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
(2) Immunoematologia e Medicina Trasfusionale AUP Federico II, Napoli
(3) II Ematologia Azienda Ospedaliera Cardarelli, Napoli
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) βββββ -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 lesions 1,2 .
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) 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 1 st , 2 nd and 5 th 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 degranulation 25 , 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.
Ricevuto: 29 giugno 2001 - Accettato: 28 settembre 2001
Corrispondenza:
Dott. Saverio Misso
Via San Francesco d'Assisi, 5
81100 Caserta
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)
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S. Misso et al.
Tabella I: studied cluster of differentiation (CD) and respective antigens
Cluster Antigen Clone Ligand
CD31
CD36
CD41
PECAM-1 gp IIIb gpIIb/IIIa complex gpIX
WM59
FA6-152
P2
--
Thrombospondine
Fibrinogen
CD42a
CD42b
SZ1
SZ2
Vwillebrand
Vwillebrand gp Ib gpIIIa CD61
CD62
CD63
P-selectine gp53
SZ21
AK-6
LP9
--
--
--
Function
Adhesion Molecule
Receptor
Receptor 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 ± 80x10 3 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 10 11 cells for each procedure. Samples were taken and evaluated such as described for PRP-PLT.
Flow cytometry analysis
FC analysis was performed according to the following method 13-15 . Platelet concentrates were diluted 1:10 with
Phosphate Buffered Saline (PBS) in order to obtain a final number of platelets between 0.5 and 1x10 6 /µL. 100
µ
L of this suspension were incubated for 15 m' at room temperature with 10
µ
L of a specific monoclonal antibody
(CD
31
, Cd
36
, CD
41
, CD
42a
, CD
43b
, CD
61
, CD
62p
, CD
63
).
Following incubation, 100
µ
L 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
TGF
βββββ
-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).
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.
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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-PLT and in APH-PLT (p < 0.05), even if more in the first one.
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 stress 18,19,24,26 . 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 degranulation 16,21,22,24 .
Our results show that the two methods of platelet 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
224

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.
Bibliography
1) Bode AP: Platelet activation may explain the storage lesion in platelet concentrates. Blood Cells, 16, 109, 1990.
2) Cox AD: A study of platelet activation: the role of membrane glycoproteins of the integrin and selectin superfamilies.
Thesis, University of London 1991.
3) Borzini P: I CD (Cluster of Differentiation) delle piastrine.
Platelet Forum, News Quarterly, 1(3), 14, 1997.
4) Pagliaro P: La citofluorimetria a flusso nello studio immunologico e funzionale delle piastrine. Platelet Forum,
News Quarterly; 3(5), 2, 1999.
5) Michelson AD: Flow cytometry: a clinical test of platelet function. Blood, 87, 4925, 1996.
6) Goodall AH: Platelet activation during preparation and storage of concentrates: detection by flow cytometry. Blood
Coagulation and Fibrinolysis, 2, 377, 1991.
7) Fijnheer R, Modderman PW, Veldman H et al.: Detection of platelet activation with monoclonal antibodies and flow cytometry: changes during platelet storage. Transfusion, 30,
20, 1990.
8) Stenberg PE, McEver RP, Shuman MA et al.: A platelet alphagranule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol, 101, 880, 1985.
9) Frojmovic MM : Flow cytometric analysis of platelet activation and fibrinogen binding. Platelet, 7, 9, 1996.
10) Ruf A, Patscheke H: Flow cytometric detection of activated platelets: comparison of determining shape change, fibrinogen binding, and P-selectin expression. Sem Thromb Hemostatis,
21, 146, 1995.
11) Michelson AD, Benoit SE, Kroll MH et al.: The activation-induced decrease in the platelet surface expression of the glycoprotein
Ib-IX complex is reversible. Blood, 83, 3562, 1994.
12) Metcalfe P, Williamson LM, Reutelingsperger CPM et al.: Activation during preparation of therapeutic platelets affects deterioration during storage : a comparative flow cytometric study of different production methods. Br J Haematol, 98, 86, 1997.
13) Goding JW: Monoclonal Antibodies: Principles and Practice,
2nd edn. Academic Press, London, 1986.
14) Ault AK, Mitchell J: Analysis of platelets by flow cytometry. In:
Darzynkiewicz Z, Robinson JP, Crissman HA (Eds): Methods in Cell Biology. Vol.42 Flow Cytometry, Part B, pag. 275.
Academic Press, San Diego, CA, 1994.
15) Barklay AN, Birkeland M.L, Brown MH: The Leukocyte Antigen
Facts Book. Academy Press, London, 1993.
16) Home S, Sweeney JD, Sawyer S, Elfath MD: The expression of p-selectin during collection, processing, and storage of platelet concentrates: relationship to loss of in vivo viability.
Transfusion, 37, 12, 1997.
17) Estebanell E, Diaz-Ricart M, Lozano M et al.: Cytoskeletal reorganization after preparation of platelet concentrates, using the buffy coat method, and during their storage.
Haematologica, 83, 112, 1998.
18) Holme S: Storage and quality assesment of platelets. Vox
Sang, 74 (Suppl. 2), 207, 1998.
19) Steffan A, Pradella P, Abbruzzese L et al. : Controllo di qualità nei concentrati piastrinici : validazione di un nuovo tipo di sacca. La Trasf del Sangue, 43, 345, 1998.
20) Engelfriet CP, Reesing HW, Snyder EL et al.: The official requirements for platelet concentrates. Vox Sang, 75, 308,
1998.
21) Rinder HM, Ault KA: Platelet activation and its detection during the preparation of platelet transfusion. Transf. Med.Rev, 12,
271, 1998.
22) Dumont LJ, Van den Broeke T, Ault KA: Platelet surface Pselectin measurements in platelet preparations: an international collaborative study. Trans Med Rev 13, 31, 1999.
23) Van der Planked MG, Vertessen F, Mortelmans E et al.: The evolution of platelet procoagulant activity of remnant platelets in stored platelet concentrates prepared by the platelet-rich plasma method and the buffy coat method. Ann Hematol, 78,
1, 1999.
24) Matsubayashi H, Weidner J, Miraglia CC, McIntyre JA:
Platelet membrane early activation markers during prolonged storage. Thromb. Res, 93, 151, 1999.
25) Fujihara M, Ikebuchi K, Wakamoto S, Sekiguchi S: Effects of filtration and gamma radiation on the accumulation of RANTES and trasforming growth factor-beta in apheresis platelet concentrates during storage. Transfusion, 39, 498, 1999.
26) Lonzano ML, Rivera J, Bermejo E et al.: In vivo analysis of platelet concentrates stored in the presence of modulators of 3'5' adenosine monophosphate, and organic anions.
Transfus Sci, 22, 3, 2000.
226