRP.1 However, at a microscopic level in a study of PRP treated fibroblasts, the PRP treated groups showed more proliferation and differentiation of fibroblasts into myofibroblasts which are essential for wound healing and increased contraction of the wound during healing time.2 PRP stimulates endothelial cells near their application site and favours the proliferation and formation of new capilliaries.3 Doctors report a high satisfaction and return rate from patients.

What is PRP?

Generation of PRP involves centrifugation of autologous blood to separate the plasma and buffy coat portion of the blood, which contains high levels of platelets. Most people have a baseline blood platelet count of 200,000 (+/- 75,000) and although the ideal concentration of PRP is at the moment unclear, most PRP systems produce a substance containing concentrations of growth factors that are three- five times that in normal plasma. A count of 1,000,000/?L or 338% more than the normal total blood platelet count has been proposed as being the ideal.4 Studies have shown that PRP with a low platelet concentration does not work and too high concentrations have an inhibitory effect on cell growth. The concentration of PRP for bone regeneration was found to be best around 1,000,000/ ?L.5

Left: Before; Right: After
A separate study found that higher leukocyte content increases inflammation and reduces tissue regeneration so it is important to exclude as many of these cells as possible from the PRP.6 Platelet Poor Plasma (PPP) is blood plasma with a very low number of platelets, usually <10,000. This can be found in the top fraction of the centrifuged blood. PPP is still a useful fraction of blood: it has been shown that both PRP and PPP, when activated with calcium and thrombin, can induce proliferation of dermal fibroblasts.7

The Growth Factors

PRP contains high concentrations of growth factors and more than 800 different proteins and it appears that the growth factors work together synergistically.8 Growth factor secretion begins 10 minutes after clotting with more than 95% of pre-synthesised growth factors secreted within an hour, however platelets continue to synthesise growth factors for at least seven days.9 Ideally the PRP must be used on the application site 10 minutes after activation to harness the growth factors. After the death of the platelet, the macrophage takes over wound healing by secreting some of the same growth factors.4 

PRGF and TGF- ß1

IMPORTANT COMPONENTS OF PRP

  • Three isomers of platelet derived growth factors PDGF PDGF-? ?, PDGF- ? ? and PDGF- ? ? 
  • Vascular endothelial growth factor VEGF
  • Two of the transforming growth factors TGF- ?1, TGF- ?2
  • Epithelial growth factor EGF 


The major effects of PRP are derived from PRGF and TGF- ?1, which are concentrated in the alpha granule of the platelet and released during platelet activation. Both PRGF and TGF- ?1 stimulate cell proliferation and differentiation resulting in tissue formation.10

Could it be dangerous?

Although no undesirable effects have been reported, hypotheses exist as to the over expression of growth factors and their receptors related to tumour formation and dysplastic tissue. However it is thought that the circumstances leading to neoplasm growth require more continuous doses of growth factors over time than those in PRP, which degrade in seven-ten days.11 Nonetheless, the use of PRP should be avoided in patients with pre-cancerous lesions, in areas of epithelial dysplasia and those with a history of exposure to carcinogens.12

Types of kits

Several commercially available methods to obtain PRP are currently used in the clinical setting and there are many kits, centrifuges and vials available. The centrifugation process should be sterile and suited to platelet separation without lysing or damaging the platelets. Not all systems have been created to produce sufficiently viable platelet and this has led to criticism regarding the efficacy of PRP. When anti-coagulated blood is centrifuged, three layers form due to the differing densities of the blood components; the bottom layer consists of red blood cells, the middle of platelets and white blood cells, and the top plasma layer.3 It is important to reduce platelet fragmentation during centrifugation. Integrity of the membrane can be preserved by the use of acid citrate dextrose type A anticoagulant and low gravity forces during centrifugation.4 Dr Daniel Sister, anti-ageing and hormone specialist at BeautyWorksWest and pioneer of Dracula therapy, speaks on considerations when applying PRP. “The type of harvesting kit, centrifugation methods and time play a crucial role,” he says. “Plasma is autologous, so most new systems now come without the gel-like separation, because plasma is not totally hermetical and filaments of the gel get mixed with the plasma. All systems must be closed.” “There is also the cost per ml of active plasma to consider,” he says. “The new systems harvest 20cc in one kit and are more efficient, have better ration of growth factors and are cheaper than previous systems.” In the UK the most widely available kits available include Angel, Regen, Tropocells, BTI Technologies, Dracula and Selphyl.

Left: Before; Right: After
The Angel system is unique amongst the described systems in that the outcome is customisable. The haematocrit level can be adjusted to the desired percentage (usually 2-7% in aesthetics), which will determine the amount of white blood cells in the PRP. In order to activate the platelets the most commercially used method is to add thrombin or calcium to the PRP, although it can be used without activation as in vitro the platelets are activated by contact with collagen. Addition of calcium replenishes that which was bound by the acid citrate dextrose type A anticoagulant. Previously, bovine thrombin was used as an activator which was associated with the risk of life threatening coagulopathies via immunologic problems and factor V deficiencies; however the adverse reactions reported were related to the source and quantity of the thrombin used.13 The addition of calcium is now considered to be a safer option.2



ANGELREGENSELPHYLTROPOCELLSBTIDRACUT SKIN REJUVENATION
Amount of blood taken50ml8ml18-36ml10ml or 30ml54ml20ml
Method of taking bloodNeedle and syringe. Has a specific kit with ACDA anticoagulantButterfly needle and vacutainerClosed systemButterfly set for 10ml blood draw. Needle and syringe could be used for 30ml blood drawButterfly cannula with vacutainer vialsNeedle and syringe
Sterile blood tubes?No tubes. Uses dedicated sterile hospital quality setYesYesYesYes, endotoxin freeYes
Size of tubesN/A8ml10ml15ml or 50ml9ml20ml
Amount of PRP obtained6-10ml4-5ml9-18ml10ml blood draw = 6-2ml dedpending on platelet conc needed. 30ml blood draw = 18-6ml conc platelets24ml dependent on haemocritMinimum 6ml
Centrifuge time17mins5mins6mins10mins8mins8mins
Centrifuge speedFirst cycle: 3700rpm

Second cycle: 2700rpm

3200rpmFirst cycle: 3800rpm

Second cycle (optional): 1800rpm

1500 RCF (or g) Only one cycle580g1800
Spinning angle of centrifugeN/A45°90°90° preferred or 45°n/a under patent30°
Activation substanceNone- self activating when injected with small lumen needleCalcium gluconate (optional)Calcium chlorideNone, activation in situCalcium chlorideCalcium gluconate optional
Number of treatments3 treatments, 4-6 weeks apart and then every 6-18 months3 treatments every 4 weeks apart and then every 6 months3 treatments every 6 weeks3 treatments every 4-6 weeks and then every 6-18 months3 treatments at 3-4 weeks apart for 3 months, top up every 6-12 monthsTwice a year
Method of separating PRPLED cellular photospectometry and fractionationThixotropic gelMechanical with sodium citrateCycloaliphatic polymer inert gel, 100% biocompatibleManually using a plasma transfer deviceMechanical filter


How is it used?

The indications for PRP are numerous. There is evidence for its use to diminish dark circles around the eyes, hair growth and cutaneous regeneration.14,15 PRP is most often performed as a superficial treatment. It can be used intradermally with a 30g needle, in a linear thread, cross-hatching or using the fan technique. It can be used like mesotherapy, in a micro injection or micro papular technique with a 32g needle. Additionally, it is being used supraperiosteally to assist with slowing of bone remodelling and ageing, although at present there is no strong evidence base for this.16
PRP is being used in combination with fat to improve the survival of grafted fat with good results.17 

Conclusions

Standardisation of PRP preparations is urgently needed to best compare systems. General opinion is that the majority of clinical studies do not have the statistical power to give conclusive results. Human trials do not take into account whether the platelets have been effectively concentrated, whether the PPP is discarded or not, whether early activation occurred or whether there were purification problems. Regarding skin rejuvenation, larger scale studies and randomised controlled trials which would decisively say which methods are most effective have not yet been performed. However, clinical experience suggests that in general, PRP, for skin rejuvenation and other aesthetic indications such as hair loss, can be a useful and attractive treatment. 

Mr Dennis Wolf,

Surgeon, The Private Clinic

I use the Tropocells kit as it has a few basic components; it is efficient and reliably provides concentrated PRP (four-five times the usual amount from 10ml venous blood). The gel plug separates the PRP from the erythrocyte and granulocyte content, which is thought to have a catabolic effect by releasing metalloproteinases. The filter sleeve prevents any contamination. Depending on what areas the patient would like treated Iaspirate10ml or 20ml venous blood.
After centrifugation at 1500g for 10 minutes I can remove some PPP thereby increasing the concentration of the platelets in the remaining plasma. My protocol for facial rejuvenation consists of treating the peri-ocular region, malar region, temple, peri-oral region and nasolabial folds. Peri-ocular and temple region I treat very superficially. The malar region I treat superficial and deep. The peri-oral region I treat superficially and the nasolabial folds deep. For the hands and face I use a 2:1 ratio of fat to PRP.

Dr Adam Thorne,
Cosmetic dentist, Harley Street Dental Group - BTI PRP system

We’ve been using PRP techniques at Harley Street Dental Studio for some time now. We use it for faster wound healing, when placing implants or to regenerate bone following tooth extraction, around implants, in bony defects, bone graft placement or after extraction of cysts. A subfamily of TGF is bone morphogenic protein (BMP), which has been shown to induce the formation of new bone. When added to the site with bone substitute particles it allows us to grow bone more predictably and faster than before.

Dr Terry Loong,

Aesthetic doctor, The Skin Energy Clinic - Regen PRP system

Having used other PRP systems, I love the Regenlab system. It has a red ATS (autologous thrombin serum) which allows thrombin to be extracted from the blood, mixing it with the liquid PRP, activating the coagulation cascade, creating a gel-like substance which provides a matrix when injected, slowly releasing the growth factors. This provides more controlled and targeted delivery. The gel-like matrix acts as a natural alternative to filler, perfect for treatment under the eyes, fine lines and wrinkles, and as skin boosters.

Dr Rita Rakus,

Aesthetic doctor, Dr Rita Rakus Clinic - Angel PRP system

The Angel Lift is a highly effective treatment; it is a two-step procedure that combines PRP with a fractional laser system. Targeting the signs of ageing inside and out, The Angel Lift reduces fine lines, wrinkles and blemishes on the skin surface as well as addressing the structure of the layers beneath to produce visible natural results. With medical grade credentials, the Angel PRP machine offers optimum performance, ensuring it delivers the best possible results.” 

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