Storage induced variations in the expression of

Storage induced variations in the expression
of some clusters of differentiation (CD)
in platelet concentrates from apheresis
or platelet rich plasma
Saverio Misso(1), Bianca Feola(1), Vincenzo Perri(1), Antonio Minerva(1),
Claudio Marotta(1), Claudio Falco(2), Giorgio Fratellanza(2), Daniela Graziano(2),
Pietro Concilio(2), Antonio Orlando Spada(3),Vincenzo Mettivier(3), Salvatore Formisano(2)
(1)
Immunoematologia e Servizio Trasfusionale Azienda Ospedaliera, Caserta
Immunoematologia e Medicina Trasfusionale AUP Federico II, Napoli
(3)
II Ematologia Azienda Ospedaliera Cardarelli, Napoli
(2)
I concentrati piastrinici subiscono, durante la
conservazione, modifiche biochimiche e strutturali che
potrebbero influenzare la loro funzionalità. La
citofluorimetria a flusso è una delle tecniche più
interessanti capaci di valutare tali modifiche mediante
lo studio dei Cluster of Differentiation (CD) presenti sulla
superficie piastrinica. A tal fine, abbiamo valutato
l'espressione di alcune di queste molecole e la
concentrazione plasmatica del Transforming Growth
Factor (TGF) β-1 (una citochina prodotta dagli α-granuli)
in concentrati piastrinici ottenuti da donazione di sangue
intero e da aferesi in I°, III° e V° giorno di conservazione.
Parole chiave: piastrine, citometria a flusso, lesioni da
conservazione, attivazione piastrinica
Key words: platelets, flow cytometry, storage lesions,
platelet activation
Introduction
Platelet concentrates undergo, during storage, to
biochemical and structural changes that give rise to
variation of their functionality named storage lesions1,2.
They are probably due to different causes as: activation
during preparation and/or storage, stress, proteases action
and so on.
Flow cytometry (FC) gave in the last ten years an
important contribution to the study of platelets. It has a
particular interest between methods able to evaluate platelet
functionality 3-5,18-20.
New policlonal and monoclonal antibodies (mabs)
Ricevuto: 29 giugno 2001 - Accettato: 28 settembre 2001
Corrispondenza:
Dott. Saverio Misso
Via San Francesco d'Assisi, 5
81100 Caserta
conjugated with fluorescent markers are now available.
They allow methodological improvements by transferring
to FC techniques previously used in fluorescent
microscopy. Storage lesions and platelet functionality can
be both evaluate by studying surface receptors named
clusters of differentiation (CD) 6-9,17,21.
A large number of molecules are expressed on platelet
membrane: ABO antigens, HLA molecules, and others.
Some of them are specific, some can be also found on others
cells and tissues.
CD include both specific and non-specific molecules.
Their presence on platelets can be detected by combined
use of monoclonal antibodies and FC techniques.
There is a large number of studies about platelet
changes during storage 10, 11,24 but not so many about the
effects induced by preparation methods 12,23.
Aim of our study was to evaluate the behaviour of
some membrane molecules in platelets concentrates
obtained from apheresis (APH-PLT) and from platelet rich
plasma (PRP-PLT), 1 hour after donation and at the 1st, 2nd
and 5th day of storage. Variations in some morphological
indexes (MPW and PDW) and in Transforming Growth
Factor (TGF) β-1, a cytokine that is normally stored in
α-granules and is released in plasma as a consequence of
platelet degranulation25, were also studied.
Materials and methods
40 platelet concentrates from PRP and 15 from apheresis,
all obtained from volunteer donors between 21 and 43 years
of age, were evaluated.
PRP-PLT
Whole blood was collected into triple bags (Baxter
Healtheare Ltd, Newbury, Berks, Nebraska, USA) containing
LA TRASFUSIONE DEL SANGUE vol. 46 - num. 4 luglio-agosto 2001 (221-226)
221
S. Misso et al.
Tabella I: studied cluster of differentiation (CD) and respective antigens
Cluster
Antigen
Clone
Ligand
CD31
CD36
CD41
WM59
FA6-152
P2
-Thrombospondine
Fibrinogen
Adhesion Molecule
Receptor
Receptor
CD42a
PECAM-1
gp IIIb
gpIIb/IIIa
complex
gpIX
SZ1
Vwillebrand
CD42b
gp Ib
SZ2
Vwillebrand
CD61
gpIIIa
SZ21
--
CD62
CD63
P-selectine
gp53
AK-6
LP9
---
gpIb/IX
factor
gpIX/Ib
factor
ethero-dimer
with CD41 or CD51
PLT-WBC interaction
lysosome protein
citrate-phosphate-dextrose anticoagulant supplemented
with adenine 1 (CPD-A1) and SAG-Mannitol.
It was centrifuged within 3 hours from collection for 15
minutes (m') at 1,100 RPM at a temperature of 22 °C using
a Heraeus Cryofuge 6000i centrifuge (Heraeus Instruments
GmBH, Hnau, Germany).
PRP was then transferred in a satellite bag and ricentrifuged for 15 m' at 3,100 RPM. Surnatant platelet poor
plasma was removed by transferring in the third bag in
order to reduce the concentrate volume to 50 mL.
Platelets were manually re-suspended after 1 hour and a
representative sample was taken. Platelet count and MPW and
PDW evaluation were done using a Coulter Max-M automatic
cell counter (IL-Instrumentation Laboratory SpA-Milano).
Part of the sample was collected to be used for flow
cytometry analyses at time zero of storage.
Concentrates were then stored in continuous shaking
at 22 ± 2 °C in order to be investigated on the next days.
APH-PLT
Platelet concentrates were obtained using a COBE
Spectra SRL System cell separator (Gambro Bct, Inc.
Lakewood, CO, 80215 USA).
Volunteer donors had a pre-donation count of
250 ± 80x103 platelets/mL. A mean of 3,500 ± 500 mL of
blood was processed using 350 ± 50 mL of ACD-A as
anticoagulant. Time required was 80 ± 20 m' and the number
of collected platelets was about 4.5 x 1011 cells for each
procedure. Samples were taken and evaluated such as
described for PRP-PLT.
TGF β-1 DOSAGE
TGF β-1 was dosed on plasma obtained by centrifuging
platelet suspensions at 2,000 RPM for 15 m' and stored at -80
°C. The test was performed using a solid phase ELISA
method from Amersham International plc (Amersham Place
Little Chalfont, Buckinghamshire, England).
222
Function
Flow cytometry analysis
FC analysis was performed according to the following
method13-15. Platelet concentrates were diluted 1:10 with
Phosphate Buffered Saline (PBS) in order to obtain a final
number of platelets between 0.5 and 1x106/µL. 100 µL of
this suspension were incubated for 15 m' at room
temperature with 10 µL of a specific monoclonal antibody
(CD31, Cd36, CD41, CD42a, CD43b, CD61, CD62p, CD63).
Following incubation, 100 µL of 0.5-1% buffered paraformaldehyde were added and a second incubation was
done for 15 m' at room temperature.
1 mL of PBS was then added and results were acquired
to flow cytometer.
Ortho Diagnostic System Cytotron Absolute flow
cytometer was used (Ortho-Clinical Diagnostics inc.
Raritan, NJ, 08869 USA).
Monoclonal antibodies (CD31, CD36, CD41, CD42a,
CD42b, CD61 and CD63) were produced by Immunotech
(13276 Marseille Cedex 9, France) and (CD62p) by Serotec
(Serotec Ltd 22 Bankside, Station Approach, Kidlington,
Oxford OX5 1JE, England).
For statistical analysis t-Student test was used. Table I
shows studied clusters of differentiation, respective clone,
ligands and functions.
Results
Figures 1-4 show expression of Clusters of
Differentiation as % of positive cells.
CD62p is less expressed than others on platelet surface
and is represented as % of positive fluorescence of
platelets. Each value of CD expression was compared to
the basal value obtained on samples collected from the
same donors before donation and in minimal trauma
conditions.
Lesioni da conservazione nei concentrati piastrinici
Figura 1 - CD62p expression as % of positive cells 1 hour after platelets separation compared with donors in basal
conditions and negative control
Figura 2 - Expression of CD62p in platelet concentrates obtained by apheresis and platelet rich plasma at different
days of storage
Unmarked platelets were used as negative control. Both
methods of preparation cause platelets activation, showed
by increased expression of CD62p (also named p-selectine)
after 1 hour (p < 0.05).
Activation is higher in PRP-PLT (> 40% of positive cells)
than APH-PLT (> 20% of positive cells) (Figure 1).
On the other hand, storage has the most significant effects
on platelets produced by apheresis, as showed by the expression
of p-selectine measured at day 1, 3 and 5 (Figure 2).
CD42b (gp Ib) also undergo changes. Its expression after
1 hour (Figure 3) has a slight reduction (p > 0.05), that become
of a considerable degree (p < 0.05) during the storage with a
small difference in favour of PRP-PLT (Figura 4).
No changes were observed in the expression of others
Clusters of Differentiation (CD31, CD36, CD41, CD42a, CD61
and CD63) and in morphological indexes (MPW - PDW)
either after 1 hour or during the next days.
Variations of TGF β-1 (Figures 5 and 6) seem to be of
particular interest. It is significantly increased (p<0.05) after
1 hour only in PRP-PLT.
On the contrary, its level rises during the next days both in
PRP-PLTandinAPH-PLT(p<0.05),evenifmoreinthefirstone.
Discussion
Platelets can be activated by physiological agonists,
as ADP, thrombin, collagen, TX-A2, platelet activation
223
S. Misso et al.
Figura 3 - CD 42b expression as % of fluorescence 1 hour after platelets separation compared with donors in basal
conditions and negative control
Figura 4 - CD 62p expression as % of positive cells 1 our after platelets separation compared with donors in basal
conditions and negative control
factor, epinephrine, or by non-physiological factors and
other causes as manipulation during preparation, ACD
release by erythrocytes, storage and stress18,19,24,26. It is
well known that platelet activation causes changes in
expression of structural membrane glycoproteins and
particularly: decrease of gpIb (CD42b) due to redistribution
of canaliculi system, increase of gpIIb/gpIIIa complex
(CD41/CD61) and externalization of p-selectine (marked by
CD62) coming from α- granules as a consequence of platelet
degranulation16,21,22,24.
Our results show that the two methods of platelet
224
production both cause, though in different ways, a variation
in platelet status; furthermore persistence of activation and
subsequent deterioration/senescence that occur during
storage are significantly different. These variations are
showed by the different expression of CD62p and CD42b
in PRP-PLT compared to APH-PLT as immediately after
preparation as during storage. P-selectine, still expressed
at day 1 in PRP-PLT and in APH-PLT (43% and 25%
respectively), slightly increases also at day 3 and 5. We
observed a continuous and constant reduction in
expression of CD42b during all the time of storage in platelets
Lesioni da conservazione nei concentrati piastrinici
Figura 5 - Value of TGF β-1 (ng/mL) in platelet concentrates obtained by apheresis and platelet rich plasma at different
days of storage
Figura 6 - Value of TGF β-1 (ng/mL) 1 our after platelets separation compared with donors in basal conditions and
negative control
produced with both methods, and a significant increase of
TGF β-1 both at day 1 and at day 5. In conclusion, it is
possible to affirm that changes in expression of CD42b
and of CD62p and increase of TGF β-1 are the results of a
large number of events that occur during platelets
preparation and storage, and flow cytometry study, using
a small panel of fluorescently-labelled reagents
comprising mabs to p-selectin (CD62p) and gpIb
(CD42b), offers a rapid and simple assay without artefact
and without loss of platelet subpopulations during
separation and provides an informative method for
evaluating new preparative procedures or for quality
assessment of platelet concentrates. Further clinical
studies are now needed to determine whether the
changes seen in vitro give a measure of the effectiveness
of the different platelet preparation in vivo.
Abstract
Platelet concentrates undergo, during storage, to
biochemical and structural changes that can influence
225
S. Misso et al.
their functionality. Flow cytometry is one of the most
interesting techniques able to evaluate redistribution of
Clusters of Differentiation on the platelet surface.
We studied changes in the expression of some of these
molecules and changes in plasma concentration of TGF
β-1 (a cytokine that is produced by α granules) depending
on the way of preparation of platelet concentrates (by
blood donation or by apheresis) and induced by storage.
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