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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