Title: Platelet rich plasma: a review for radiologists.

Abstract

Platelet rich plasma (PRP) injection is now offered by musculoskeletal

radiologists for a wide variety of indications. While its extraction from

whole blood was initially described in the context of autologous blood

product recovery in surgery, targeted local infiltration with PRP sees

increasingly widespread use in the musculoskeletal sphere. A growing

evidence base now supports its application at numerous anatomical sites

for the treatment of both acute and chronic musculoskeletal pathologies.

This article will review the principle indications for PRP use and the

evidence underlying the practice, the technique of injection and technical

aspects of the preparation of PRP, as well the mechanism of action and

the profile of adverse effects associated with its use. This article will

review the biological properties of PRP, methods of production and

administration, the evidence for the practice and the risks and adverse

effects associated with its use.

History of the practice

The treatment of musculoskeletal complaints by means of image guided

local injection or infiltration has been described since the advent of

musculoskeletal ultrasound in the mid 20th Century [Dussik]. Since that

time, multiple agents have been administered by this method including

local anaesthetics, corticosteroids, polidocanol, glycerol [refs]. The

mechanism of action of these agents has been generally thought to

1
modulate inflammation, either through suppression (corticosteroids) or

provoking it in order to stimulate the healing process (polidocanol,

glycerol).

In 1987 Ferrari [ferrari] et al demonstrated that the collection and

readministration of autologous blood products including PRP was safe and

effective in the context of cardiac surgery. In the mid 1990s oral and

maxillofacial surgeons began to utilise PRP [Whitman 97, marx 98]

intraoperatively. PRP began to be used by musculoskeletal specialists as

an alternative to other injectates for the treatment of a wide variety of

acute and chronic musculoskeletal pathologies [ehrenfest 2014].

PRP biology

Whole blood is composed of a suspension of red blood cells, white blood

cells and platelets in plasma. Platelets, which are formed through the

fragmentation of megakaryocytes [patel], range in size from 2 4m.

They are anucleate and typically survive in circulation for 10 days

[george] until senescent individuals are phagocytosed in the spleen and in

the liver [ghoshal]. Normal platelet concentration in whole blood ranges

from 150,000450,000 /l [Harrison]. Aside from their role in thrombus

formation, platelets are known to express high levels of growth factors

and inflammatory mediators [table 1] which have functions and effects on

inflammation, cell recruitment and migration, tissue healing and

regeneration. Platelets circulate in an inactivated discoid form until

activated, for example by exposure to the collagen in damaged vascular

endothelium, when they change into a compact spherical shape with

2
many pseudopods [George]. Activation of the platelets in PRP for clinical

use may be allowed to occur naturally through exposure to the patients

own tissues upon administration, or it may be achieved ex vivo by the

addition of agents such as thrombin, calcium chloride or collagen. The

process of ex-vivo activation has been demonstrated experimentally to

influence the level of secretion of platelet growth factors and cytokines

[cavallo] although clinical significance of this has not been proven.

Obtaining PRP in clinical practice

General principles

PRP is prepared through the separation of whole blood, usually through

centrifugation, such that a heavy bottom layer of red blood cells and the

platelet poor supernatant may be discarded, retaining the so-called buffy

coat which is rich in platelets and white blood cells. This supernatant may

be directly injected or may be activated before use by adding thrombin or

calcium chloride to the specimen. Ehrenfest et al have classified PRP

preparations into four groups [ehrenfest 2009] according to leucocyte

content and fibrin content [table 2] while Mishra et al described a similar

classification which also considers pre-adminstration platelet activation

and categorises the platelet concentration as greater than or less than 5

times the baseline concentration [mishra] . P-PRP and L-PRP are the

preparations generally used in musculoskeletal practice as a low density

fibrin content is required in order to produce an injectable liquid form of

PRP. A consensus does not yet exist regarding what platelet concentration

3
constitutes platelet rich plasma. Marmotti [marmotti] defined PRP as

containing at least 200% of the peripheral blood platelet count, while

Marx [marx] has proposed a minimum concentration of 1,000,000 / l,

approximately 5 times the normal platelet concentration. In contrast,

Anitua has suggested that a lower cut off point (above 300,000 / l) can

be defined as PRP [anitua], however a proven dose dependent relationship

between efficacy and platelet concentration has not been demonstrated in

the musculoskeletal sphere.

Sample double centrifugation protocol for PRP preparation

A generalized protocol for PRP preparation is given.

1. Obtain informed consent from the patient

2. Obtain 30ml of anticoagulated whole blood by venepuncture
3. First spin: low speed centrifugation to separate the whole blood into

three layers
o Top layer platelets, plasma and some white blood cells
o Middle layer Buffy coat rich in white blood cells
o Bottom layer - Red blood cells

4. Transfer the top layer (to obtain P-PRP) or top and middle layers (for

L-PRP) to second tube

5. Second spin: The transferred supernatant is centrifuged at high

speed to obtain a platelet pellet, and a supernatant containing

platelets and plasma.

6. The upper 1/2 to 2/3 of the new supernatant consists of platelet

poor plasma and should be discarded.

7. Resuspend the platelet pellet in the remaining plasma to obtain PRP.
8. This specimen should be used immediately.

4
Published variations on this technique typically vary at step 3 and 4 in

terms of centrifugation speed and duration and in respect of the inclusion

or otherwise of the buffy coat for transfer to the second tube. Landesberg

et al [landesberg] found that centrifugation above 250g produced a pellet

that could not be resuspended, however Perez et al [perez] in an

extensive experimental study of centrifugation techniques in PRP

preparation found that pellets could be resuspended even after

centrifugation at up to 1600g for ten minutes. The reasons for inability to

resuspend platelet pellets may be related to the equipment used, the

anticoagulant used, and the method of supernatant transfer between

tubes. Clinicians should be aware of this possibility with their own systems

in order to avoid the need for repeated venepuncture.

Obtaining the product.. include definition of platelet rich plasma, normal

concentrations, significance of thrombocytopaenia

FDA regulation.

Under FDA 21 CFR 1271 of the Code of Regulations [ref FDA regs] some

autologous blood product preparations such as PRP are not regulated in

the typical manner with regard to clinical trials [Beitzel]. PRP preparation

systems generally, however, are regulated by the FDA, as typical

5
preparation systems were designed for application in the orthopaedic

setting to mix bone graft. In this regard their use for the preparation of

pure PRP is considered an off label use.

Evidence include mention of duration of action here?

Guidelines?

Adverse effects, risks and pitfalls

Aside from normal exposure prone procedure precautions, care must be

taken when preparing the product that labelling and patient identification

procedures are obeyed. This is of particular concern in high throughput

settings where multiple patient specimens may be prepared

concomitantly. PRP is prepared as an autologous blood product and if

administered to another patient can theoretically provoke a severe type ii

hypersensitivity reaction. The authors recommend consultation with a

haemovigilance expert for advice on patient safety in this regard.

Increased pain after administration is common and expected as PRP

provokes inflammation at the treatment site [ajr Lee]. Some authors

recommend that no NSAIDS be used in the 2 weeks prior to the procedure

as cyclooxygenase inhibition reduces platelet aggregation and this may

adversely affect the release of growth factors from the platelet alpha

granules [schippinger].

6
As an autologous blood product, allergic reaction is very unlikely, but a

florid inflammatory response to the treatment may be seen in some cases

[kaux] and clinicians should be aware of this as a possible differential

diagnosis in any adverse reaction.

Evidence for the practice

General considerations

It is intrinsically difficult to perform high quality clinical trials on any

treatment where an invasive intervention is to be tested. Blinding and

placebo control are problematic, particularly in the case of PRP, where the

putative mechanism of action of the treatment stems from the provoked

inflammatory response and subsequent healing, such that the injection of

a placebo may theoretically provoke an inflammatory response and exert

a treatment effect. In most cases, the evidence base for PRP treatment of

a given pathology is based on small randomised controlled trials, or

retrospective cohort studies with no control groups. Mautner et al

[mautner] for example, retrospectively reviewed 180 patients with chronic

tendinopathies at a variety of anatomical locations and found that over

50% of patients treated with ultrasound guided PRP injection reported a

moderate improvement in symptoms. A systematic review and meta-

analysis of PRP in sports medicine by Gholami et al [gholami] found 12

randomised controlled trials which were of heterogenous design and small

patient numbers. They concluded that the evidence for an PRP in sports

medicine remains unclear and requires large well designed randomised

7
controlled trials to clarify. the true efficacy of PRP in the musculoskeletal

sphere remains unknown.

Upper limb

Lateral epicondylitis

Lower limb

Hamstring tendinopathy

Patellar tendinopathy

Achilles tendinopathy

Evidence for the efficacy of PRP in achilles tendinopathy is controversial. A

large well designed clinical trial has not yet been reported in the

literature. Several case series have been published finding a beneficial

effect of treatment [monto, filardo]. Krogh et al [krogh] found no

difference in outcome between PRP and saline injection in a small

randomised controlled trial of 24 patients. However, in that study, follow

up was limited to 3 months and in both treatment arms an injection of 10

to 15 ml of lignocaine solution was injected into the peritenon which may

itself have provoked an inflammatory response and thus exerted a

treatment effect. In another randomised controlled trial and one year

follow up of 54 patients with chronic mid portion achilles tendinopathy, de

Vos and de Jonge et al found no difference in outcome between those

treated with standard therapy plus PRP injection and those treated with

8
standard therapy and placebo saline injection [de Vos, de jonge].

Similarly, Boesen et al [boesen], in a three arm randomised controlled trial

of 60 patients, compared PRP, high volume injection and sham treatment

of achilles tendinopathy. They found that both high volume injection and

PRP delivered statistically significant improvements in pain and function at

follow up. Notably, the mean reduction in VISA-A score (Victorian Institute

of Sports AssessmentAchilles) was 13-14 points in the PRP arm of the

trial by Boesen at al, while Krogh et al found a mean change of only 3.4

points. Filardo et al [Filardo] in a published case series of 34 tendon

injections, argue that applicability of the de Vos study to the younger

sporting population may be limited due to the higher mean age of the

patients included in it (49 and 50 years in the PRP and placebo groups

respectively). They also point out that the actual platelet concentration

administered to the treatment group was unknown, and that the use of an

inactivated PRP may limit the efficacy of the injection in vivo, as the

injectate may actually extrude from the tendon injection site before it is

fully activated, thus limiting the treatment effect. Lastly, the

administration of a placebo injection may itself provoke bleeding and local

inflammation, thus mimicking the treatment putative effect derived from

PRP, given that significant improvements were observed in pain and

activity scores in both PRP and placebo injection groups.

Technical consideration

9
Conclusion

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Figures

Figure 1.

Figure 2

13
Tables

Table 1 Platelet associated mediators

PDGF Stimulates cell

growth,
differentiation, cell
migration [andrae]
TGF-Beta Leukocyte and fibroblast
migration, granulation
tissue formation,
angiogenesis [pakyari]
FGF Angiogenesis,
granulation tissue
formation and tissue
remodelling [eming]
IGF-1 Recruitment and
proliferation of bone
marrow stem cells,
stimulates proteoglycan
and collagen synthesis
[provenzano]
CTGF Connective tissue
formation, fibroblast
chemotaxis,
extracellular matrix
formation [cicha]
EGF Accelerated
epithelialization
[nanney], regulates
collagenase secretion
[everts]
HGF Mitogenic, morphogenic

14
 and antiapoptotic
properties [matsumoto]
VEGF Epithelialization,
collagen deposition,
chemotaxis and
angiogenesis [bao]

PDGF platelet derived growth factor, TGF-Beta transforming growth

factor beta, FGF fibroblast growth factor, IGF-1 insulin like growth
factor 1, CTGF connective tissue growth factor, EGF epidermal growth
factor, HGF hepatocyte growth factor, VEGF vascular endothelial
growth factor.

Table 2. Classification of PRP preparations

Preparation Abbreviation Fibrin network Leukocytes

density present
Pure platelet P-PRP Low No
rich plasma
Leukocyte and L-PRP Low Yes
platelet rich
plasma
Pure platelet P-PRF High No
rich fibrin
Leukocyte and L-PRF High Yes
platelet rich
plasma

Acknowledgements

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