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Schattauer 2011

Original Research

Concentration of platelets and

growth factors in canine autologous
conditioned plasma
M. Stief1; J. Gottschalk2; J.-C. Ionita3; A. Einspanier2; G. Oechtering1; P. Bttcher1
1Department

of Small Animal Medicine, University of Leipzig, Leipzig, Germany; 2Institute of Physiological Chemistry,
University of Leipzig, Leipzig, Germany; 3Large Animal Clinic for Surgery, University of Leipzig, Leipzig, Germany

Keywords
Platelet-rich plasma, dog, thrombocyte,
growth factor

Summary
Objectives: To report the concentration of
blood cells and selected growth factors in canine autologous conditioned plasma (ACP).
Methods: The density of blood cells in whole
blood (WB), ACP and standard plasma preparation (SP) of 10 healthy mature dogs was
determined. In both ACP and SP, the concentration of insulin-like growth factor-1 (IGF-1),
epidermal growth factor, vascular endothelial
growth factor, platelet-derived growth factorAA, platelet-derived growth factor-AB, platelet-derived growth factor-BB, transforming
growth factor-1 (TGF-1), and transforming
growth factor-2 was measured using the
ELISA technique. In another ten dogs, ACP was
prepared using an ultra-soft spinning protocol, and again blood cell density was compared to that obtained in WB.
Results: The density of platelets in ACP was
significantly higher than that in SP (p =

Correspondence to:
Dr. Peter Bttcher, Dr. med. vet., Dipl. ECVS
Klinik fr Kleintiere
Universitt Leipzig
An den Tierkliniken 23
D-04103 Leipzig
Germany
E-mail: boettcher@kleintierklinik.uni-leipzig.de
Phone: +49 341 97 38 700
Fax: +49 341 97 38 799

Introduction
For treatment of various acute and chronic
sport injuries, platelet-rich concentrates
(PRC) have incrementally gained in importance in both humans and animals (14).

0.0002), but there was not any significant difference between ACP and WB, nor between
WB and ACP prepared using softer centrifugations. Interestingly, only for IGF-1, PDGFBB, and TGF-1 could reliable measurements
be obtained, showing a significant increase in
PDGF-BB and TGF-1 concentrations in ACP
compared to SP (p = 0.001, p = 0.0028). Regarding IGF-1 content, there was not any significant difference between ACP and SP.
Clinical significance: Canine ACP prepared
according to the manufacturers recommendations, or by using a softer spin does not
show the same specifications as human ACP,
which shows a doubling in platelet count
compared to WB. Even though canine ACP has
a similar number of platelets per injected volume and consequently, probably the same
amount of injected growth factors than WB,
application of canine ACP would not be associated with the proinflammatory potential
reported for WB, as it is almost free of erythrocytes and nucleated cells.

Financial support:
Arthrex provided financial support for the ELISA.
Vet Comp Orthop Traumatol 2011; 24: 122125
doi:10.3415/VCOT-10-04-0064
Received: April 20, 2010
Accepted: September 6, 2010
Pre-published online: January 11, 2011

Basically, PRC is defined as a substrate which

contains a platelet concentration above
baseline, however there are no consistent
recommendations on the fold-increase of
platelets which has to be achieved to potentially enhance wound healing (5, 6). The pla-

telet granules are rich in numerous growth

factors, which play an essential role in tissue
healing, such as transforming growth factorbeta (TGF-), vascular endothelial growth
factor (VEGF), and platelet-derived growth
factor (PDGF) (7). Therefore, any substrate
containing a high number of platelets can be
considered to be rich in those growth factors
as well (8).
Intra-articular application of autologous PRC has been suggested to have preventive effects against osteoarthritis (OA)
progression by stimulation of cartilage anabolism (9, 10). In people, clinical symptoms associated with OA are lowered following intra-articular application, allowing PRC therapy to be a potential alternative to hyaluronic acid or corticosteroid
injections in the treatment of OA (1113).
Even though similar evidence regarding the
treatment of canine OA using intra-articular administration of PRC are lacking, favourable results in horses suggest that this
kind of biological treatment could also be
beneficial to dogs (14, 15).
With the release of a specially designed
double syringea, bedside preparation of
autologous PRC in the form of autologous
conditioned plasma (ACP)b, has become
available to veterinary clinicians. However,
the protocol for ACP preparation provided
by the manufacturer has only been validated in human patients so far (11, 16). The
concentration of platelets in human ACP is
nearly two times higher compared to baseline, and the concentrations of PDGF-AB,
epidermal growth factor (EGF), VEGF, and
PDGF-BB are significantly elevated when
compared to traditionally prepared plasma,
the latter being virtually free of any type of
a

ACP Double Syringe System: Arthrex Inc, Naples,

FL, USA
ACP: Arthrex Inc., Naples, USA

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M. Stief et al.: Canine autologous conditioned plasma

cell (11). This enrichment in platelets seen

in human ACP is the result of one-step centrifugation at low speed (350 g RCF
[relative centrifugal force]) during five
minutes, thus allowing removal of erythrocytes and nucleated cells while retaining the
majority of the platelets. Whether the same
quality of cytologic separation occurs when
using canine blood samples is currently unknown, but differing results may be expected, considering the difference in sedimentation of human and canine platelets
(17, 18).
The aim of the present study was to
evaluate blood cell density and concentration of growth factors in canine ACP
samples prepared using the currently recommended protocol for human ACP preparation. As platelet enrichment using that
specific protocol proved to be unsatisfying
during the course of this study, it was decided as a further aim to investigate an
ultra-soft spin protocol for canine ACP
preparation in order to obtain a platelet
concentration increase comparable to that
seen in human ACP (11).

Materials and methods

Samples and processing
Group 1 consisted of 10 sound dogs, ranging
in size from medium to large breed (body
weight >18 kg), with a mean age of 4.6 years
(range = 0.5 10 years). There were five females and five males. Ten millilitre blood
samples were collected from each dog from
the cephalic vein with minimal trauma and a
large-gauge needle to minimise platelet activation. Three separate samples were obtained from each dog: one EDTA-stabilised
whole blood sample (WB), one sample for
standard plasma (SP), and one sample for
ACP preparation. Standard plasma was produced by centrifuging citrate-stabilised WB
(citrate to blood 1:10) at 2000 g for 10 minutes. The ACP was prepared utilising a
double syringea, containing 1 ml acid citrate
dextrose solution A (ACDA) (citrate to blood
1:10). The double syringes were centrifuged
at 1500 rpm (350 g, soft spin) for five minutes.
Group 2 consisted of another group of
10 sound dogs, which were obtained at a

later stage, ranging in size from medium to

large breed (body weight >18 kg). There
were four females (2 spayed) and six entire
males with a mean age of 4.3 years (range =
1.5 9 years). Two blood samples (WB,
ACP) per dog were collected.
The ACP was prepared using ACDA
stabilised WB with a citrate concentration
of 1:6 instead of 1:10, and subsequently
centrifuged at 700 rpm (75 g, ultra-soft
spin) for 15 minutes. Standard plasma was
not obtained.

Cell density and concentration of

growth factors
Number of platelets, nucleated cells, and
erythrocytes were automatically counted
using a blood cell counterc in all three
samples (WB, ACP, SP) of group 1, and in
the two samples (WB, ACP) of group 2. In
group 1, the platelet count in ACP and SP
was verified manually with a haemacytometerd. Subsequently, each plasma
sample of group 1 (SP, ACP) was partitioned into eight aliquots, then frozen and
kept at 80C until further analysis.
Concentration of growth factors in both
the SP and ACP samples of group 1 were assayed by use of an ELISAe. The following
eight growth factors were investigated: insulin-like growth factor-1 (IGF-1), EGF,
VEGF-canine, PDGF-AA, PDGF-AB,
PDGF-BB, transforming growth factor-1
(TGF-1), and TGF-2. With the exception
of VEGF-canine, all ELISA kits were validated for human application only. Moreover, IGF-1, VEGF-canine, PDGF-AA, and
PDGF-BB were not validated for the use
with citrate stabilised blood samples.

Data analysis
Descriptive statistics were performed using
commercial softwaref testing for normaldistribution of data using the D'Agostinoc

d
e

pocH-100iV DIFF Hmatologiesystem: Scil Animal

Care company, Viernheim, Germany
Fein-Optik, Bad Blankenburg, Germany
Quantikine Immunoassay R&D Systems Inc., Wiesbaden, Germany
MedCalc, v. 9.4.2.0: MedCalc Software, Mariakerke, Belgium

Pearson Omnibus Test. As the assumption

of normality regarding cell density was rejected, median cell counts and their associated interquartile ranges (IQR) were calculated. The IQR is the distance between the
25th percentile and the 75th percentile. The
IQR is essentially the range of the middle
50% of the data. Growth factor concentrations were expressed as mean and standard deviation.
Comparison of cell density between
WB, SP and ACP was carried out based on
paired-data analysis by means of the Wilcoxon rank sum test. Comparing growth
factor concentrations between ACP and SP
was performed by means of paired T-Test.
Comparison of platelet count in ACP prepared at 350 g and 75 g was based on unpaired data analysis using the Signed rank
sum test. For all tests, significance was set at
an value of 0.05.

Results
Autologous conditioned plasma of group 1
prepared at 350 g showed only marginal
concentrations of leukocytes (median 0.10
G/l [G = Giga]; IQR: 0.03 0.25) and erythrocytes (median 0.01 T/l [T = Tera]; IQR:
0.00 0.01) (!Table 1). Manually counted
concentrations of platelets highly correlated with the results obtained using the
cell counter (Spearmans rho = 0.948; p
<0.001 ; R2 = 0.9754), thus only platelet
concentrations obtained using the cell
counter were finally analysed and reported
hereby.
Median platelet count in group 1 for
WB, ACP, and SP are reported in !Table 1.
Calculation of the Wilcoxon rank sum test
did not show any significant increase in
platelet count in ACP (293.50 G/l) compared to WB (271.50 G/l) (p = 0.3594).
With a more than 10-fold increase, concentration of platelets in ACP was significantly
higher than in SP (p = 0.002) in group 1.
In group 2, lowering the spin speed at
which ACP was centrifuged while increasing the duration of centrifugation (700
rpm [75 g] for 15 minutes) concurrently
did not result in any significant change in
the median platelet concentration of ACP
(277.00 G/l; IQR: 197.75 395.00) or
WB (255.50 G/l; IQR: 205.50 273.50)

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M. Stief et al.: Canine autologous conditioned plasma

Table 1

Cell density within whole blood, autologous conditioned plasma (ACP), and standard plasma for group 1 (n = 10) and within whole blood and
ACP for group 2 (n = 10). All values are expressed as median and associated interquartile range (IQR).

Whole blood
PLT (G/l)

Standard plasma#

Autologous conditioned plasma*

RBC
(T/l)

WBC (G/l)

PLT
(G/l)

WBC
(G/l)

RBC
(T/l)

PLT
(G/l)

WBC
(G/l)

RBC
(T/l)

Group 1

271.50a
9.35
(244.75-338.75) (8.45-9.95)

6.78
(6.65-7.17)

293.50a
0.10
(221.00-321.75) (0.03-0.25)

0.01
29.00b
0.00
(0.00-0.01) (23.00-55.25) (0.00-0.00)

0.00
(0.00-0.00)

Group 2

10.25
255.50a
(205.50-273.50) (8.70-11.50)

6.08
(5.91-6.54)

277.00a
0.00
(197.75-395.00) (0.00-0.35)

0.02
(0.01-0.05)

Key: PLT = platelets; WBC = white blood cells; RBC = red blood cells; G = Giga; T = Tera; *= Autologous conditioned plasma was centrifuged at 350 g
for five minutes for group 1, and at 75 g for 15 minutes for group 2; # = Standard plasma was centrifuged at 2000 g for 10 minutes; a, b = Concentrations
of platelets having different superscripts are significantly different (p 0.05).

(!Table 1) (p = 0.1934). Also, the platelet

count in ACP group 1 and 2 did not show
any significant difference (p = 0.7394). The
quality of cytologic separation in ACP prepared by either method of centrifugation
were similar, both being virtually free of
cells except for platelets.
!Table 2 summarises the concentrations of the eight growth factors in both
SP and ACP for group 1. Overall, measurable concentrations only of IGF-1, PDGFBB and TGF-1 were detected, whereas no
signal could be detected in SP or in ACP for
EGF, VEGF-canine, PDGF-AA, PDGF-AB
and TGF-2. The factor of enrichment
comparing ACP to SP was 1.0 for IGF-I, 6.1
for PDGF-BB and 1.6 for TGF-1, with
the latter two growth factors being signifi-

cantly enriched in ACP (p <0.001, p =

0.0028). The concentration of TGF-1 in
ACP exceeded the measurement range of
the ELISA kit in all 10 ACP samples. Therefore, we observed a significant increase in
TGF-1 concentration in ACP by at least
1.6-fold, however, analysis in diluted ACP
samples are required for future studies.

Discussion
This is the first study as far as we know which
evaluates platelet count and growth factor
concentration in canine ACP. Even though
the presented findings have to be regarded as
preliminary due to the limited sample size
and unexpected shortcomings during

Table 2 Concentration of growth factors measured in standard citrate plasma and autologous
conditioned plasma using Quantikine ELISA kitsd. Values are expressed as mean standard deviation.
Autologous
conditioned
plasma*

Standard
plasma#

Factor of
enrichment

Insulin-like growth factor-1 (ng/ml)

68.5 35.6

67.9 32.1

1.0

Epidermal growth factor

No signal

No signal

Vascular endothelian growth factor - canine

No signal

No signal

Platelet-derived growth factor-AA

No signal

No signal

Platelet-derived growth factor-AB

No signal

No signal

Platelet-derived growth factor-BB (pg/ml)

251.0 132.8

1540.8 553.8 6.1

Transforming growth factor-1 (pg/ml)

1239.9 590.8

>2000.0
(not available)

>1.6

Transforming growth factor-2

No signal

No signal

Key: * Autologous conditioned plasma centrifuged at 350 g for five minutes.

# Standard plasma centrifuged at 2000 g for 10 minutes.

measurement of growth factor concentrations, the study may serve as a guide for
clinicians, allowing them to appreciate the
potential biological character of ACP. When
prepared as currently recommended by the
manufacturer using 350 g for five minutes,
ACP did not reach significantly higher platelet counts than unprocessed whole blood.
This contrasts with the results obtained
when using human blood samples, in which
a two-fold increase in platelet count could be
detected (11). When comparing conventionally prepared canine plasma to canine
ACP, platelet count in ACP was about
10-fold higher. This significant enrichment
in platelets is the reason for the elevated concentration of -granule-derived growth factors observed in canine ACP compared to SP,
supporting the previously reported correlation between platelet count and concentration of platelet-derived growth factors
(8). Insulin-like growth factor-1 was not
elevated in ACP compared to SP, which is an
expected finding when preparing PRC (11,
20). Insulin-like growth factor-1 is primarily
excreted by the liver into the blood plasma,
being independent from the present number
of platelets (21). The inclusion of IGF-1 into
to the study protocol was thought to serve as
a negative control, differentiating platelet related and unrelated growth factor enrichment.
The problems encountered during
growth factor assay, which resulted in unreliable measurements for EGF, VEGFcanine, PDGF-AA, PDGF-AB and TGF-2
both in SP and ACP, may have been related
to species differences when using human
ELISA kits as well as when using kits not

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M. Stief et al.: Canine autologous conditioned plasma

validated for citrate plasma. Another

source of interference might have been the
handling and storage of the samples until
final growth factor analysis, especially
freezing at 80C, which may have affected
the integrity of the platelets and their
growth factors. In the future, more elaborated analysis of these growth factors need
to be carried out immediately after retrieval of the samples using canine specific assays validated for citrate plasma samples.
Finally, analysis of the growth factor concentration in ACP compared to WB instead
of SP would be desirable, but due to the interference of the ELISA technique by the
cellular components present in WB, so far
there are no direct assays established.
Even though there are not any consistent
recommendations on the fold-increase of
platelet concentration that has to be achieved to potentially enhance wound healing, invivo as well as in-vitro studies investigating
the biological effect of PRC as well as a twofold increase in platelet count and more
compared to WB might be necessary (6, 7).
Even with the ultra-soft spin protocol, this
goal was not attained in the majority of ACP
samples (only 3 out of 10 samples reached
values 1.4, data not shown). In addition,
when using this protocol, in most cases only
a small volume of supernatant (1 3 ml)
could be obtained compared to the 3 5 ml
obtained using the standard protocol as recommended by the manufacturer. Such low
volumes might not provide the absolute
number of platelets needed for clinical application, especially when injected intra-articularly, necessitating pooling of more than
one sample, and increasing the cost and
work-load. The reason for the observed individual variation in the amount of ACP gathered remains unknown. The lack of enrichment in platelet count in canine ACP
compared to human ACP may be explained
by divergences in specific gravity, size and
deformability of the cells as well as the viscosity of the medium, and has to be considered when attempting to interpolate the
observed clinical efficacy of human ACP to
canine ACP (11, 16, 19).
In summary, use of canine ACP as a substitute for WB would probably not result in

the application of a platelet-rich concentrate as both substrates appear to have the

same amount of platelets per volume. But
being virtually free of erythrocytes and nucleated cells, ACP injected intra-articularly
would not be associated with the reported
proinflammatory potential for whole
blood as leukocytes and erythrocytes may
initiate inflammatory processes within the
joint cavity, potentially leading to cartilage
destruction and ongoing pain. As this was a
laboratory study, we cannot conclude on
the clinical effect of canine ACP in the
management of wound healing and OA related disability. However, it might be suggested that canine ACP has a different biological effect than canine PRC.
Conflict of interest
Arthrex provided financial support for the
ELISA.

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