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Clinical Biochemistry 42 (2009) 1452 – 1460

The effect of anticoagulants on the quality and biological efficacy

of platelet-rich plasma
Hua Lei, Lai Gui ⁎, Ran Xiao
Department Six of Plastic Surgery Hospital, Peking Union Medical College, China Academy of Medical Sciences, No.33 BaDaChu Road,
Shijingshan District, Beijing, 100144, China
Received 2 February 2009; received in revised form 12 June 2009; accepted 18 June 2009
Available online 26 June 2009

Abstract

Objectives: This study investigated the effect of anticoagulants on platelet-rich plasma (PRP) quality to determine the appropriate
anticoagulants for PRP production.
Design and methods: This study was carried out at the Plastic Surgery Hospital of Peking Union Medical College. The microstructure of
platelets collected with heparin, citrate, acid citrate dextrose (ACD) and citrate-theophylline-adenosine-dipyridamole (CTAD) was observed. The
extent of spontaneous activation of platelets was detected by measuring sP-selectin in plasma. The amount of TGF-β1 released from PRP and the
effect of PRP on cell proliferation were also studied.
Results: ACD and CTAD were superior to heparin and citrate in maintaining the integrity of platelet structures and preventing the platelet
spontaneous activation. ACD-PRP and CTAD-PRP released more TGF-β1 and significantly enhanced the proliferation of human marrow stromal
cells compared to heparin-PRP and citrate-PRP.
Conclusions: The PRP quality was closely related to the type of anticoagulants. ACD and CTAD are appropriate anticoagulants for PRP production.
© 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Keywords: Platelet-rich plasma; Anticoagulant; Platelet activation; Growth factor; Biological effect

Introduction (platelet-derived growth factor [PDGF], transforming growth

Platelets are derived from the cytoplasm of megakaryocytes.
The normal platelets are small, disc-shaped cells without a
nucleus, normally measuring 1 to 2 μm in diameter. The resting
platelet is divided into three zones. The peripheral zone consists
of fluffy glycocalyx coat, cytoskeleton and platelet membrane,
responsible for adhesion and aggregation. The sol–gel zone
contains the connecting system called the open canalicular
system and the dense tubular system, responsible for contrac-
tion and support microtubule system. The dense tubular system
is very rich in calcium and rich in phospholipase A2, cyclo-
oxygease and thromboxane synthease. The organelle zone
contains alpha-granules, dense granules, lysosomal granules,
glycogen granules [1]. Alpha-granules contain growth factors
⁎ Corresponding author.
E-mail address: guilaizhuren@163.com (L. Gui).
0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2009.06.012
factor-beta1 [TGF-beta1], insulin-like growth factor [IGF] and
epidermal growth factor [EGF]), but also other molecules such
as beta-thromboglobulin (beta-TG), platelet factor 4 (PF4), low
affinity platelet factor 4 (LA-PF4), fibrinogen, fibronectin,
thrombospondin, albumin, high-molecular-weight kininogens,
coagulation factor V, coagulation factor VIII, plasminogen,
histidine-rich glycoprotein, protein S, heparinase, elastase, an
inhibitor of collagenase, osteonectin, Vitronectin, etc [2].
Platelet-rich plasma (PRP) is a concentration of platelets in a
small volume of plasma. Upon activation, platelets in PRP
release the above-mentioned growth factors via exocytosis.
Theoretically, the local application of PRP might deliver a
higher level of growth factors to specific sites to stimulate tissue
healing [3,4]. For example, recent findings have suggested that
PRP not only enhances bone regeneration when repairing bone
defects and elevating the sinus floor [5–9] but also promotes
and accelerates osseointegration when placed in the preparation
site for a dental or bone implant [10,11].
 H. Lei et al. / Clinical Biochemistry 42 (2009) 1452–1460
Since the regenerative potency of PRP undoubtedly depends
on the level of growth factors released by platelets, the viability
of platelets determines the quality of PRP. If platelets are
activated during the PRP production process, growth factors in
platelets will be released into plasma most of which is discarded
during PRP preparation, which consequently decreases the
efficacy of PRP when applied to surgical sites. It is therefore
obvious that less platelet activation and more growth factor
retention in platelets are the keys to improving the potency of
PRP.
In hematological studies, the effect of anticoagulants on
platelet activation is an area of concern: if anticoagulants cannot
prevent, or may even promote, the spontaneous activation of the
ex vivo platelets, this would affect the hematologic variables
being measured, which in turn could interfere with the clinical
evaluation and management of patients at risk from thrombotic
and bleeding disorders. Therefore, it is important to seek an
appropriate anticoagulant to avoid platelet spontaneous activa-
tion in samples used for pathophysiological studies [12–16].
Unfortunately, in both the clinical application and the basic
research of PRP, the issue of anticoagulant choice has not been
given sufficient attention. Various kinds of anticoagulants are
used in PRP preparation [5,8,10,11,17–20], and there have been
no reports investigating the association between anticoagulant
choice and the biological effects of PRP. Therefore, we
conducted a comparative study to investigate the effect of
anticoagulants on PRP quality and biological efficacy, to clarify
the appropriate anticoagulant choice in PRP production.
We studied anticoagulants commonly used in PRP prepara-
tion or hematology, including heparin, citrate, acid citrate
dextrose and citrate-theophylline-adenosine-dipyridamole. The
quality of PRP undoubtedly depends on platelet status, so
changes in platelet morphology and the extent of spontaneous
activation of platelets collected using different anticoagulants
were investigated. In addition, the amount of growth factor
released from activated PRP and the biological effect of PRP on
cell proliferation were studied.
This study was carried out at the research center of Plastic
Surgery Hospital of Peking Union Medical College. 30
volunteer donors provided their whole blood and 3 volunteer
donors provided their bone marrows for our study.
Materials and methods
Special reagents and apparatus
Sodium citrate (SC) 0.129M; heparin sodium (HS) 1000 U/
mL; ACD, composed of 0.48% (w/v) citric acid, 1.32% (w/v)
sodium citrate and 1.47% (w/v) glucose; CTAD, composed of
0.11 mM/L citrate, 15 mM/L theophylline, 3.7 mM/L adenosine
and 0.198 mM/L dipyridamole; and activating solution, consist-
ing of 10,000 U of human thrombin (Sigma, China) dissolved in
1 mL 10% CaCl2. Unless otherwise stated, the chemicals were
obtained from Sigma (Steinheim, Germany). The instruments
used were a laboratory-standard centrifuge (KUBOTA 5500,
Japan), transmission electronic microscope (TEM) (EM 301,
Phillips, Eindhoven, Netherlands), microcentrifuge (Hitachi
1453
CT15E, Japan) and microplate spectrophotometer (SpectraMax
190, Sunnyvale,USA).
Collection of blood and experimental protocol
The study was conducted with the informed consent of
volunteer subjects and approval from the Ethics Committee of
the Peking University Health Science Center, Beijing, China.
The 30 volunteer donors, 10 male and 20 female, aged between
18 and 41 with an average of 26-years-old, were enrolled in this
experiment. All donors were on no medications, including
aspirin and other non-steroidal anti-inflammatory drugs, during
the preceding 2 weeks. The experimental protocol is summar-
ized in Fig. 1. The first 1–2 mL of blood was discarded to avoid
the effects of traces of thrombin generated during venipuncture.
The 16 mL whole blood anticoagulated by SC was drawn
from each donor and centrifuged at 302 g for 15 min at 20 °C.
The plasmas of three donors in each experiment were mixed and
centrifuged at 1209 g for 15 min at 20 °C. The upper, platelet-
poor plasma (PPP) was named SC-PPP and used to resuspend
the platelet pellets.
To study platelet spontaneous activation and growth factor
(TGF-β1) release from activated PRP, 28 mL whole blood
samples from each donor were drawn into four 10 mL syringes
which contained 1 mL ACD, CTAD, SC and HS, respectively,
with 7 mL of blood going into each syringe. The whole blood in
the 12 syringes from 3 donors was transferred to 12 conical
bottom centrifuge tubes (15 mL, Corning, USA) and centri-
fuged at 302 g for 10 min at 20 °C. Yellow plasma from 3 tubes
of each anticoagulant group was pooled in one 15 mL centrifuge
tube. Following manual platelet counting, the pooled plasma
volume from each group was adjusted to ensure that the total
platelet count of each of the four groups was equal. The adjusted
pooled plasma of each group was quartered and aliquots were
centrifuged at 1209 g for 10 min at 20 °C at 2 h, 6 h, 12 h and
20 h post-venipuncture. All samples were stored at room
temperature before centrifugation. After all of the upper PPP
was transferred to another tube, the platelet pellet and a few of
the red cells at the bottom of tubes were resuspended by 1.0 mL
SC-PPP; the resulting mixture was platelet-rich plasma (PRP).
The PPP and PRP samples were coagulated by adding 30 μL
activation solution, permitted to clot overnight at 4 °C, stirred
by pipette and centrifuged at 2150 g for 10 min at 4 °C. The
clear supernatants were named PPP-serum and PRP-serum and
were transferred to microcentrifuge tubes (Eppendorf, China)
and stored at − 70 °C until used.
For TEM observation of platelets, the pooled plasma of each
anticoagulant group was quartered without counting the
platelets or making volume adjustments. Aliquots were
centrifuged at 1209 g for 10 min at 20 °C at 2 h, 6 h, 12 h
and 20 h post-venipuncture. The upper PPP was discarded,
while the platelet pellet was processed for TEM analysis. All
samples were stored at room temperature before centrifugation.
For the preparation of PRP culture medium, the volume of
pooled plasma from each of the four groups was adjusted to
produces equal total amounts of platelets. The adjusted, pooled
plasma was not quartered and was directly centrifuged at 1209 g
1454 H. Lei et al. / Clinical Biochemistry 42 (2009) 1452–1460

Fig. 1. The experimental protocol. ACD, acid citrate dextrose; CTAD, citrate-theophylline-adenosine-dipyridamole; HS, heparin sodium; SC, sodium citrate; WB,
whole blood; P, plasma; PP, platelet pellet; PPP, platelet-poor plasma; PRP, platelet-rich plasma; TEM, transmission electron microscopy. The plasmas of three donors
anticoagulated by one kind of anticoagulant were mixed. For the measurement of sP-selectin in PPP-serum and TGF-β1 in PRP-serum, the volumes of four kinds of
pooled plasmas were adjusted to assure the equal platelet amount; the adjusted pooled plasmas were quartered and aliquots were centrifuged at four time points to
obtain PPP and PRP. For TEM observation, the pooled plasma of each anticoagulant group was quartered without counting the platelets and centrifuged at four time
points to obtain platelet pellets. For the preparation of cell culture medium, after the volume adjustment, the pooled plasmas were centrifuged to obtain platelet pellets
which were resuspended in SC-PPP to form PRP.

for 10 min at 20 °C. The upper PPP was discarded and the lower
platelet pellet and a few red cells were resuspended in 1.5 mL
SC-PPP to form PRP, which was immediately activated by
adding 30 μL activation solution and stored overnight at 4 °C to
allow fully clotting. 4.5 mL serum-free Dulbecco's modified
minimum essential medium (DMEM, Gibco BRL, Grant Island,
NY, USA) was added to the activated PRP, the mixture was
centrifuged at 2150 g for 15 min at 4 °C, and the clear
supernatant (named ACD-PRP, CTAD-PRP, NC-PRP or HS-
PRP medium) was transferred to another tube and stored at 4 °C
until used for cell culture. The TGF-β1 concentration in the
PRP medium was measured.
 H. Lei et al. / Clinical Biochemistry 42 (2009) 1452–1460
Platelet processing for TEM
Platelet pellets were fixed with 2.5% glutaraldehyde in
phosphate buffer for 24 h, and then post-fixed with 1% osmium
tetroxide. Specimens were dehydrated in a series of graded
alcohols and embedded in Epon 812 following standard
methods. Ultrathin sections were stained with uranyl acetate
and lead citrate before being examined and photographed in the
TEM at 80 Kv accelerating voltage.
Measuring the level of spontaneous platelet activation
P-selectin is a component of platelet alpha-granules that is
expressed on the platelet surface membrane and shed into the
plasma (as soluble P-selectin, sP-selectin) following platelet
activation [21]. We measured levels of sP-selectin in plasma
collected using the four anticoagulants at 2 h, 6 h, 12 h and 20 h
post-venipuncture to evaluate the extent of spontaneous platelet
activation in vitro. The PPP-serum samples were thawed and
centrifuged at 12,000 g for 10 min in a microcentrifuge
immediately before assay at room temperature. The sP-selectin
was determined using commercially available ELISA kits
(Quantikine, R&D System, Minneapolis, MN, USA) according
to the manufacturer's instructions. Measurements were per-
formed in duplicate, and no unexpected scattering of the data
(b10%) was observed. This experiment was repeated for 10
times and 3 donors were included each time.
Measuring the amount of TGF-β1 released from activated PRP
TGF-β1 is one of main growth factors released by platelets
[3,4]; therefore, the amount of TGF-β1 released from activated
PRP was measured as an indicator of PRP quality. The PRP-
serum samples were thawed and centrifuged at 12,000 g for
10 min in a microcentrifuge immediately before assay at room
temperature to analyze growth factor content. The TGF-β1
concentration was determined using commercially available
ELISA kits (Quantikine, R&D System, Minneapolis, MN,
USA) according to the manufacturer's instructions. Both active
and latent forms of TGF-β1 can be detected with this ELISA
assay. Measurements were performed in duplicate, and no
unexpected scattering of the data (b 10%) was observed.
Because the volume of each PRP sample was equal, the
resultant TGF-β1 concentration in the PRP-serum represented
the total amount of TGF-β1 released from the PRP. This
experiment was repeated for 10 times and 3 donors were
included each time.
Cell isolation
Human bone marrow stromal cells (hBMSCs) were isolated
from whole bone marrow aspirates from the iliac crest of donors
using a modification of the method previously reported [22]. In
brief, heparinized bone marrow was fractionated over a Ficoll-
PaqueTM Plus solution (Amersham Biosciences, Uppsala,
Sweden). The mononuclear cells were plated at a concentration
of 2 × 106 nucleated cells/cm2 and incubated in a basic culture
1455
medium composed of DMEM supplemented with 10% fetal
bovine serum (FBS; Hyclone, Logan, UT, USA), 100 U/mL
penicillin, and 100 mg/mL streptomycin at 37 °C and 5% CO2.
hBMSCs were isolated from bone marrow cells by removing
unattached cells when the medium was exchanged for the first
time, 5 days after plating. hBMSCs were incubated and allowed
to proliferate until 80% confluent. The cells were trypsinized
and subcultured as passage 1. The cells used in the experiments
were passage 2.
Cell culture
The passage 2 hBMSCs were seeded in 96-well plates
(Corning Life Sciences, Acton, MA, USA) at a density of
1 × 104 cells/well and were incubated in 100 μL DMEM
containing 10% FBS. Medium was removed after 24 h and
replaced with PRP culture medium. The cells were divided into
five different groups: (i) ACD group, cultured in ACD-PRP
medium; (ii) CTAD group, cultured in CTAD-PRP medium;
(iii) SC group, cultured in SC-PRP medium; (iv) HS group,
cultured in HS-PRP medium; and (v) control group, cultured in
SC-PPP medium composed of 4.5 mL DMEM and 1.5 mL SC-
PPP-serum. This test was with 10 samples from each group. The
hBMSCs were grown for 72 h without the medium being
changed. The assessment of cell proliferation was conducted at
4 h, 24 h, 48 h, and 72 h.
Assessment of cell proliferation
The cell numbers were determined using the 3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenylterazolium bromide (MTT)
assay at 4 h, 24 h, 48 h and 72 h. The MTT assay was carried out
using a cell proliferation kit protocol (Promega, US). When
cells had been incubated with 15 μL dye solution for 4 h,
tetrazolium salts were transformed by active enzymes in the
cells into intracellular formazan deposits. The formazan salts
were dissolved with 100 μL Trizol and the absorbance was
determined at 570 nm. The amount of color produced was
directly proportional to the number of viable cells.
Three donors' BMSCs were studied in this experiment. Each
donor's BMSCs were cultured for three times. The PRP
medium was made of three donors' pooled plasmas each time.
Statistical analysis
Data are presented as the median ± SD. Single-factor analysis
of variance (ANOVA) was used in conjunction with a multiple
comparison test (Tukey's test) to test for differences among the
groups. Differences were considered to be statistically sig-
nificant when the p values were b0.05.
Results
Characterization of platelet microstructure by TEM
In order to understand the effects of different anticoagulants
and the passage of time, platelets collected in ACD, CTAD, HS,
1456 H. Lei et al. / Clinical Biochemistry 42 (2009) 1452–1460

and SC anticoagulated whole blood were analyzed by TEM at
2 h, 6 h, 12 h and 20 h post-venipuncture at room temperature.
The overall platelet size, shape and intracellular contents were
observed as previously described [14].
At 2 h post-venipuncture, all platelets had some pseudopo-
dias and, consequently, irregular shapes. Most CTAD platelets
had a discoid shape and smaller size, whereas ACD, HS and SC
platelets had a more rounded shape and larger size. CTAD and
ACD platelets had more alpha-granules and dense granules with
fewer vacuoles than HS and SC platelets (Fig. 2).
At 6 h, ACD and CTAD platelets became bigger and more
rounded with increased numbers of vacuoles, but did not
display a significant decrease in alpha-granules and dense
granules. The characteristics of HS and SC platelets were not

Fig. 2. Representative TEM microphotos of platelet samples collected with ACD, CTAD, HS or SC at 2 h, 6 h, 12 h and 20 h post-venipuncture (original
magnification × 10,000). ACD platelets show gradual large and rounding contours over time, with increased vacuoles and decreased alpha-granules and dense-bodies;
at 20 h some of them had fuzzy intracellular structures. CTAD platelets had a small size and discoid shape at 2 h, then became large and rounded at 6 h; at 20 h, alpha-
granules and dense-bodies decreased and vacuoles increased, but the intracellular structures were still intact. HS and SC platelets show large contours from 2 h and had
significantly fewer alpha-granules and dense-bodies than ACD and CTAD platelets in all photos. At 12 h, half the HS platelets were undergoing lysis and at 20 h almost
all of them hadcollapsed. A few SC platelets began to lyse at 12 h and the majority of SC platelets were lysed at 20 h.
 H. Lei et al. / Clinical Biochemistry 42 (2009) 1452–1460
obviously different from the 2 h samples except that they had
fewer pseudopodias (Fig. 2).
At 12 h, CTAD platelets had similar characteristics to the 6 h
sample. The ACD sample had one or two platelets beginning to
lyse and missing all intra-platelet structures, but most ACD
platelets were not obviously different to the 6 h sample. The SC
sample showed some lysed platelets, but most platelets were
intact just containing less alpha-granules and dense granules. In
the HS sample almost half the platelets were lysed and the
remaining intact platelets contained very few alpha-granules
and dense granules (Fig. 2).
At 20 h, the number of alpha-granules and dense granules in
ACD platelets decreased and the intra-structures of some
platelets became fuzzy, but the platelet membranes were intact.
CTAD platelets not only maintained intact cell membranes and
clearly-visualizable intra-structures, but still contained some
alpha-granules and dense granules, although more and larger
vacuoles appeared. However, the majority of HS and SC
platelets were lysed and the cell structures were hard to visualize.
sP-selectin concentration in PPP
The sP-selectin concentrations in different PPPs at different
time points are shown in Fig. 3. At the four time points, the
lowest concentration was measured in the ACD-PPP and the
highest in the HS-PPP or the SC-PPP.
At 2 h post-venipuncture, the sP-selectin concentrations in
the four kinds of PPP were similar (p N 0.05). At 6 h post-
venipuncture, the sP-selectin concentration in the SC-PPP was
approximately 8.4 fold (p b 0.05), 4.7 fold (p b 0.05), and 1.7
fold (p N 0.05) higher than that in the ACD-PPP, the CTAD-PPP,
and the HS-PPP respectively; there was no significant
difference among the ACD-PPP, CTAD-PPP and HS-PPP. At
Fig. 3. The sP-selectin concentration in different PPP at 2 h, 6 h, 12 h, 20 h post-
venipuncture. The CD, CTAD, HS, and SC-PPP are platelet-poor plasmas
prepared using ACD, CTAD, HS, or SC, respectively. The sP-selectin
concentrations in ACD-PPP and CTAD-PPP were significantly less than those
in SC-PPP at 6 h, 12 h, 20 h post-venipuncture (p b 0.05) and those in the HS-
PPP at 12 h, 20 h post-venipuncture (p b 0.05). From 2 h to 20 h post-
venipuncture, the sP-selectin concentrations in the HS-PPP and SC-PPP
significantly increased (p b 0.05), whereas those of the ACD-PPP and CTAD-
PPP did not significantly increased (p N 0.05).
1457
Fig. 4. TGF-β1 concentration in the supernatants of different activated PRPs.
The TGF-β1 concentration represents the total amount of TGF-β1 released from
PRP. The CD, CTAD, HS, and SC-PRP are platelet-rich plasmas prepared using
ACD, CTAD, HS, or SC, respectively. The ACD-PRP and CTAD-PRP released
significantly more TGF-β1 than the HS-PRP and SC-PRP at all time points
(p b 0.05), while the differences between ACD-PRP and CTAD-PRP (p N 0.05)
and between HS-PRP and SC-PRP (p N 0.05) were not significant. From 2 h to
20 h, the TGF-β1 concentration in four groups of PRP-serums all decreased (the
ACD-PRP decreased 26.43%, the CTAD-PRP 28.5%, the HS-PRP 33.7%, the
SC-PRP 43.0%), but these decreases were not significant (p N 0.05).
12 h post-venipuncture, the sP-selectin concentration of the SC-
PPP was approximately 14.5 fold (p b 0.05) and 5.2 fold
(p b 0.05) higher than that in the ACD-PPP and the CTAD-PPP,
respectively; the HS-PPP was approximately 12.0 fold
(p b 0.05) and 4.4 fold (p b 0.05) higher than the ACD-PPP
and the CTAD-PPP, respectively; there was no significant
difference between the ACD-PPP and CTAD-PPP and between
the HS-PPP and SC-PPP. At 20 h post-venipuncture, the sP-
selectin concentration of the SC-PPP was approximately 12.1
fold (p b 0.05) and 3.9 fold (p b 0.05) higher than that in the
ACD-PPP and the CTAD-PPP; the HS-PPP was approximately
13.8 fold (p b 0.05) and 4.4 fold (p b 0.05) higher than the ACD-
PPP and CTAD-PPP, respectively; there was no significant
difference between the ACD-PPP and CTAD-PPP and between
the HS-PPP and SC-PPP. From 2 h to 20 h, the sP-selectin
concentration of ACD-PPP changed very little; the sP-selectin
concentration of the CTAD-PPP, HS-PPP and SC-PPP
increased by 2.5 fold (p N 0.05), 5.2 fold (p b 0.05) and 6.4
fold (p b 0.05) respectively.
Amount of TGF-β1 release from activated PRP
The TGF-β1 concentration in PRP-serum, indicating the
amount of TGF-β1 release from activated PRP, is shown in
Fig. 4. At all time points, the CTAD-PRP and ACD-PRP
released significantly more TGF-β1 than the HS-PRP and SC-
PRP (p b 0.05). There was no significant difference between the
ACD-PRP and CTAD-PRP and between the HS-PRP and SC-
PRP at all time points. At 2 h, 6 h, 12 h, 20 h post-venipuncture,
the TGF-β1 concentrations in the ACD-PRP and CTAD-PRP-
serum were approximately 3.0 fold, 3.2 fold, 3.3 fold and 3.1
1458 H. Lei et al. / Clinical Biochemistry 42 (2009) 1452–1460
Fig. 5. Effect of different PRP mediums on the proliferation of hBMSCs. During
the 72 h period, the number of hBMSCs increased 4.4 fold in the ACD-PRP
(p b 0.05) and CTAD-PRP groups (p b 0.05), 3.3 fold in the SC-PRP group
(p b 0.05), 3.2 fold in the HS-PRP group (p N 0.05), and 2.6 fold in the control
group (p b 0.05). At 72 h, the numbers of hBMSCs in the ACD-PRP and CTAD-
PRP groups were similar (p N 0.05), while they were approximately 1.4 fold
more than those in the SC-PRP and HS-PRP groups (p b 0.05), and 2.0 fold more
than that in the control group (p b 0.05); the SC-PRP and HS-PRP groups had 1.3
fold more hBMSCs than the control group (p b 0.05).
fold higher than that in the HS-PRP-serum (p b 0.05), and 1.9
fold, 2.1 fold, 2.0 fold and 2.5 fold higher than that in the SC-
PRP-serum (p b 0.05), respectively. From 2 h to 20 h, all kinds
of PRP-serum samples had a decrease in the TGF-β1
concentration (the ACD-PRP group decreased 26.43%, the
CTAD-PRP group 28.5%, the HS-PRP group 33.7%, the SC-
PRP group 43.0%), but these decreases were not significant
(p N 0.05).
Biological effects of activated PRP medium on hBMSC
proliferation
During the 72 h period, the hBMSCs cultured with all kinds
of mediums proliferated significantly (p b 0.05). The hBMSCs
cultured with the SC-PPP medium showed the smallest increase
in proliferation (2.6 fold), whereas the hBMSCs incubated with
the different PRP mediums had more rapid increases in
proliferation (4.4 fold in the ACD-PRP and CTAD-PRP groups,
3.3 fold in the SC-PRP group, 3.2 fold in the HS-PRP group)
Table 1
The concentration of TGF-β1 in the PRP mediums and the control medium.
ACD-PRP CTAD-PRP
TGF-β1 (ng/mL) 30.3124 ± 1.1230 27.4150 ± 2.0311
The data are shown as means ± SD. PRP, platelet-rich plasma; ACD, acid citrate dextrose; CTAD, citrate-theophylline-adenosine-dipyridamole; HS, heparin sodium;
SC, sodium citrate.
(Fig. 5). Considering the different proliferation rates, at 72 h, the
numbers of proliferating hBMSCs in the ACD-PRP and CTAD-
PRP groups were similar (p N 0.05), while they were approxi-
mately 1.4 fold greater than those in the SC-PRP and HS-PRP
groups (p b 0.05) and 1.8 fold greater than that in the control
group (p b 0.05); the SC-PRP and HS-PRP groups had 1.3 fold
more hBMSCs than the control groups (p b 0.05). The
concentration of TGF-β1 in the activated PRP medium and
control medium is shown in Table 1.
Discussion
ACD is the anticoagulant used by blood banks to store viable
platelets for platelet transfusions because ACD can maintain
platelet viability for up to 6 h [12]. Some researchers suggest
clinicians should use ACD to produce PRP [2]. Therefore, we
selected ACD as a positive control anticoagulant against which
to evaluate other anticoagulants. We found that ACD
maintained the structural integrity of platelets in PRP for up
to 12 h, at which point one half of the HS platelets and a few of
the SC platelets began to lyze (Fig. 2). Additionally, the ACD
platelets contained more alpha-granules and dense granules than
the HS and SC platelets at all time points (Fig. 2), which agrees
with the findings that the level of the ACD platelet spontaneous
activation was significantly lower than the HS and SC platelet
(Fig. 3) and the ACD-PRP released significantly more TGF-β1
than the HS-PRP and SC-PRP during the first 12 h post-
venipunure (Fig. 4). The superior effects of ACD on
maintaining platelet viability may be due to its glucose and
low citrate (1.32%) concentrations [23]. Glucose is necessary
for platelet energy metabolism during the in vitro storage
period, and depletion of glucose and subsequent significantly
reduced levels of adenine nucleotides have been associated with
loss of platelet viability [24–26]. The low citrate concentration
helps avoid excessive lactate production and a fall in pH, which
is associated with substantial loss of platelet viability [27].
Heparin is not used in coagulation studies because it
activates platelets in vitro, which potentially interferes with
the determination of hemostatic parameters [16]. However,
heparin is usually used in collecting whole blood for PRP
production [5,11,28], so it was also included in this study.
Unfortunately, TEM microphotos showed that the platelets in
the HS-PRP contained fewer alpha-granules and dense granules
and began lysis earlier than other groups (Fig. 2). The higher sP-
selectin level was found in the HS-PPP at 6 h, 12 h and 20 h
post-venipuncture (Fig. 3), which may indicated that HS
stimulated or could not prevent platelet spontaneous activation.
In accordance with these results, the HS-PRP released the least
TGF-β1 at any time point (Fig. 4).
HS-PRP SC-PRP Control
9.1927 ± 1.9017 11.3083 ± 1.3235 0.5878 ± 0.1945
 H. Lei et al. / Clinical Biochemistry 42 (2009) 1452–1460
For many years, citrate was the anticoagulant preferred by
most investigators undertaking platelet studies [29], but some
researchers have suggested that it causes platelet activation and
platelet aggregation, rendering it unsuitable for functional
platelet assays [30]. As sodium citrate is the commonly used
anticoagulant in PRP preparation for clinical or research
applications [6,17,18,31,32], we studied its suitability for PRP
production and found that the SC platelets showed fewer alpha-
granules and dense granules, earlier lysis (Fig. 2) and more
spontaneous activation (Fig. 3) and the SC-PRP released
significantly less growth factor (Fig. 4) than the CTAD and
ACD-PRP (p b 0.05).
Adding a mixture of three platelet antagonists – theophyl-
line, adenosine and dipyridamole (the mixture is known as
CTAD) – to citrate can inhibit platelet activation in vitro for at
least 4 h [33], therefore CTAD has been an optimal antic-
oagulant for assessing platelet activation states in vivo, which
offer advantages in clinical hematologic investigations [13].
However, so far, CTAD has not been used in PRP production.
We wanted to determine the feasibility of using CTAD as an
anticoagulant to produce PRP and to compare its effects with
ACD. Our results showed that CTAD could maintain platelet
integrity in vitro for at least 20 h (Fig. 2), which was 8 h longer
than ACD. Charaf et al. reported that CTAD platelets acquired a
rounder shape at 60 min but later tended to return to their basal
shape, which they maintained until 24 h [14]. However our
TEM microphotos showed that CTAD platelets had discoid
shapes at 2 h, then became larger and rounder at 6 h, and kept
the round shape till 20 h (Fig. 2). This may be because our
platelet sample was collected by the two-step centrifuge
method, which might mechanically damage platelets, whereas
Charaf et al. collected platelets directly by 1.5% heated
glutaraldehyde without centrifuge. We found the CTAD-PRP
released as much TGF-β1 as the ACD-PRP (Fig. 4). Moreover,
CTAD resulted in significantly less platelet spontaneous
activation than HS and SC (p b 0.05), and a little more than
ACD (p N 0.05) (Fig. 3).
Previous studies have used the plasma sP-selectin level to
detect the degree of platelet spontaneous activation in vivo
[34–36] or artificial activation during platelet pheresis [37], and
demonstrated good results. In this study, we took sP-selectin as
an indicator of platelet spontaneous activation in vitro in order
to assess PRP quality. Our findings suggest it is an applicable
index of PRP quality. It is because when the number of alpha-
granules and dense granules in all platelet samples gradually
fell (Fig. 2) and four groups of PRP released less and less TGF-
β1 (Fig. 4) from 2 h to 20 h post-venipuncture, the sP-selectin
level in plasma increased, especially in HS-PPP and SC-PPP.
A final purpose to improve the quality of PRP is to maximize
the biological effect of PRP. To further verify the conclusions
from our TEM findings and the assessment of the sP-selectin
level in plasma and the amount of growth factor release from
PRP, we used a PRP medium made of activated PRP
supernatant and DMEM to culture hBMSCs. The biological
effects of PRP were reflected in the hBMSC proliferation rate.
The ACD and CTAD-PRP mediums significantly stimulated
cell proliferation compared with the SC and HS-PRP medium
1459
(Fig. 5). The various PRP samples had the same number of
platelets, plasma quality and quantity, activation method, and
preparation procedure but different platelet viability status; we
therefore conclude that the biological effect of PRP depends
mainly on the platelet viability which we found to be associated
with the type of anticoagulant used.
Our methods of PRP medium preparation and cell culture
were similar to those in the study of Han et al. except that they
used citrate-phosphate-dextrose as an anticoagulant [38]. Their
results suggested when the TGF-β1 concentration in a PRP
medium was less 50 ng/mL, different PRP mediums did not have
significant abilities to stimulate the proliferation of human
periodontal ligament cells. However, in our study, the ACD and
CTAD-PRP mediums containing 30 and 27 ng/mL TGF-β1
significantly stimulated the proliferation of hBMSCs compared
with the HS-PRP and SC-PRP medium containing 9 and 11 ng/
mL TGF-β1. Perhaps the response of different cell lineages to
PRP is different, or perhaps different anticoagulants used in PRP
preparation or particular components in different PRPs – such as
other growth factors [39–41], cytokines [42] and fibrinogens
[39] – regulate hBMSC proliferation differently. Future studies
should be conducted to examine these assumptions.
In summary, we demonstrated that anticoagulants influence
the quality of PRP which in turn is directly associated with its
biological effects. Compared with heparin and citrate, the
compound anticoagulants ACD and CTAD maintained platelet
integrity for a longer time, reduced platelet spontaneous
activation, increased the amount of growth factor released
from PRP and, consequently, improved the efficacy of PRP in
stimulating cell proliferation. These findings provide convin-
cing evidence and useful data for clinicians and researchers who
use PRP to facilitate tissue regeneration or wound healing.
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