Transfusion and Apheresis Science 50 (2014) 207–209
Contents lists available at ScienceDirect
Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
Platelet activation by riboﬂavin and UV light: Is it really the other side of the coin? Gianpaolo Del Proposto a,⇑, Alessandro Lanti a,1, Eleonora Fiorelli b,2, Gloria Palazzo b,3, Giorgia Guiducci b,4, Francesca Messina a,5, Alessandra De Masi b,6, Angelo Salvatore Ferraro a,7, Oana Marilena Chiru b,8, Paola Cerrone a,9, Maddalena Antonelli b,10, Gaspare Adorno a,b,11 a b
SIMT Tor Vergata Hospital, Rome, Italy Immunohematology Section, Tor Vergata University, Rome, Italy
a r t i c l e
i n f o
Article history: Received 13 January 2014 Accepted 31 January 2014
Key words: Platelet collections Riboﬂavin UV light Annexin V Glycoproteins IIb/IIIa
a b s t r a c t Introduction: Nowadays transfusion safety is still put at risk by contamination of pathogens. The Mirasol PRT System blocks the replication of pathogens and white blood cells. Our goal was to quantify the activation of platelets after treatment with the Mirasol device. Materials and methods: From September to December 2013, 131 platelet collections were studied using a simple ﬂow cytometric strategy. Results: There was a signiﬁcant correlation between the percentage of platelet activated before and after the treatment. Conclusion: Our results induced us to think that the activation of platelets after treatment was acceptable. Ó 2014 Elsevier Ltd. All rights reserved.
Corresponding author. Tel.: +39 06 20900651, mobile: +39 339 2966447. E-mail addresses: [email protected]
(G. Del Proposto), [email protected]
(A. Lanti), eleﬁ[email protected]
(E. Fiorelli), gloriapalaz [email protected]
(G. Palazzo), [email protected]
(G. Guiducci), [email protected]
(F. Messina), [email protected]
(A. De Masi), [email protected]
(A.S. Ferraro), oanaaﬂ@gmail.com (O.M. Chiru), [email protected]
(P. Cerrone), [email protected]
(M. Antonelli), [email protected]
(G. Adorno). 1 Tel.: +39 06 20900651, mobile: +39 393 7960705. 2 Tel.: +39 06 20900651, mobile: +39 392 1978080. 3 Tel.: +39 06 20900651, mobile: +39 328 1397165. 4 Tel.: +39 06 20900651, mobile: +39 328 7037777. 5 Tel.: +39 06 20900651, mobile: +39 331 7751619. 6 Tel.: +39 06 20900651, mobile: +39 320 9733991. 7 Tel.: +39 06 20900651, mobile: +39 349 5967348. 8 Tel.: +39 06 20900651, mobile: +39 347 9829394. 9 Tel.: +39 06 20900651, mobile: +39 331 4894955. 10 Tel.: +39 06 20900651, mobile: +39 333 3922731. 11 Tel.: +39 06 20900651, mobile: +39 392 5648067. http://dx.doi.org/10.1016/j.transci.2014.01.020 1473-0502/Ó 2014 Elsevier Ltd. All rights reserved.
Blood transfusion can be life-saving in patients with either massive blood loss or in those unable to produce blood due to defective hematopoiesis Although serology and molecular biology constitute valid diagnostic tools, transfusion safety is still put at risk through contamination by viruses, bacteria and parasites, due to the ‘‘window period’’ between infection and onset of disease and due to the emergence of new pathogens (e.g. West Nile Virus). Nowadays, there are different systems for the inactivation of pathogens in blood components. The Mirasol PRT System for platelets and plasma (Caridian BLT, Lakewood CO, USA) uses riboﬂavin and ultra violet (UV) light to induce irreversible damage to nucleic acids, so blocking the replication of pathogens and white blood cells that may be present in the transfusion bag. However, treatment with riboﬂavin and UV light activates some of the platelets. But exactly how many? Platelet activation causes degranulation of the alpha granules which induces the exposure of
G. Del Proposto et al. / Transfusion and Apheresis Science 50 (2014) 207–209
Table 1 Platelet activation and MPV before and after Mirasol treatment. Platelet collection
Platelets activated %
Before Mirasol After Mirasol
3.36 ± 0.91 3.14 ± 0.53
1180 ± 193 1035 ± 173
0.8 ± 0.5 1 ± 0.7
8.7 ± 0.84 8.59 ± 0.83
From left to right are shown total platelets, platelets 103/ll, percentage of activated platelets and mean platelet volume ‘‘MPV’’ before and after Mirasol treatment. The values are expressed as mean and standard deviation.
phosphatidylserine (FS) on the external side of the membrane. Phosphatidylserine is a membrane phospholipid with a high afﬁnity for Annexin V (a procoagulant calcium dependant protein, contained in the delta granules). Various resultant changes of platelet functionality induce a block of the membrane aminophospholipidic transferase with a lack of the normal membrane phospholipid asymmetry and exposure of phosphatidilserine on the external membrane side [1,2]. Therefore the extra membrane exposure of phosphatidylserine can be useful to check the metabolic state of platelets . Our goal was to quantify the activation of platelets after treatment with the Mirasol device, using a simple ﬂow cytometric strategy. 2. Materials and methods From September to December 2013 in the Immunohematology and Transfusion Medicine Service (SIMT) of the Tor Vergata Hospital (Rome) we studied 131 platelet collections from healthy donors (105 male; 26 female; age 39 ± 9) collected using the Amicus cell separator (Baxter). Immediately after collection the platelets were transferred to Illumination/Storage Bags, the riboﬂavin was added (0.035 l at a concentration of 500 lmol/l in 0.9% saline) and they were placed in the Illuminator and exposed to UV light at 37 °C according to the manifacturer’s instructions. After the inactivation, the collections were stored with continous agitation at 22 °C. Before and after treatment with riboﬂavin and UV light, aliquots of platelets were taken into tubes with EDTA. These samples were analysed with the SYSMEX XT 1800automatic analyzer, stained with monoclonal antibodies against CD41a FITC and Annexin V PE (both Becton & Dikinson BD) and studied with a cytometer FACScanto II (BD). In detail the monoclonal antibody versus CD41a FITC recognizes the glycoproteins IIb/IIIa normally present on the platelet membrane.This complex is a receptor for ﬁbronectin, ﬁbrinogen, von Willebrand factor, vitronectin and thrombospondin and mediates platelet aggregation. The second monoclonal antibody binds to extracellular annexin V, which is a marker of cellular stress. For the analysis we used a double platform strategy. 3. Results There were correlations between platelet activation before and after treatment with riboﬂavin and UV light (R = 0.81; ⁄P < 0.05), mean platelet volume ‘‘MPV’’ before and after (R = 0.94; ⁄P < 0.05), between platelets 103/ll and activated platelets before treatment (R = 0.12; ⁄P not
signiﬁcant), and after treatment (R = 0.13; ⁄P not signiﬁcant) (Table 1). Namely, the decrease in platelets 103/ll and the increase in activated platelets after the treatment were very low.
4. Discussion Donor screening and serologic and molecular testing help to make blood a safer product for transfusion, reducing the risk of transfusion-transmitted infectious agents such as HBV, HCV and HIV. However, the routine serological tests, currently in use do not identify various parasitic diseases. Bacteria may be present in asymptomatic donors or, more frequently, contaminate the blood, during collection. The necessity to reduce the risk of bacteria contamination of platelet products is important because platelets are stored at room temperature, a favourable condition for bacterial proliferation, and also because platelets are frequently given to patients with impaired immune systems. The Mirasol PRT System for platelets and plasma is a new technology that uses the synergy of riboﬂavin and UV light to inactivate blood pathogens. Riboﬂavin is a water soluble, rapidly excreted vitamin that cannot be stored by the body. In the Mirasol device, the riboﬂavin is added to the platelet collection before exposure to UV B light. Photolysis then results in the formation of metabolites of riboﬂavin, that have also been detected in normal non-illuminated human blood at low concentrations [3,4]. At present, the upper limit of the no toxic effect level of riboﬂavin remains unknown and its elimination occurs mostly through the kidney. Riboﬂavin can quickly traverse the lipid membrane and bind the nucleic acids in nonspeciﬁc manner. Upon exposure to UV light, the riboﬂavin irrevocably alters the nucleic acids of pathogens and white blood cells, through oxidation of guanine residues and generation of reactive oxygen species. In conclusion, based on our experience, the treatment with riboﬂavin and UV light activated very few platelets in the collections. Moreover the differences between MPV and swirling phenomenon before and after treatment with the Mirasol device were irrelevant (data not shown).
References  Schroit AJ, Zwaal RFA. Tranbilayer movement of phospholipids in red cell and platelet membranes. Biochim Biophys Acta 1991;1071:313–9.  Tait JF, Smith C, Wood BL. Measurement of phosphatidylserine exposure in leukocytes and platelets by whole-blood ﬂow cytometry with Annexin V. Blood Cells Mol Dis 1999;25(5):271–8.
G. Del Proposto et al. / Transfusion and Apheresis Science 50 (2014) 207–209  Reikvam H, Marschner S, Oveland Apelesh T, Goodrich R, Hervig T. The Mirasol Pathogen Reduction Technology and quality of platelets stored in platelet additive solution. Blood Transfus 2010;8:186–92.  Marschner S, Goodrich R. Pathogen reduction technology treatment of platelets, plasma and whole blood using riboﬂavin and UV light. Tranfus Med Hemother 2011;38(1):8–18.
 Del Proposto G, Cerrone P, Sansone L, Antonio N, Sinopoli S, Lanti A, et al. Platelet storage and ﬂow cytometry, an interesting couple. Trasfus Af Sci 2012;47:121–2.