PRP- Introducing a

clinically successful

service to your practiceAndy Armitage BSc BVM&S MRCVS

What is Platelet rich plasma?

Autologous (self-derived)

conditioned plasma that contains

high concentrations of platelets

Platelets contain large quantities

of bioactive proteins and growth

factors

GF are contained in the alpha

granule portion of the cell

These are released when the

platelet is activated (usually at a

site of injury)

How does PRP work?

GF contained in platelets:

Initiate and accelerated muscle,

tendon, ligament and cartilage

repair and regeneration

Decrease inflammatory mediators

in OA

Reduce pain and improve articular

function in OA

Platelets exposed to an area of

damage or to fibrinogen become

activated.

Activation causes the platelet to

change shape so that the alpha

granule releases its GF’s

These GF play an essential role in

tissue repair and regeneration by

their ability to attract other cell

types to that area

Cellular migration in response to injury

Injury leads to increases in Hypoxia inducible factor (HIF1)

HIF1 increases levels of CXCL12 (or SDF-1 alpha, Stromal-derived factor 1 alpha)

CXCL12 is a massive chemokine drawing regenerative cells towards it

PRP introduces additional CXCL12, as well as other factors, into the region of damage

In addition to recruitment PRP enhances proliferation and differentiation of cells involved in tissue regeneration

Roles of other alpha granule growth

factors Transforming growth factor β (TGF-β)

Growth and regeneration of epithelial cells and vascular endothelial cells, promotion of wound healing

Platelet derived growth factor (PDGF-AB and BB)

Cell growth, generation and repair of blood vessels and collagen production

Insulin-like growth factor (IGF-1)

Stimulates cell growth and proliferation, promotes extracellular matrix formation, and a potent inhibitor of apoptosis

Vascular endothelial growth factor (VEGF)

Growth and generation of vascular endothelial cells and therefore promotes angiogenesis into the lesion.

Epidermal growth factor (EGF)

Promotion of epithelial cell growth, angiogenesis and promotion of wound healing

Fibroblast Growth Factor (FGF)

Tissue repair, cell growth, collagen production, hyaluronic acid production

PRP Matrix formation

In addition to growth factor

release platelets mediate

conversion of Fibrinogen to fibrin

Fibrin forms a matrix which can be

used by other cells as a scaffold

Activated platelets bind strongly to

damaged collagen

Crosslinked fibrin matrix forms

around bound activated platelets

This provides a scaffold for other

cell types such as stem cells

PRP research: Evidence based medicine

for tendon injuries

Rha DW, Park GY, Kim YK, Kim MT, Lee SC. Comparison of the therapeutic effects of ultrasound-guided platelet-rich plasma injection and dry needling in rotator cuff disease: a randomized controlled trial. Clin Rehabil. 2013 Feb;27(2):113-22. doi: 10.1177/0269215512448388. Epub 2012 Oct 3.

Ahmad Z, et al. Exploring the application of stem cells in tendon repair and regeneration. (2012) Arthroscopy. 28(7):1018-29.

Chen L, et al. Synergy of tendon stem cells and platelet-rich plasma in tendon healing. (2012) Journal of Orthopedic Research. 30(6): 991-7.

De Almeida AM, et al. Patellar tendon healing with platelet-rich plasma: a prospective randomized controlled trial. (2012) American Journal Sports Medicine. 40(6):1282-8.

Finnoff et al. Treatment of chronic tendinopathy with ultrasound-guided needle tenotomy and platelet-rich plasma injection. (2011) PM&R. 3(10):900-11.

Mont RR, et al. Platelet rich plasma treatment for chronic Achilles tendinosis. (2012) Foot Ankle International. 33(5):379-85.

Nixon AJ. Cell and gene-based approaches to tendon regeneration. (2012) Journal of Shoulder Elbow Surgery. 21(2):278-94.

PRP research: Evidence based medicine

for Osteoarthritis

Ahadi T., et al. Platelet-rich plasma versus hyaluronic acid. (2012)

Arthroscopy. 28(11):1585-6

Anitua E., et al. A biological therapy to osteoarthritis treatment using

platelet-rich plasma. (2013) Expert Opinion Biologic Therapy. 13(8):1161-72.

Pourcho AM, et al. Intra articular platelet-rich plasma injections in the

treatment of knee osteoarthritis: review and recommendations. (2014)

American Journal Physiology, Medicine and Rehabilitation. 93(11 Suppl

3):S108-21.

Zhu Y, et al. Basic science and clinical application of platelet-rich plasma

for cartilage defects and osteoarthritis: a review. (2013) J. Osteoarthritic

Cartilage. 21(11):1627-37

PRP research: Evidence based medicine

for bone growth/reconstruction

Loquercio G, et al. Autologous platelet gel improves bone reconstruction of

large defects in patients with bone giant cell tumors. (2015) In Vivo. 29(5):

533-540.

Eskan MA, et al. Platelet-rich plasma-assisted guided bone regeneration for

ridge augmentation: a randomized, con trolled clinical trial. (2014) Journal

of Periodontology. 85(5): 661-8.

Cho AR, et al. The incorporation of platelet-rich plasma into calcium

phosphate cement enhances bone regeneration in osteoporosis. (2014) Pain

Physician. 17(6): E737-45

How is PRP obtained?

There are a number of PRP systems available in the UK

Many are designed for human applications and have not been validated in veterinary species

Unlike pharmaceuticals regulated by the VMD, in which the precise contents, concentrations and potency are clearly known, PRP preparations have no such guarantee

Am J Vet Res 2015 Sep;76(9):822-7 Characteristics of canine PRP prepared with five commercially available systems. Franklin SP, Gamer BC, Cook JL.

Results: The various PRP concentrating systems differed substantially in the amount of blood processed, method of PRP preparation, amount of PRP produced, and platelet, leukocyte and erythrocyte concentrations or reductions for PRP relative to results of whole blood

Conclusions and clinical relevance: The characteristics of PRP products differed considerably, investigators evaluating the efficacy of PRP need to specify the characteristics of the product they are assessing. Clinicians should be aware of the data (or lack of data) supporting use of a particular PRP for a specific medical condition.

PRP processing: Goals

The goal is to obtain the highest concentration of platelets and growth

factors, whilst removing the red and white blood cells, which can be

deleterious

Generally accepted:

Ideal platelet concentration is a 3-7 fold increase compared with whole blood

No Neutrophils

No Red blood cells

Increased monocyte concentration

Unknown optimal lymphocyte concentration

PRP composition:

RBC concentration

Prefer decreased concentration

RBCs damage cartilage and

synovium directly via iron-

catalysed formation of ROS

RBCs increase concentrations of

unwanted inflammatory mediators

(IL-1 and TGF-α)

RBCs cause significantly more

synoviocyte death when compared

to LR-PRP, LP-PRP, and PBS

Neutrophil concentration Prefer decreased concentration post

spin

Multiple studies show that neutrophils increase concentrations of unwanted inflammatory mediators (IL-1β, TNF-α, IL-6 and IL-8)

Increased concentrations of neutrophils in PRP is positively correlated with an increased MMP-9 concentration which degrades collagen and other extracellular matrix molecules. Matrix metalloproteinases play a significant role in OA

PRP composition:

Monocyte concentration

Prefer increased concentration

post spin:

Monocytes are associated with an

increase in cellular metabolism and

collagen production in fibroblasts

Decreased release of anti-

angiogenic cytokines interferon-γ

and IL-12

Lymphocyte concentration

Preferred concentration unknown

Platelets have been shown to

activate peripheral blood

mononuclear cells (Lymphocytes,

monocytes and macrophages) to

help stimulate collagen production

This is mediated by an increase in

IL-6 expression

PRP system validation in dogs:

Canine Platelet-Rich Plasma Systems: A Prospective study. Carr, B.J; Canapp, S.O;

Mason, D.R; Cox, C and Hess,T. Front. Vet. Sci., 05 January 2016

PRP multicentre analysis Results

Each system produced a different

final product that varied greatly in:

Platelet concentration

RBC concentration

Neutrophil concentration

Monocyte concentration

Lymphocyte concentration

The purpose of this study was to

prospectively analyse and compare

key parameters of the PRP product

from five commercial canine PRP

systems in healthy, adult canines.

No claims regarding the efficacy of

the PRP therapy in dogs or the

efficacy of the PRP formulations

evaluated can be deduced from

this study

Platelet concentration:

Red blood cell concentration:

Neutrophil concentration

Monocyte concentration

Lymphocyte concentration

Conclusions

The systems with the highest

platelet yield were the:

SmartPRep®2 ACP

Companion CRT pure PRP system

However the SmartPRep®2 ACP

failed to reduce neutrophil

concentrations

The Companion CRT Pure PRP

system yields a 550% mean

increase of platelets, whilst

removing greater than 95% of the

RBCs, 19% of the WBCs, and 85% of

neutrophils

The Companion CRT Pure PRP

system produces PRP that best fits

our current thinking of an ideal

PRP product

CRT system validation: Average cell numbers,

n=77 (Cases at Greenside, Unpublished data)

Concentration factor x 6.3

92% Reduction

In Neutrophils Conclusion:

The CRT system is

consistent and

reliable

The Companion CRT PRP system

Step 1:

Open sterile kit containing everything you need for PRP processing

Fill and prime the 60 ml syringe with anticoagulant (ACD-A)

10 ml of ACD-A for 50mls of blood

5 ml of ACD-A for 25mls of blood

Aseptic prep of blood sample site

Collect 25 or 50 ml of blood from the jugular vein with supplied 60 ml syringe and butterfly catheter

Can be done conscious or sedated

Step 2Fill concentrating device with 60 ml of anticoagulated blood

Step 3

Use scales to counterbalance the

concentrating devices

Step 4Place the concentrating device and counterbalance in the centrifuge and spin for 1 minute at 3600 RPM

Step 5Carefully remove the concentrating device from the centrifuge

There will be two or sometimes three layers visible

Step 6Aspirate the platelet plasma suspension until the disc touches the RBCs and a “flash” of RBCs are seen in the line

Transfer this into the final concentrating device

Step 7Counterbalance the concentrating devices and place in centrifuge

Spin for 5 minutes at 3800 RPM

Step 8Remove from centrifuge and draw off plasma to leave 4 ml in the concentrating device

Re-suspend platelet pellet from the bottom of the concentrating device using gentle swirling and agitation

Step 9Remove all remaining fluid from the concentration device

This should give you 4ml of Pure PRP

Step 10Perform CBC to confirm PRP composition (IDEXX Procyte ideal due to low sample volume requirement)

Use 3 way tap to dispense PRP into individual syringes ready for injection!

Typical CBC of a PRP sample

What conditions can be treated with

PRP?

Osteoarthritis

Tendon injuries

Ligament injuries

Burn wounds

Open wounds

Bone fractures

Post surgical

Orthopaedic e.g. TTA, Disc surgery

Soft tissue e.g. ligament repair

Diagnosis and appropriate application

Very important to have a correct

diagnosis

Rule out concurrent associated soft

tissue or orthopaedic problems

PRP must be applied to the site of

damage in order for it to be

beneficial

Joint injections e.g in OA

US guided soft tissue injections e.g

shoulder tendinopathies

Direct application e.g mixed with

Manuka honey for dermal lesions

Treatment guidelines

PRP therapy is often performed as a series of one to three injections with at

least two weeks in between

About 50% of dogs require more than one injection for significant improvement

Sedation or GA is usually required depending on location of the injection

Clinician must be proficient in intra-articular injections. Ensure joint fluid is

aspirated before introducing PRP to confirm correct needle placement

For soft tissue injuries, ultrasound guidance is essential to ensure accuracy of

the injection

PRP can be combined with other therapies such as stem cell therapy for

advanced disease processes

Post implantation care

Joint “flare”

Exercise restriction

Rehab/Physio

Post implantation analgesia

NSAID?

Laser therapy

Cold compression

Paracetamol and Codeine 5 day course

What are the benefits of incorporating

PRP into the clinic?

Benefits to the clients/patients:

A drug free very effective treatment option for a number of conditions

Evidence based. A lot of human research articles. Mounting evidence in the

veterinary field

Minimally invasive patient side therapy

No drug side effects

More options in arthritis management

Affordable treatment option and is covered by most insurance companies

PRP preparation is fast, easy and can be done patient side

PRP processing in about 15 minutes

What are the benefits of incorporating

PRP into the clinic?

Benefits to the veterinary practice:

Easy and quick to perform

Offers an alternative therapy

Is a good starting point to adding regenerative therapies to your practice

Minimal time investment to maximise profit

Provides an additional revenue stream

Cost of equipment paid for after 10 procedures

Attracts new clients

Case study: Domino

6 year old Male(N) Boxer

Chronic RF lameness exacerbated by exercise

Conservative management with rest and NSAID failed to improve lameness

Stance analysis showed a marked reduction in weight bearing in the right thoracic limb compared to the left

Supraspinatus muscle atrophy on the right with a positive bicipital test

Domino: Further investigations

Radiography Musculoskeletal ultrasound

Domino

Diagnosis

Bicipital tendonitis with concurrent

supraspinatus tendinopathy

Radiography not helpful in

diagnosis of this case

Treating one tendinopathy without

treating the other would have

resulted in treatment failure

Treatment PRP was produced using the

Companion CRT Pure PRP system

Ultrasound guided injections into the tendon lesions

IA injection of PRP as the bicipital tendon is contained within the joint capsule

Pardale V 5 days post implantation

Lead exercise for 2 weeks followed by gradual increase in exercise over the following 6 weeks

Domino: Treatment outcome

6 week check up

No lameness

Mild stiffness after rest which

resolved quickly with exercise

Stance analysis showed equal

forelimb weight distribution

No pain on bicipital test

Repeat ultrasound examination

revealed reduction in fibrosis and a

more normal linear fibre pattern

Repeat US examination

Where do you start?

Budget: Buy outright vs lease or rental. Single use kits

Joint/tendon injections

Training if required

Online resources e.g. joint injection guides and webinars

Courses / Cadaver practice

Joint injections are not difficult and the presence of joint fluid ensures you are in the right location

Educational courses: Companion Regenerative therapies

http://www.litecure.com/regenerative/education-training/tiered-training/

Where do you start?

How do you sell it to your clients?

Regenerative medicine vs masking clinical signs with drugs

Drug free, side effect free, treatment option

Improved healing times so that their animals can get back to normal activity

quicker

Client education

Evening talks

Facebook case studies

Website testimonials

Incorporating PRP into treatment

protocols

Senior patient clinics: OA treatment option. Provide drug free pain relief

Wound management: Reduced healing time, increases client and patient

satisfaction. Also reduces costs of bandage changes and dressings in chronic

wounds

Tendinopathies: Potential cure vs reduction of inflammation with steroid

injections

Orthopaedic surgery: Osteotomies, fractures, and non unions. Repair of

damaged cartilage following injury e.g. Cruciate tear and surgery

Any procedure where speeding up healing would be beneficial

Implementation of PRP at

Greenside Vets

Regenerative medicine referral clinic

More chronic and end stage cases

Combination therapies: Stem cell therapy and PRP

Synergistic action which speeds treatment outcomes

Elbow dysplasia and OA: ROM changes

Lumbosacral disease: Speed of analgesic effects

Longevity of response?

Implementation of PRP at

Greenside Vets

Orthopaedic surgeries

Joint fluid replacement following intra-articular surgery especially cruciate surgery

with meniscal damage

Osteotomies e.g TTA Speed of healing and return to function

Questions?

Email: andy@greensidevetpractice.co.uk

www.stemcellsscotland.co.uk