Platelet function and constituents of platelet rich plasma.

74 Clinical Sciences

Platelet Function and Constituents of Platelet Rich
Plasma

Affiliations

Key words

▶
● platelet rich plasma
▶
● blood
▶
● autologous
▶
● therapy
▶
● p-selectin
▶
● viable

M. H. Pelletier1, A. Malhotra1, T. Brighton2, W. R. Walsh1, R. Lindeman2
1
2

Surgical & Orthopaedic Research Labs, University of New South Wales, Randwick, Australia
Department of Haematology, Prince of Wales Hospital, Randwick, Australia

Abstract

▼

Platelet Rich Plasma (PRP) therapies require
blood to be processed prior to application, however, the full assessment of the output of platelet
sequestration devices is lacking. In this study the
products of the Autologous Fluid Concentrator
(Circle BiologicsTM, Minneapolis, MN) and the
Gravitational Platelet Separation System (GPS,
Biomet, Warsaw, IN, USA) were evaluated in
terms of platelet viability and PRP constituents.
The AFC and GPS produced 6.4 ( ± 1.0) ml and
6.3 ( ± 0.4) ml of PRP, with platelet recovery of
46.4 % ( ± 14.7 %) and 59.8 % ( ± 24.2 %) producing fold increases of platelets of 4.19 ( ± 1.62)

Introduction

▼
accepted after revision
May 10, 2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0032-1316319
Published online:
August 14, 2012
Int J Sports Med 2013; 34:
74–80 © Georg Thieme
Verlag KG Stuttgart · New York
ISSN 0172-4622
Correspondence
Dr. Matthew H Pelletier, PhD
Surgical & Orthopaedic
Research Labs
University of New South Wales
Level 1
Clinical Sciences Bld
2031 Randwick
Australia
Tel.: +61/293/822 687
Fax: +61/293/822 660
m.pelletier@unsw.edu.au

Clinical use of Platelet Rich Plasma (PRP) has
been reported since the early 1990s for oral and
maxillofacial surgery as a source of growth factors to increase the rate and degree of bone formation at early time points [31]. Since then PRP
has found an increasing use in a variety of clinical
applications. Primarily PRP has been used to support hard and soft tissue healing and formation
[5, 9, 35, 44], but has also extended to other uses
such as on nervous tissue [10, 42], in facial plastic
and cosmetic surgery [6, 28, 39], burns [36] and
chronic leg ulcers [46]. The autologous nature of
PRP also alleviates concerns of transmissible diseases or immunogenic reactions [34].
The potential clinical benefit of PRP relies on the
maintenance of intact platelet α-granules. Upon
activation, platelets degranulate and secrete an
array of mitogenic and chemotactic growth
factors, which include platelet derived growth
factor (PDGF-αα, PDGF-αβ, PDGF-ββ), transforming growth factor (TGF-β1, TGF-β2), vascular
endothelial growth factor (VEGF), fibroblast
growth factor (FGF), interleukin-1 (IL-1), and epi-

Pelletier MH et al. Platelet Function and Constituents … Int J Sports Med 2013; 34: 74–80

and 5.19 ( ± 1.62), respectively. Fibrinogen concentration was increased above baseline PPP
produced with the AFC. pH was lower for both
of the processed samples than for whole blood.
White Blood Cell count was increased around 5
fold. Functional tests showed preserved viability with both devices. This represents essential
knowledge that every treating physician should
have before they can confidently administer PRP
therapy produced by any method. These are the
first published results of platelet function for the
GPS system and the first performance results of
the AFC system. The PRP produced is classified
according to broad classifications as LeukocytePRP (L-PRP) for both devices.

dermal growth factor (EGF). These secreted proteins have important roles in healing and tissue
regeneration [1, 5, 27, 40]. Although there are
many proteins that may promote wound healing
in PRP, such as fibrinogen, the basic premise for
PRP remains the delivery of a soup of plateletderived proteins at concentrations above baseline [13–15, 50].
PRP can be generated by centrifugation of anticoagulated whole blood and collected with pipette
or syringe, but interest in PRP therapies has led to
the development of several commercial systems
aimed at improving ergonomics, decreasing variability, and improving platelet recovery in a
closed system. While these devices do produce
PRP with a concentration of platelets above circulating baseline blood levels [29], the product
varies between devices, leading Ehrenfest et al.
[11] to propose a classification to highlight their
properties. Specifically, the classification takes
account of differences in production including
whether or not the buffy coat (which contains a
near 7-fold increase in leukocytes [7]) is collected, fibrin and fibrinogen characteristics,
collection efficiency, as well as practical consid-

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Authors

Clinical Sciences 75

Fig. 1 Allocation of collected blood.

Whole Blood in 2 x
30ml syringes loaded
w (ACD-A) 10 % for
each device

1 ml: Whole Blood
(Heparin syringe)

4 ml: Whole Blood
(EDTA vacutainer)

4.5 ml: Whole Blood
(Li-Heparin vacutainer)

3.5 ml: Whole Blood
(Citrate vacutainer)

AFC or GPS
Processing

pH

Full Blood Count

Biochemistry

Fibrinogen

PPS; GPS
c-PPP; AFC

0h p-selectin resting
0h p-selectin ADP
0h Hypotonic Stress
0h Aggregation

4h p-selectin ADP
4h Hypotonic Stress
4h Aggregation

pH
Fibrinogen
Full Blood Count

0h p-selectin ADP
0h Hypotonic Stress
0h Aggregation

erations such as size, weight of the centrifuge, and ease of use.
The development of this classification will hopefully resolve
variable and sometimes conflicting results regarding PRP therapies in the literature [19, 28, 30, 37, 38, 44, 47].
Here we present a first evaluation of the Autologous Fluid Concentrator (AFC) (Circle BiologicsTM, Minneapolis, MN), a platelet
sequestration device which has recently been granted approval
by the Food and Drug Administration for use in the United States,
and a paired comparison of an established device (GPS, Biomet,
Warsaw, IN, USA).

Materials and Methods

▼

Both devices yield PRP, with the GPS system additionally producing Platelet Poor Plasma (PPP) and the AFC offering the option of
PPP or Concentrated Platelet Poor Plasma (c-PPP). An evaluation of
c-PPP, PRP, and whole blood included blood count, platelet count,
pH, white cell count, platelet recovery, and fibrinogen concentration. Platelet function testing on obtained PRP was performed in a
smaller subset of samples at 0 and 4 h to investigate platelet reactivity and short-term stability. These tests included assessments
of p-selectin levels while resting and upon activation with Adenosine Diphosphate (ADP), reaction to hypotonic shock and agonistinduced light transmission aggregometry.

4h p-selectin ADP
4h Hypotonic Stress
4h Aggregation

tested in this study utilised a plasma concentration chamber at
the rear and a chamber that, when viewed from the front, resembled an hourglass. Inside the hourglass chamber there is a collection tube with a moveable collection area. The GPS system
uses a buoy of specific density within a cylindrical chamber
▶
(● Fig. 2).
Blood and ACD-A filled syringes were emptied into the devices
which were centrifuged at 3 200RPM for 15 min at room temperature. Following centrifugation the plasma, red cell and buffy
▶
coat layers were clearly evident (● Fig. 3).
PPP was drawn off with syringes connected to respective ports
and was further processed by 3 filter passes for the AFC resulting
in (c-PPP). PRP was drawn off with a moveable collection window between 2 O-ring seals with the AFC and via a separate port
with the GPS device.
For the collection of PRP, the selector valve was turned back to
“draw”, the collection window was moved so that the top seal
was level with the top of the buffy coat and all available fluid was
drawn off using a 10 ml syringe connected to port B. A portion of
both PRP, PPP and, where processed, c-PPP was placed in capped
test tubes for FBC (n = 59) and fibrinogen concentration (n = 59)
and a Li Heparin syringe for pH (n = 59). A portion of PRP was
also placed in a capped tube for tests of platelet function (n = 12
for each test).

Blood assays
Subjects
This study conforms to ethical standards in sport and exercise
research as described by Harriss, et al. [21]. Volunteers were
recruited at the University of New South Wales and Prince of
Wales Hospital following approval of the Human Research Ethics
Committee. A total of 64 subjects were recruited for the study.
Volunteers were excluded if taking anticoagulant, anti-platelet,
or anti-inflammatory medication, or aspirin. Subjects were deidentified by assigning a number relating to all subsequent blood
test results.

Blood collection
Blood was collected from the medial cubital vein with a 19 G
needle by trained phlebotomists. Blood was collected in a 4 ml
EDTA Vacutainer for Full Blood Count (FBC), a 4.5 ml Li-Heparin
Vacutainer for standard biochemistry, a 3.5 ml Citrate Vacutainer
for fibrinogen concentration, 1 ml heparin syringe for pH, and for
each device 2 × 30 ml syringes preloaded with acid-citrate-dex▶
trose formula A (ACD-A) anticoagulant, 10 % (● Fig. 1).

Platelet sequestration
The centrifuge and AFC devices arrived in sterile packages ready
for use. The AFC is a 2 chamber modular system. The devices

All blood assays were performed at the South Eastern Area Laboratory Services (SEALS) which is accredited by the National
Association of Testing Authorities (NATA), Australia’s government-endorsed, international laboratory accreditation body.
59 matched samples of whole blood, PRP and PPP were evaluated for pH, fibrinogen concentration and FBC. pH was measured
on an ABL800 FLEX Radiometer Blood Gas Analyser (Radiometer,
Copenhagen). Full Blood Count was performed on a Roche Sysmex Model XE-2 100 (Sysmex Corp., Kobe, Japan). Biochemistry
assays were performed on a Beckman Coulter UniCel DXC880
blood analyzer (Beckman Coulter, USA). Fibrinogen assays were
performed on an STA R Evolution Coagulation Analyzer (Stago,
USA). Platelet recovery was calculated from platelet counts as
100 % *(platelet count of PRP * volume of collected)/(Platelet
count of whole blood * volume of whole blood). Platelet concentration factor was calculated as Platelet count of PRP/whole
blood platelet count.
Surface P-selectin expression (CD62p antigen), as a measure of
platelet activation, was assessed by flow cytometry in resting
and activated state samples. Approximately 1 ml of PRP from
each device was set aside for p-selectin assays. Phosphate buffered saline (2.92 μl) or ADP agonist (40 μM) was added to 70 ml
subsamples of PRP. These samples were incubated for 15 min at



pH
Fibrinogen
Full Blood Count

PRP



Fig. 2 Left to Right; Circle Biologics and Biomet
devices, before use (top row) and after centrifugation (bottom row).

Fig. 3 Close up of buffy coat interface, Circle
Biologics (Left) and Biomet (Right). Black arrows
indicate the PPP portion, the White arrows indicate
the buffy coat, and the double white arrows indicate the red cell pack.

37 °C. 4 × 5 μl aliquots were taken and incubated with CD41aPerCP antibody and either 1μl CD62-PE or 1 μl mouse IgG1-PE
(isotope control) (DB Biosciences, San Jose, CA). These samples
were incubated in the dark at room temperature for 20 min.
600 ml of Ringer’s solution was added following incubation.
Samples were analyzed in a BD FACS Canto II flow cytometer
(Becton, Dickinson and Company, USA). For determination of %
p-selectin expression, forward and light scatter and fluorescence
were acquired using logarithmic scale. A total of 20 000 platelet

events were gated. The isotope matched control antibody was
used to set threshold for CD62P positivity.
Hypotonic shock response (HSR) measures the platelet’s ability
to recover its normal volume after swelling when exposed to a
hypotonic environment. HSR is an optical method and PRP
required an additional soft spin (900RPM/10 min) to remove
intervening red cells for output from the AFC device. The resulting PRP was removed from the red cells with a pipette and platelet concentrations adjusted to < 500 × 109/ml and allowed to rest



AFC

GPS

Whole Bld
fibrinogen (g/L)
pH
platelet concentration ( × 10^9/L)
WBC ( × 10^9/L)

c-PPP

PRP

PPP

PRP

2.96 (0.73)
7.34 (0.03)
222 (45.7)
6.36 (1.42)

3.30 (0.73)+*
7.13 (0.03)+*
18.7 (12.7)+*
0.04 (0.135)+

2.97 (0.65)
7.02 (0.06)+*
926 (378)+*
32.2 (11.7)+*

2.95 (0.65)*
7.11 (0.04)+*
11.0 (6.2)+*
0.01 (0.027)+

2.94 (0.64)
7.08 (0.05)+*
1149 (497.7)+*
31.0 (8.9)+*

Table 1 Blood Assays. Data
summary for whole and processed
blood samples showing mean
(standard deviation).

*difference compared to whole blood value, + difference between devices, p < 0.05

AFC 0 h
HSR ( %)
aggregation ( %)
p-selectin resting ( %)
p-selectin activated ( %)

AFC 4 h

GPS 0 h

GPS 4 h

54.0 (5.0)
95.2 (7.3)
7.7 (4.2)
25.4 (8.1)*

54.1 (6.6)
96.5 (7.1)
6.4 (3.0)+
24.3 (9.2)*+

50.6 (6.8)
97.4 (3.4)
12.4 (6.5)
26.5 (6.1)*^

55.6 (6.1)
98.5 (2.7)
9.8 (2.5)+
31.9 (3.6)*+^

Table 2 Platelet Function Testing. Data summary for platelet
function assays showing mean
(standard deviation).

for 30 min. 6 × 150 μl of PRP from each device was transferred
into the wells of a microtitre plate. 3 × 150 μl of saline was added
to PRP samples and the microtitre plate was placed in a BioTeck
EL808 Ultra Microplate Reader (BioTek, Instruments, Inc.,
Winooski, VT). 150 μl of H2O were added with a micropipette to
the remaining 3 samples simultaneously, the lid was closed and
reading commenced. Readings of light transmission was performed at 405 nm for 10 min at 15 s intervals. Results were
recorded with this protocol via Gen5 Data Analysis Software
(Generation 5, Toronto, Canada). Light transmission (T) was converted to optical density according to OD = − log10 T or T = lO − OD.
The optical density (OD) of the saline samples acted as the baseline for measurements. The difference in the peak OD and the
baseline divided by the difference in OD at 10 min and baseline
produced a percent recovery.
Aggregation was performed following preparation to remove
red cells as above. 240 μl samples of PRP, PPP and c-PPP were
tested in a 4 station AggRAM aggregometer (Helena Laboratories, Gateshead, UK). All samples were stirred with magnetic stir
bars at 600 rpm. Following a quality check, the OD was measured in the matched samples of PPP, which acted as a control.
The PRP was then placed in the aggregometer. Continuous reading of OD continued for 10 min while 10 ul of collagen aggregant
was added to achieve a final concentration of 20 μg/ml and final
volume of 250 μl. Results of all tests were compared with paired
t-tests using Tukey’s criteria and a significance level of 0.05.

Results

▼

Of the 64 blood samples collected for the study, none were
excluded based on irregular biochemistry results, one was lost
to human error, and post processing assays were precluded by
insufficient volume in 4 samples resulting in a sample size of
n = 59 for fibrinogen, cell count and pH tests and n = 12 for all
platelet function assays. 54.1 ( ± 0.2) ml (AFC) and 55.7 ( ± 1.5) ml
(GPS) of whole blood plus ACD-A was loaded into the device and
6.4 ( ± 1.0) ml for the AFC and 6.3 ( ± 0.4) ml for the GPS of PRP
was collected. Platelet recovery from whole blood samples for
the AFC and GPS was 46.4 % ( ± 14.7 %) and 59.8 % ( ± 24.2 %),
respectively, representing concentration factors (fold increase)
of 4.19 ( ± 1.62) and 5.19 ( ± 1.62). Fibrinogen concentration was
increased above baseline for c-PPP (AFC only) but not PRP. pH
was lower for both of the processed samples than for whole
▶
blood. Results are summarized in ● Table 1.

▶
The results of platelet function assays are summarized in ● Table 2.
HSR tests at 0 h and 4 h showed platelet recovery of around 54 %
with no differences detected between time points or device.
Tests of the aggregation of PRP revealed total aggregation at
10 min was above 95 % at both time points with no differences
found between time or device. Upon in vitro activation with collagen there was a significant increase in surface expression of
CD62p (P selectin) confirming the collected platelets are functional and capable of activation at 0 and 4 h for both devices.

Discussion

▼

The current study demonstrates that both the AFC and GPS
devices produces platelet and leukocyte enriched plasma in a
closed system with functional viable platelets in volumes suitable for clinical use. Although platelet concentration has been
previously reported, platelet viability has not. Additionally the
AFC performance has not been reported.

Platelet function
Damage to platelets during processing can lead to the premature
release and subsequent loss of growth factors [1]. Viable platelets exhibit several behaviours when activated. Some of these
behaviours can be monitored such as shape change, aggregation
and surface marker exposure. Platelet aggregation is considered
the gold standard in evaluating platelet function [41]. Mean
platelet aggregation above 95 % in this study represents the
retention of activity. Likewise HSR has been indicated as a very
sensitive marker for viability [22], and predictor of in vivo survival [23]. During these tests platelet shape recovery was apparent. Granular release is closely related to p-selectin expression
[18]. Identifying p-selectin on the surface of activated platelets
allows them to be quantitatively assessed as a percentage of all
platelets present. While absolute numbers of p-selectin assays
are shown to vary a great deal between laboratories [12], it is a
useful measure to determine activation for matched samples
within a study centre. The results of the current study showed
that platelets could be activated beyond the resting state, indicating preserved function.
The broad classification of PRP encompasses a range of constituents at varying concentrations. The basic definition dictates that
it contains a platelet concentration that is increased above
circulating levels. Marx et al. [29] describes PRP as a platelet
concentration in a 5 mL volume with 1 000 000 platelets/μL



*difference between resting value, + difference between devices, ^ difference between time points


Fibrin
While platelet concentration and growth factor potential are
obviously essential aspects of PRP, fibrin (the activated form of
fibrinogen) is thought to directly induce angiogenesis by providing a matrix scaffold supporting cell migration and providing
chemotactic activity. The binding of growth factors such as
PDGF and VEGF to fibrin also supports wound healing [8, 25].
The fibrinogen concentration could therefore be an important
feature not always addressed when comparing different PRP
products.
Passing the PPP repeated through the filter increased the concentration of fibrinogen, with each successive pass decreasing
the overall volume as water was removed. Our conservative
processing with the AFC showed an increased concentration
compared to whole blood. This process can be repeated any
desired number of times further increasing fibrinogen concentration producing a product that could be used as a scaffold and
when added to PRP may alter the velocity of aggregation [24],
however, this was not examined in the current study.

pH
Although there was a difference between the whole blood pH
and that of the processed samples, all groups were greater than
6.9 which is well above the level of 6.2 that would indicate a loss
of platelet function [22, 33]. The increase in the acidity is likely
due to the addition of citrate to the blood from the ACD-A.

Leukocytes
Platelets have a known direct and significant role in immunity
and host defence against pathogenic microorganisms [4, 45].
Moojen, Everts et al. [32] demonstrated the antimicrobial potential of PRP in vitro. In addition to platelets, the PRP product collected from the buffy coat layer has been reported to contain
around a 7-fold increase in leukocytes [7]. The output of both
the devices produced around a 5 fold increase above circulating
levels.
The collection of leukocytes with the PRP product is an area of
uncertainty. Anitua et al. [2] studied a concentrated platelet
product, Preparation Rich in Growth Factors (PRGF), which
avoids leukocyte content with the intent of avoiding the proinflammatory effects of leukocytes. An increase in leukocytes,
combined with the view of platelets themselves as innate
inflammatory cells with acute host defence functions [16],
would suggest the PRP product may also be useful against postoperative infections. The inclusion of leukocytes during processing may also have beneficial effects. Neutrophils have been
suggested to produce additional VEGF [48], a protein known to
promote angiogenesis [2, 50]. Autologous Conditioned Serum
(ACS) is created by incubating whole blood with glass beads, and
has been shown to increase the concentration of relevant growth
factors considerably without unwanted side effects in human
and animal tests [49, 50]. A portion of the increase in growth
factor concentration may be attributable to the inclusion of leukocytes during the activation process. Understanding whether
or not to include, and the extent to which WBCs are concentrated in the PRP is likely to be relevant for future studies. It has
been suggested that PRP products be described as Leukocyte
Platelet Rich Plasma (L-PRP), and Pure Platelet Rich Plasma
(P-PRP) to differentiate between the 2 PRP variations [11].

Classification
It is commonly understood that not all PRPs are produced equal
[30], and as such, PRP from different methods and devices need
to be well characterised in order for the final product to have the
anticipated effect. According to the specifications laid out by
Ehrenfest et al. [11] the product of the ACP device is classified as
L-PRP as it contains leukocytes, just as the GPS output is. The
volume collected would be classified as small ( < 25 % blood volume) except that it can be varied by adding fibrin-rich PPP in the
case of the AFC. Platelet and Leukocyte concentration were good
(40–80 % of all available) and platelets were healthy after collection. The systems we tested could be activated with any agonist
so fibrin is delivered unaltered, however the ability to concentrate the fibrin in the PPP with the option to add this to PRP may
alter the classification.
One aspect that should be noted regarding the output of the AFC
is that the output can be purposefully modified by the user by
changing the height of the collection window while drawing. The
protocol used in the present study collected a portion of the red
cell pack, the buffy coat and the plasma portion resulting from a
single spin. The PRP included some red cells which, while biologically may have little impact in common use, but may be of
interest for future studies depending on the application. It is possible that the protocol could be altered to allow collections
excluding red cells, and possibly the leukocyte layer, although
this was not studied in the present study. The location of the
drawing window also dictates that a full blood draw be used with
the device so that the buffy coat layer is in the drawing area.



(approximately a 4–5 fold increase). Gociman et al. [17] summarized the reported platelet enrichments of 11 platelet-concentrating devices. These results showed fold increases ranging
from 1.34 to 6.07. In Gociman’s study the GPS produced a 4.86
fold increase on platelet count, which aligns well with the 5.19
increase seen here. The AFC was capable of increasing platelet
concentrations by a factor of 4.19 which places it roughly in the
middle of Gociman’s results. While many devices report fold
increases of 3–7 fold [3, 6, 43], it is important to note that highly
concentrated platelet preparations may have an inhibitory effect
on healing [20, 47].
The devices were capable of recovering 46.4 % (AFC) and 59.8 %
(GPS) of all available platelets which is in line with previous publications. Leitner [26] compared 4 devices, and reported results
of 17 % for the Vivostat PRF Preparation Kit (Vivosolution A/S, Birkeroed, Denmark), 70 % for the Harvest SmartPReP 2 APC 60 Process (Harvest Technologies Corp., Plymouth MA, USA) 67 % for the
PCCS Platelet Concentrate Collection System (3i PCCS, 3i Implant
Innovations, Palm Beach Gardens FL, USA) and 66 % for the Fibrinet Autologous Fibrin & Platelet System (Cascade Medical Enterprises Ltd, Plymouth, UK). Everts et al. [14] reported results for
the Autologous Growth Factor Filter (Interpore Cross, Irvine CA,
USA) at 32 %, the Electa Cell-Separator (Sorin Group, Irandola,
Italy) at 48 % but reports a lower value for the GPS Platelet Separation System (Biomet Biologincs, Inc., Warsaw IN) at 36 %.
Although the ideal concentration of platelets within PRP remains
unknown, the effect of PRP is likely to be linked closely to platelet concentration. Leitner et al. [26] has shown that PDGF release
is closely related to platelet count. Likewise Everts et al. [14]
demonstrated the need for devices to maintain platelet viability
during preparation to maximise growth factor release upon activation when required. Regardless of the device or method used,
platelet recovery and viability are 2 important factors when
evaluating the PRP product.

It is anticipated that the full characterisation of PRP produced by
the untested device and correlation with the existing device will
allow future studies to accurately assess what is being implanted.
Having this information widely available will also aid the practitioner in determining if the output is appropriate for the needs
of their patients.

Acknowledgements

▼

The work presented in this study was supported by funding
from Circle Biologics, Minneapolis, MN. No individual author
received funding from Circle Biologics or other sources in relation to this study.

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