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Vox Sanguinis (2008)

2008 The Author(s)


Journal compilation 2008 Blackwell Publishing Ltd.
DOI: 10.1111/j.1423-0410.2007.01027.x

ORIGINAL PAPER

Platelet-rich plasma and platelet gel preparation using


Plateltex

Blackwell Publishing Ltd

L. Mazzucco, V. Balbo, E. Cattana & P. Borzini


Blood Transfusion Centre and Biotechnology Laboratory, Ospedale SS Antonio e Biagio, Alessandria, Italy

Background The platelet gel is made by embedding concentrate platelets within a


semisolid (gel) network of polymerized fibrin. It is believed that this blood component
will be used more and more in the treatment of several clinical conditions and as an
adjunctive material in tissue engineering. Several systems are available to produce
platelet-rich plasma (PRP) for topical therapy. Recently, a new system became
commercially available, Plateltex. Here we report the technical performance of this
system in comparison with the performance of other commercially available systems:
PRGF, PRP-Landesber, Curasan, PCCS, Harvest, Vivostat, Regen and Fibrinet.
Material and Methods Both the PRP and the gel were prepared according to the
manufacturers directions. The blood samples of 20 donors were used. The yield, the
efficiency, and the amount of platelet-derived growth factor AB (PDGF-AB), transforming growth factor , vascular endothelial growth factor and fibroblast growth
factor were measured in the resulting PRP. The feature of the batroxobin-induced
gelation was evaluated.
Results The yield, the collection efficiency and the growth factor content of Plateltex
were comparable to those of most of the other available systems. The gelation time
was not dependent on the fibrinogen concentration; however, it was strongly influenced by the contact surface area of the container where the clotting reaction took
place (P < 00001).

Received: 11 September 2007,


revised 28 November 2007,
accepted 29 November 2007

Conclusions Plateltex provided platelet recovery, collection efficiency and PDGFAB availability close to those provided by other systems marketed with the same
intended use. Batroxobin, the enzyme provided to induce gelation, acts differently
from thrombin, which is used by most other systems. Platelets treated with thrombin
become activated; they release their growth factors quickly. Furthermore, thrombin
platelet interaction is a physiological mechanism that hastens the clot-retraction rate.
On the contrary, platelets treated with batroxobin do not become activated; they are
passively entrapped within the fibrin network, and their growth factor release occurs
slowly. In these conditions, the clot retraction takes longer to occur. According to
these differences between thrombin and batroxobin, it is expected that batroxobininduced PRP activation will tailor slow release of the platelet content, thus, providing
longer in loco availability of trophic factors. In selected clinical conditions, this durable
anabolic factor availability might be preferable to quick thrombin-induced growth
factor release.
Key words: batroxobin, platelet gel, platelet-rich plasma.

Correspondence: Laura Mazzucco, Blood Transfusion Centre and Biotechnology Laboratory, Ospedale SS Antonio e Biagio, Via Venezia 16, 15100 Alessandria,
Italy
E-mail: lmazzucco@ospedale.al.it

2 L. Mazzucco et al.

Introduction
Platelet-rich plasma (PRP) represents an emerging biotechnology in current tissue engineering and cellular therapy; it
is likely that platelet gel will be used widely in future for soft
and bony tissue repair [1]. Several devices to produce PRP
and platelet gel have been commercially released for topical
therapy. There is increasing evidence regarding the clinical
use of platelet gel [27]. Somehow more intriguing is the
concern about the equivalence or the differences among the
methods or the devices available to produce the PRP [814].
Prior to clinical use of a new device, its technical performance
must be tested and, if possible, the results should be compared to those of other products with the same intended use.
The aim of this report is to evaluate the efficiency of PRP
preparation using the Plateltex kit (Plateltex, Bratislava,
Slovakia) and to compare the results with those obtained
from other similar devices.
Increasing consent is accumulating that PRP products
must achieve a platelet count of at least 1 109/ml to be in
the therapeutically effective range, indicating approximately
a 45 times the baseline value [1,15,16].
Taking this enrichment as a prerequisite of the study, in
this report the performances of the Plateltex platelet gel
preparation kit have been tested. The technical end-point of
the study were: (i) the platelet yield, (ii) the collection efficiency, (iii) the gelation time, (iv) the concentration of some
platelet-derived growth factors (PDGF). The kinetics of the
gelation and some properties of the gelation-inducing enzyme,
batroxobin, were evaluated as well.

Material and methods


For clinical use, the Plateltex instruction sheet maintains
that platelets must reach the concentration of 12 109/ml.
To achieve this result, a two-step procedure is indicated. First,
a soft spin centrifugation to obtain the PRP. Second, a highspin centrifugation for the pelleted platelets to be brought to
the desired final concentration.
The blood samples from 20 consenting blood donors were
used for the technical analysis. To prepare the PRP and to
induce its gelation, the materials provided by the manufacturer were used and the provided instructions followed.
The peripheral blood was collected in four ACD-A (anticoagulant citrate-dextrose, formula A)-containing tubes. Sixteen
millilitres of blood were drawn from each donor (8 ml per
tube, each containing 09 ml of ACD-A solution; final blood:
ACD ratio 10 : 1). The PRP was collected after centrifugation
at 180 g for 10 min with a common bench centrifuge (ALC
4237; Thermo Scientific Co., Waltham, MA, USA) equipped
with a swing-out 090 rotating system. The platelet count
was performed using ABX Micros 60 cytometer (Horiba ABX,
Shefford, UK). Keeping into account the platelet concentration,

the PRP was centrifuged at 1000 g for 10 min, appropriate


amount of platelet pooz plasma (PPP) was removed accordingly
to adjust the platelet concentration to 2 109/ml.
Gelation of the PRP was induced adding the activation solution to the PRP. Briefly, 1 ml of calcium gluconate was harvested
from the provided vial. This was injected into the sterile vial
containing 5 BU of lyophilized purified batroxobin, the fibrinogenactivating enzyme that clives fibrinopeptide A, thus, inducing
fibrin polymerization and quick formation of three-dimensional
platelet-embedding fibrin mesh. One millilitre of calciumenriched batroxobin solution provides enough material for
PRP gelation to occur without noticeable PRP dilution.
The gelation time was measured considering two macroscopic end-points: the appearance of visible fibrin polymerization and the occurrence of the gel solidification (the gel
remaining attached to the surface of the Petri dish after
turning it upside down). The kinetics of the gelation was also
determined by serial measurements of the optical density
(wavelength 405 nm) in 96-well culture trays.
PDGF-AB, transforming growth factor (TGF-), fibroblast
growth factor (FGF) and vascular endothelial growth factor
(VEGF) concentration were quantified using enzyme-linked
immunosorbent assay (ELISA) methods (all human ELISA kits
were obtained from R&D Systems, Wiesbaden, Germany).
The analyses were then performed as instructed by manufacturer. Accordingly, PRP samples adjusted to 2 109 platelets/
ml were pretreated as directed through the manufacturers
instructions. Standards and the test samples were dispensed
into the wells of growth factor antibody-coated plates. After
incubation and removal of unbound substances, an enzymecoupled secondary antibody was added. After washing, the
substrate solution was added. After stopping the colourimetric
reaction, the optical density was measured at the appropriate
wavelength.
The results obtained using Plateltex were compared to the
results obtained from other devices, taking these data from
the literature. For such comparison, the results of the following systems were selected: PRGF kit G.A.C. Medicale, San
Antonio, Vitoria, Spain; PRP according to Landesber [14];
AG Curasan Autologous Growth Factors PRP kit, Kleinostheim, Germany; PCCS Platelel Concentrate Collection
System, 3i Implant Innovation Iberica S.I., Barcelona, Spain
and Palm Beach Garden, FL, USA; Harvest SmartPReP 2 APC60
Process Kit, Harvest Technologies, Plymouths, MA, USA;
Vivostat, PRF Preparation Kit, Vivolution A/S, Birkeroed,
Denmark; Regen PRP Kit, ReGenLab SA, Mollens, Switzerland; and Fibrinet Autologous Fibrin & Platelet System,
Cascade Medical Enterprises Ltd, Plymouth, UK.

Statistical analysis
The mean and the standard deviation were determined when
relevant to evaluation of the end-points of the study. To

2008 The Author(s)


Journal compilation 2008 Blackwell Publishing Ltd., Vox Sanguinis (2008)

PRP preparation using plateltex 3

Table 1 Volume, platelet count, yield and collection efficiency after platelet-rich plasma (PRP) sequestration obtained through soft-spin centrifugation of the
whole blood performed according to the manufacturers directions. The volume of the concentrated platelets following high-speed centrifugation (desired final
concentration 2 109/ml) and that of the platelet-poor plasma are reported in the last two columns (right)
Whole blood

PRP

Volume (ml)

Donor

ml

l 103
Platelet/

Platelet
absolute 109

ml

l 103
Platelet/

Platelet
yield 109

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Mean
SD

16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16

188
176
234
254
192
207
321
198
222
284
310
254
262
266
170
327
231
325
256
244

30
28
37
41
31
33
51
32
36
45
50
41
42
43
27
52
37
52
41
39

90
80
83
100
96
104
94
94
106
98
94
86
84
80
102
94
94
96
86
86

256
288
319
368
278
288
452
265
272
333
458
341
365
395
214
454
320
456
356
296

23
23
26
37
27
60
42
25
29
33
43
29
31
32
22
43
30
44
31
25

Collection
efficiency
(%)

2 109
platelet/ml

Harvested
PPP

77
82
71
91
87
90
83
79
81
72
87
72
73
74
80
82
81
84
75
65
79
68

12
12
13
18
13
15
21
13
14
16
22
15
15
16
11
21
15
22
15
13
16
034

78
68
70
82
83
89
73
81
92
82
72
71
69
64
83
73
79
74
71
73
76
073

PPP, platelet-poor plasma.

determine whether gelation significantly depended on the


surface contact area, the gelation times obtained in wells of
different sizes were analysed by a two-tailed unpaired t-test
with Welchs correction, assuming that the two populations
might have different variances (GraphPad Prism, version 4,
GraphPad Software; GraphPad Software Inc., San Diego, CA,
USA).

Results
The amount and the platelet concentration of the PRPs
harvested from each blood sample are reported in Table 1.
The mean collection efficiency was 79 68% (range 6591%).
The final volume of the recovered hyperconcentrated platelets
(2 109/l) and the amount of the platelet-poor plasma
harvested after the high-spin centrifugation are also reported.
The kinetics of the gelation was evaluated by light transmission analysis. The results are given in Fig. 1. Figure 1
depicts the increasing turbidity of the PRP solution after the
addition of (i) calcium gluconate, (ii) batroxobin, or (iii) a
combination of both according to the Plateltex instructions.

Macroscopically, gelation occurred after 78 24 seconds;


tensile strength for user-friendly manipulation or suturability
of gel intervened after 81 11 min. Gelation time was not
dependent on fibrinogen concentration, such as depicted in
Table 2, but was strongly dependent on the surface contact
area. Gelation time was measured using either 12-wells (12 ml
PRP per well; contact surface area 133 mm2) or 96-wells culture
trays (200 l per well; contact surface area 79 mm2). A significant
difference was evidenced in these experimental conditions
(P < 00001, unpaired t-test with Welchs correction; GraphPad
Prism, version 4), demonstrating that in vitro gelation time
is surface tailored (Fig. 2). The clot retraction took hours;
retraction in the batroxobin-treated samples occurred much
more slowly than that usually induced by thrombin (data not
shown).
The results of the measurements of PDGF-AB are shown in
Fig. 3. The amount of each of the other measured growth
factors was quite variable between individual samples. Median,
range, lower and upper 95% confidence interval (CI) of mean
being the following: TGF- 755 pg/ml (range 4802700;
95% CI 7711396); VEGF 360 pg/ml (range 01550; 95% CI

2008 The Author(s)


Journal compilation 2008 Blackwell Publishing Ltd., Vox Sanguinis (2008)

4 L. Mazzucco et al.

Fig. 1 Kinetics of the gelation time of a representative platelet-rich plasma


(PRP) sample. The PRP, adjusted to the platelet concentration of 2 109/ml,
was dispensed into the wells of 96-well cell culture trays. Either calcium
gluconate (1000 mg/ml) or batroxobin (5 BU/ml), or a combination of both,
were added to the PRP at a v/v ratio of 1 : 10. Serial optical density
measurements at 405 nm were performed (every third minute up to 15 min,
and every fifth minute up to 40 min). The percentage value of each
measurement is reported such as that calculated in comparison to the
maximum light absorbance taken as 100%.

Fig. 3 Platelet-derived growth factor AB (PDGF-AB) concentration in the


platelet-rich plasma (PRP) samples of 20 subjects. The PRP was prepared
according to the manufacturers direction. The platelet concentration of
each PRP sample was adjusted to 2 109/ml by additional centrifugation at
high gravitational force and an appropriate amount of platelet-poor plasma
was removed.

Table 2 Gelation time as evaluated by visual observation. The platelet-rich


plasma (PRP) and the activator as provided by the manufacturer were mixed
in 12-wells cell culture trays. The initial visible fibrin polymerization and the
stabilization of the gel were measured in seconds and min, respectively.
The fibrinogen content of each sample is also reported
Gelation time

Fig. 2 Dependency of the gelation time on the platelet-rich plasma (PRP)


contact surface area. The PRP and the activator as provided by the
manufacturer were dispensed into the wells of either 12- or 96-well cell
culture trays (PRP surface contact area: 133 and 79 mm2, respectively) and
the time for stable gelation to occur was recorded (min).

286820); and FGF 156 pg/ml (range 40520; 95% CI


135287).
The benchmark comparison for platelet recovery and the
PDGF-AB content provided by the selected PRP-preparing
devices such as presented here and in the current literature
[8,10,12,13,1719] is reported in Table 3.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Mean
SD

Fibrinogen
(mg/ml)

Starting (second)

Firm and stable (min)

231
293
287
139
278
183
244
181
139
183
264
415
268
156
183
224
307
263
577
256
254
1012

58
70
113
115
128
59
60
65
60
70
75
55
60
65
60
120
100
90
80
55
78
241

7
75
75
9
9
10
85
6
75
8
75
65
7
8
85
10
85
95
95
75
81
11

2008 The Author(s)


Journal compilation 2008 Blackwell Publishing Ltd., Vox Sanguinis (2008)

PRP preparation using plateltex 5

Table 3 Benchmark comparison of the mean platelet recovery and of the mean platelet-derived growth factor AB (PDGF-AB) content

PRGF kit
PRP (Landesberg)
AG Curasan
PCCS
Harvest
Vivostat
Regen Kit
Fibrinet
Plateltex

Processed blood (ml)

PRP volume (ml)

Platelet recovery (%)

PDGF-AB (ng/ml)

Activator

20
60
15
50
50
120
10
8
8

95 41
106 24
76 16
85 35
100 0
50 0
50 05
44 02
50 04

35 16
30 10
33 10
68 9
67 10
17 6
90 5
65 10
79 7

47 21
33 7
35 17
103 27
133 29
130 29
140 14
105 30
60 20

Autologous thrombin
Ca++, thrombin
Ca++, thrombin
Ca++, thrombin
Ca++, thrombin
Neutralization of acidified batroxobin
Autologous thrombin
Ca++, high speed centrifugation
Ca++, batroxobin

Discussion and conclusions


Albeit randomized-controlled trials are rare, PRP and platelet
gel are more and more used in several clinical settings for
hard and soft tissue regeneration with predicable therapeutic
outcomes, as corroborated by hundreds of clinical and experimental reports published through the last decade. Nevertheless, it is very difficult to compare these reports one to another
owing to a complete lack of standardization of this new
therapeutic tool and because very few papers provide full
information about platelet-rich products [9,20,21].
Efforts have been made to compare the performances of
the most frequently used PRP-preparation systems designed
for the intended use of topical tissue-healing therapy [814].
Nevertheless, most of these studies reported only about platelet
sequestration efficiency and growth factor enrichment. Very
few concerns, if any, are provided about the gelation phase
and about the means to induce ex vivo the fibrin mesh formation. In this report, we aimed to compare the technical
performances (i.e. collection efficiency, platelet and grow
factor yield) of a newly marketed system, Plateletex, with
those of other systems. Furthermore, it is aimed to extend our
analysis to the features of the gelation phase.
In Tables 1 and 3, result about platelet and growth factor
recovery and some of the data collected from current literature are reported. Despite the attempt to put the little data
available into a homogeneous format, a certain degree of
heterogeneity is inevitable. This heterogeneity depends on
the variable composition and on the intended use of each
system. As an example, the intended use of Vivostat is to
provide the surgeon with platelet-enriched fibrin glue.
Accordingly, the final biologic product contains high fibrinogen concentration, the platelet recovery being lower than
that provided by other devices. Table 3 depicts how much the
blood processing volume is heterogeneous among systems
(8120 ml). Therefore, the final PRP volume, the platelet
concentration and recovery, and the amount of the plateletderived factors are quite heterogeneous. Furthermore, it has

been demonstrated that each system exerts different strength


and biomechanical stress on the platelets, hence, influencing
the growth factor recovery and availability at the end of the
process [10]. Even so, in this heterogeneous context, it is
maintained that the PRP prepared from Plateltex shares
similar characteristics with those obtained from PCCS,
Harvest, Regen Kit and Fibrinet.
All systems concentrate platelets by means of gravitational
force, achieving heterogeneous platelet recovery (Table 3).
Gelation (i.e. formation of the fibrin mesh) is obtained by
different means as summarized in Table 3. Most systems
employ exogenous thrombin and calcium chloride (PRP
according to Landesberg [14]; Curasan; PCCS and Harvest).
Others employ ex vivo prepared autologous thrombin by
treating the autologous platelet-poor plasma with calcium
chloride (PRGF; Regen). Vivostat neutralizes with alkaline
bicarbonate the concentrated fibrin I polymer previously
obtained through acidified batroxobin solution [22]. Fibrinet
uses calcium chloride and mechanical platelet stress (additional
high-speed centrifugation). Finally, Plateltex uses calcium
gluconate and batroxobin. According to the clinical use, gelation
must occur between a few seconds and a few minutes. Although
this point is clinically relevant, there are no published data
about the gelation time, except for the users agreement that
gelation occurs more quickly when the reagents are mixed
spraying them in vivo (e.g. on the surgical field) than when
they are mixed together in vitro (e.g. to prepare medications
or bone grafts).
It has been demonstrated that (i) in vitro, batroxobin and
calcium gluconate provide the formation of a stable and
easy-to-handle gel in a mean time of 8 min (Table 2); (ii)
batroxobin and calcium gluconate act synergistically (Fig. 1);
(iii) gelation does not depend on fibrinogen concentration if
it is within the physiologic range; and (iv) gelation time is
dependent on the surface contact area. This last finding
(beside the action of tissue factors acting in vivo) is relevant
to the experience of the shorter gelation time occurring when
the reagent is sprayed directly within the wound site. Indeed,

2008 The Author(s)


Journal compilation 2008 Blackwell Publishing Ltd., Vox Sanguinis (2008)

6 L. Mazzucco et al.

a delayed PRP clot retraction in vitro has also been observed


using batroxobin compared to that induced by thrombin.
In spite of comparable gelation time, thrombin and batroxobin exert dissimilar activity on both coagulation factors and
platelets. Thrombin catalyses the release of both fibrinopeptide A and B; batroxobin only removes fibrinopeptide A [23].
Thrombin is active on several coagulation factors that are not
sensitive to the batroxobin activity [24]. Bovine thrombin is
said to be a potential inducer of bovinehuman cross-reacting antibodies to coagulation factors V and XI [25]; this
effect has not been observed through a 30-year-long clinical
use of batroxobin. Thrombin activates platelets, while
batroxobin does not activate platelets that lay trapped within
the fibrin network [26,27]. Thrombin is inhibited by heparin,
while batroxobin is not [24]. These differences explain the
delayed batroxobin-induced clot retraction [2729] and
significantly illustrate how PRP gels obtained through the
enzymatic activity of either thrombin or batroxobin do have
different inner characteristics.
It is likely that these differences have a role on the kinetics
of the growth factor release from the PRP gel. It is highly
probable that platelets from thrombin-activated PRP release
their granule content much more quickly than platelets from
batroxobin-activated PRP. It has also been shown that depending on the kind of fibrin polymerization, the lifespan and the
availability of the platelet-released cytokines vary [30].
It is not maintained that one enzyme is better than the
other one; however, it is maintained that batroxobin and
thrombin do have different properties, from each one the
most favourable one for selected clinical conditions can be
selected [31].
In conclusion, Plateltex performs similarly to the other
small blood volume-processing systems. In addition, the
characteristics of the batroxobin-activated PRP gel are therapeutically valuable in selected clinical conditions requiring
long-term availability of platelet-derived trophic factors.

Acknowledgements
The authors are indebted with Dr Michael Ellard for his kind
revision of the manuscript.

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2008 The Author(s)


Journal compilation 2008 Blackwell Publishing Ltd., Vox Sanguinis (2008)