The comparison between different systems to prepare
platelet rich plasma using equine blood
L.N. Hessel, G. Bosch and C. van der Lest
Veterinary Faculty, University of Utrecht, The Netherlands
Keywords: horse; tendon; platelet rich plasma; growth factors
Summary
Reasons for performing study: To compare different platelet rich plasma production
systems with regard to the composition of the end-product.
Hypothesis: There are differences in resulting platelet and leukocyte concentrations and
other characteristics using different techniques.
Material/methods: blood from six horses was used to produce PRP with five different
techniques. The end-products were analyzed for hematologic parameters, such as
platelet and leukocyte concentrations, and growth factor levels, namely PDGF-BB and
TGF-ß1.
Results: All systems showed an increase in platelets and growth factors compared to the
baseline, but the differences between the systems were significant. Platelets increased in
a range from 1.33 to 6.72 times compared to whole blood, PDGF-BB had a mean
baseline of 0.44 ng/mL in plasma and showed mean concentrations ranged from 0.85 to
5.27 ng/mL and mean TGF-ß1 concentration increased in a range from 0.6 to 1.70
ng/mL in comparison with a mean baseline of 0.30 ng/mL. Leucocyte concentrations
varied a lot between the systems, with a decrease in one system and a mean increase
ranging from 1.46 to 7.58 times the baseline in the other systems.
Conclusions and potential relevance: This study revealed some interesting differences in
hematologic parameters between different systems to prepare PRP. It also showed that
there a great differences in growth factor concentrations between the systems.
Leukocytes and its contribution to or negative effects on the healing process remain
unclear. The findings of this study show the characteristics of PRP using several
techniques that are commercially available. The differences between the systems will
give practitioners an insight in the system they prefer.
Introduction
The flexor tendons in the lower limbs of horses are important weight-bearing structures at rest
as well as during locomotion. Injuries to these structures heal slowly and inefficiently,
because the scar tissue that is formed in this repair is functionally deficient compared to
normal tendon. This has important consequences for the animal in terms of reduced
performance and an increased risk of re-injury (Richardson et al. 2007).
The healing of the tendon follows a sequence of events consisting of inflammation,
fibroblastic proliferation, collagen production and remodelling. Pain is most closely correlated
with the degree of inflammation, but not necessarily with the amount of damage done to the
tendon – demonstrated by lameness and pain on palpation of the affected tendon, which is
short-lived. Soon after the clinical injury, the proliferation phase begins which overlaps with
the inflammatory phase. This is associated with vascular in-growth and cellular infiltration
(Smith 2008). After this period, linearly arranged Type I collagen fibrils prevail, indicating
progressive remodelling and normalization of the healing tissue. The disorganized scar tissue
can, if given long enough, reach similar ultimate strength as the original tendon. However, it
is the abnormal composition and arrangement of matrix in fibrous scar tissue, which has poor
biomechanical properties in comparison to normal tendon, and the slow rate of healing that
are believed to be responsible for the high incidence of re-injury (Dowling et al. 2000).
Most treatments attempt to decrease inflammation within the tendon, prevent further
trauma to damaged tissues, stimulate the healing process, and prevent re-injury after the horse
resumes training. High rates of recurrence of tendon injury have stimulated researchers to
look for methods for modulating tendon healing that provide better repair in comparison to
the original tendon architecture (Nixon et al. 2008).
One of the treatments currently available is the use of platelet-rich plasma (PRP). It is derived
from the patients own whole blood, which is its most important advantage, as there is no need
for concern about transmissible diseases or immune reactions. PRP contains platelets which
upon activation release growth factors and other substances that promote tissue repair. Growth
factors like Platelet-Derived Growth Factor (PDGF), Transforming Growth Factor-ß (TGF-ß)
and Vascular Endothelial Growth Factor (VEGF) stimulate cell proliferation and
differentiation, and increased extracellular matrix production. (Hsu et. al 2004; Anitua et. al
2004). It may also contain leucocytes and other mediators which play a crucial role in
inflammatory responses. Several human studies have pointed out the key role of leucocytes in
PRP, both for their anti-infectious action and immune regulation (Dohan Ehrenfest et al.
2008). Others studies were concerned about the pro-inflammatory mediators and their
potential for inciting an undesirable inflammatory reaction in affected tendons (Scott et al.
2004; Schnabel et al. 2007; Cissel et al. 2010).
PRP is already been used in many different clinical applications, demonstrating the
effectiveness and importance of the product for a variety of medical procedures. For example,
Maia et al. (2009) treated six horses with PRP. They induced tendinopathy by injecting
collagenase in the superficial digital flexor tendon of the horses and treated them twelve days
later. The animals were subjected to daily physical examination and clinically monitored
twice per week. Thirty-six days after inducing the tendinopathy, histologic evaluation
showed that the injuries treated with PRP healed with a more uniform and organized repair
tissue compared to the control group. Another study was performed by Bosch et al. (2010).
The hypothesis that a single intratendinous PRP treatment affects on the repair tissue in
surgically induced core lesions in tendons of horses was confirmed. Rindermann et al. (2010)
used autologous conditioned plasma as therapy of tendon and ligament lesions in seven
horses. All horses revealed a positive outcome in 10 to 13 months post injury and were either
performing in their previous work-load or were back in full training.
A lot of systems that prepare PRP are commercially available. Most of them use
centrifugation, but also filtration and a manual technique are being used. In human research
much research has been conducted to detect differences between the systems. They all
showed that there are differences in number of platelets and therefore in concentration of
different growth factors, but also inclusion of leukocytes and use of activators that may lead
to different biological effects when it is used as therapy (Everts et al. 2006; Sanchéz et al.
2010; Lopez-Vidriero et al. 2010).
These human systems are also used in the veterinary field, but there is no data for the PRP
production techniques on the resulting platelet and leukocyte concentrations and other
characteristics. Therefore, the goal of this study is to compare different systems with regard to
the composition of the end-product.
Material and Methods
PRP preparation
Six warmblood horses were used with a mean age of 14 years old (range 9-17) and a mean
weight of 543 kg. (range 488-584 kg). All the horses were shaved before drawing blood, to
prevent contamination. The blood was collected aseptically from a jugular vein using a 18 G
needle. All the syringes were inverted a couple of times to ensure proper mixing with the anticoagulant before PRP was prepared. A 60 ml syringe was pre-filled with 6 ml of citrate
solution and filled up to 60 ml with whole blood. This was put in the Angel system
(Cytomedix, Gaithersburg, MD, USA) and prepared following manufacturer’s instructions.
Following centrifugation at 3200 rpm for 16 minutes, the PRP was collected in a 20ml syringe
and diluted to a volume of 6ml using plasma volume from the platelet poor plasma collection
bag.
10 ml whole blood, without an anti-coagulant, was taken from the horse for the ACP
Double Syringe System, (Arthrex, Naples, FL, USA) and also prepared following
manufacturer’s instructions. The syringe was put in a Rotofix 32A centrifuge and rotated at
1500 rpm for 5 minutes. A second syringe, already in the first was then pushed down to
collect the plasma, which is the end-product.
A 60 ml syringe with 6 ml of the attached anti-coagulant was prepared to PRP with the
E-PET (Pall Corporation, Port Washington, NY, USA) following manufacturer’s instructions.
The anti-coagulated venous blood taken from the horse is mixed with a capture solution in the
collection bag before passed through Pall’s cell harvest device where platelets are captured.
Back-flushing the harvest solution through the filter recovers the platelet concentrate into a
syringe.
Another 60 ml syringe with 6 ml of the attached anti-coagulant was used to prepare
PRP with the GPSIII (Biomet, Warsaw, IN, USA), also following manufacturer instructions.
The blood was put in the GPS tube and placed in the centrifuge. It was centrifuged for 15
minutes at 3200 rpm. Afterwards, the platelet poor plasma is extracted through a special port
and discarded from the device. While the PRP is in a vacuumed space, the device is shaken
for 30 seconds to re-suspend the platelets. Afterwards the PRP is withdrawn.
The last syringe was used to prepare PRP manually. The 60 ml of anti-coagulated
venous blood was divided over six 10 ml tubes and placed in an EBA20 centrifuge (Hettich,
Tuttlingen, Germany). This was set on 1800 rpm for 10 minutes. After the first round was
finished the buffy coat was taken out of the tubes with a pipette and placed in a new tube.
This was again centrifuged at 1800 rpm for 10 minutes and the actually PRP was the buffy
coat suspended in the supernatant. This procedure was used as described by De Mos et al.
(2009).
Hematological evaluation
Blood samples, approximately 3.5 ml, used for baseline measurements were drawn from the
horses. Another 10 ml of blood was used to prepare plasma for further analysis. After the
procedures were done, the entire PRP product was taken for hematologic evaluation by an
ADVIA® 120 Hematology System (Siemens Healthcare Diagnostics B.V., Breda, The
Netherlands) This machine counts platelets, leukocytes and erythrocytes, and it calculates the
Mean Platelet Volume (MPV) and Mean Platelet component Concentration (MPC).
After this analysis the whole blood and the PRP products where divided in
Eppendorfes. These cups were placed in a freezer with a temperature of -80 ºC till further
analysis.
Biochemical evaluation
Platelet growth factor concentrations, PDGF-BB and TGF-β1, were determined in whole
blood, plasma and five PRP samples using a sandwich enzyme immunoassay technique (R&D
Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Samples were
measured in duplicate and in appropriate dilutions to fit the respective calibration curves.
Cross reaction has been assumed on homology between equine and human protein structure
(Schabel et al. 2007)
Statistical analysis
Data were analyzed statistically using SPSS® 16.0 (SPSS Inc, Chicago, IL, USA). Data for
hematological and biochemical testing, which were non parametric and repeated
measurements, were tested using a Friedman test. The scores were compared using a
Wilcoxon Signed Ranks test. The significance level was set at P ≤ 0.05.
Results
PRP preparation
Six horses were used to determine differences between five systems that prepare PRP. After
putting the blood in a system, it did not take long before the end-product was ready,
approximately 10 to 30 minutes. The manual technique takes longer, because the blood has to
be in the centrifuge twice for 10 minutes and it takes skills and therefore time to extract the
buffy coat. This makes the manual method the most time conceiving method and the method
most prone to mistakes.
Hematological parameters
All the systems showed a significant increase in platelets compared to whole blood, except the
Angel. The Arthrex showed a minimum increase and the GPS the most (table 1). Significant
differences were seen between the GPS with all systems, between the E-PET with the Angel
and the Arthrex, and between the manual technique with the Arthrex.
Table 1 shows a decrease in MPV and a correlated increase in MPC in almost all systems.
Differences between the systems are all significant, except for the differences between the
manual technique and the Angel and the Arthrex, and between the GPS and Arthrex.
Table 1. Hematological parameters
Parameter
Platelets (%)
MPV (fL)
MPC (g/L)
Leucocytes
(%)
Whole
blood
(n=6)
Mean
Range
Mean
Range
Mean
Range
Mean
Range
9.5
8.8 – 10.2
207
198 – 219
Angel
(n=6)
Arthrex
(n=6)
E-PET
(n=6)
GPS III
(n=6)
Manual
(n=6)
232.0
47.7 - 378.7
9.6
7.4 – 10.8
197
188 – 213
146.6
36.1 - 235
133.8
74.2- 156
7.1
5.0 – 8.7
259
224 – 303
10.3
3.2 – 16.6
444.8
311 - 565
5.7
4.9 – 6.9
286
243 - 305
210.7
165 - 250
672.3
393 - 1259
7.0
5.9 – 9.2
260
226 - 287
758.2
610 - 1253
408.9
203 - 565
8.3
5.2 - 9.7
227
216 - 238
160.2
89 - 450
The GPS showed a significant increased level of leucocytes compared to whole blood. The
Angel, E-PET and manual technique also showed in increase, but this was not significant. The
Arthrex system showed a significant decrease in leukocytes. Between the systems there was a
significant difference between all systems with the Arthrex system, and between the GPS with
the Angel and the E-PET.
All hematologic parameters are shown in graphics in annex 1.
Growth factors
The counts in the growth factors concentrates obtained by the five methods were significantly
greater than in plasma, except for TGF-ß by the Arthrex system (table 2). The concentrations
of PDGF-BB were significantly different between the E-PET with the Angel, the Arthrex and
the manual technique. The GPS system also differ significantly with the Angel, the Arthrex
and the manual technique and there was a significant difference between the Angel and the
Artrex. For TGF-ß the concentrations were significantly different between all systems with
the Arthrex and between the E-PET with the Angel, the Arthrex and the GPS.
PDGF-BB and TGF-ß1 are shown in graphics in annex 2.
Table 2. Growth factor concentrations
Parameter
Plasma
Angel
(n=6)
(n=6)
0.44 ± 0.18 2.44 ± 0.62
PDGF-BB
(ng/ml)
0.30 ± 0.04 0.6 ± 0.05
TGF-ß1
(ng/ml)
Arthrex
E-PET
GPS III
Manual
(n=6)
(n=6)
(n=6)
(n=5)
0.85 ± 0.05 5.27 ± 1.47 5.14 ± 1.02 1.61 ± 0.59
0.28 ± 0.08 1.70 ± 0.38 0.68 ± 0.26 1.58 ± 0.59
Discussion
In this study it is shown that the platelet and leukocyte concentrations may differ in the PRP
end-products prepared by different systems. The production of growth factors and
prostaglandins are also influenced by the different preparation techniques. The goal of this
study was to increase knowledge on whether different PRP preparation methods result in
different growth factor release. In addition, the leukocytes and it features were investigated
because of its role in inflammatory responses.
It all starts with producing the PRP. Most systems use a centrifuge to harvest the end-product.
This means it should be done in a clinic where the proper materials are available. In the
current study we used one system that did not need a centrifuge, being the E-PET system.
This filtration system only has to be hung so that the blood can flow through the filter. This
means that the collection can be done near the stables were the patient is held, which might be
an advantage of this system.
Time is not a big issue in this matter. All systems need approximately 10 – 30 minutes
to produce the PRP. Except for the manual system, which takes about 50 minutes. This is not
only due to its long centrifuge time but also because it takes skills to extract the buffy coat. It
is also the most unreliable technique, because extracting the buffy coat is precise work and
depends on the experience of the person doing the extraction. This in contrary to the other
techniques where the materials are developed to be as precise as possible.
Although the exact mechanisms of action of PRP remain unclear, the evaluated levels of
PDGF and TGF-β in PRP can be assumed to play an important role, as both have shown to
positively influence tendon healing. PDGF plays a role in the acute phase after tendon
damage, and stimulates the production of other growth factors; TGF-β is active during the
inflammatory phase, and it has several effects in cell migration and proliferation (Bosch et al.
2010).
All systems showed an increase in platelets and growth factors compared to the
baseline, but the differences between the systems were great. The Arthrex system showed a
mean increase of platelets of 133.8%, which is a similar result to study of Rindermann et al.
(2010). The PDGF was concentrated twice and the TGF-ß was not concentrated at all. The
generally accepted increase of platelets numbers is three to five-fold the baseline. The Arthrex
system did not reach this increase, which could be explained by the method it used; it is
thought that the system uses plasma which does not contain platelets (platelet poor plasma).
The GPS system showed the largest increase in platelets (672.3%), but not in growth
factors. The PDGF concentration was very well elevated, 13-fold the plasma level, but the
TGF-ß did not show the same increase. The E-PET also showed high results with an increase
of 444.8% in platelets and a respectively 5-fold increase in PDGF and almost a 2-fold
increase in TGF-ß compared to the baseline. Both systems showed a platelet concentration
that was far above the generally accepted increase, but it is unknown whether these high
platelet levels might have negative effects.
Although the manual technique is time consuming and less reliable than other
techniques, it did show an increase in both platelets (408.9%) as growth factors (5.1 times and
4.6 times for respectively PDGF and TGF-ß). It might be used in practice, when the user is
experienced enough. The Angel system did also increase in both platelet concentration and
growth factor levels, but was significantly different with the GPS and E-PET system for the
platelets as well as PDGF.
The Mean Platelet Component (MPC) provides information on density, or granularity, of
platelets and indirectly on their structure and function. MPC is reduced when platelets
degranulate, which is an indication that the platelets have undergo activation (Diaz-Ricart et
al. 2010). Another platelet function parameter is the Mean Platelet Volume (MPV), showing
the mean of the distribution of cells by volume. Measuring platelet size is also a potential
marker of platelet reactivity. Larger platelets are denser and contain more α-granules, which
release growth factors, making these platelets more metabolically and enzymatically active
(Chu et al. 2010). In the present study only the Angel system showed an activation of
platelets.
PRP may also contain leucocytes which play a crucial role in inflammatory responses, but
there is little known about leucocytes present in PRP and their contribution to the healing
process (Everts et al. 2006). Several human studies have pointed out the key role of
leucocytes in PRP, both for their anti-infectious action and immune regulation (Dohan
Ehrenfest et al. 2008). They also mentioned the production of large amounts of vascular
endothelial growth factor (VEGF) by leucocytes, which is an angiogenesis stimulator.
Others studies were concerned about the pro-inflammatory mediators and their
potential for inciting an undesirable inflammatory reaction in affected tendons (Schnabel et al.
2007). There is data that prostaglandins degrade tendon extracellular matrix (Cissell et al.
2010) and that matrix metalloproteinases (MMP), produced by neutrophils, and reactive
oxygen species degrade matrix, and both leading to local inflammation and damaging the
surrounding tissue. (Scott et al. 2004; Kaux et al. 2010). Cytokines such as tumour necrosis
factor-alfa and interleukin 1, can upregulate MMP and cyclooxygenase 2, which have
damaging effects on the extracellular matrix and resident cells of tissues as tendons and
ligaments (Argüelles et al. 2008).
More research about the role of leucocytes and other inflammatory mediators in the
healing process of tendon tissue is needed. This is necessary to answer the question whether
or not leucocytes must be present in PRP.
In conclusion this study revealed some interesting differences in hematologic parameters
between different systems that prepare PRP. It also showed that there are great differences in
growth factor concentrations between the systems. Leukocytes and their contribution to or
negative effects on the healing process remain unclear.
This research managed to reach its goal to compare different systems regarding the
composition of the end-product. Practitioners using PRP to treat tendon injuries now have
more knowledge about the characteristics of the techniques that are commercially available
and this may help them choose for a systems they thinks is best.
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Annex 1: Hematologic parameters
Annex 2: Growth factor concentrations