BLOOD COMPONENTS In vitro study of platelet function confirms the contribution of the ultraviolet B (UVB) radiation in the lesions observed in riboflavin/UVB-treated platelet concentrates Me lanie Abonnenc,* Giona Sonego,* David Crettaz,* Alessandro Aliotta, Michel Prudent, Jean-Daniel Tissot, and Niels Lion


Platelet inactivation technologies (PITs) have been shown to increase platelet storage lesions (PSLs). This study investigates amotosalen/ ultraviolet (UV)A- and riboflavin/UVB-induced platelet (PLT) lesions in vitro. Particular attention is given to the effect of UVB alone on PLTs. STUDY DESIGN AND METHODS: Buffy coat– derived PLT concentrates (PCs) were treated with amotosalen/UVA, riboflavin/UVB, or UVB alone and compared to untreated PCs throughout storage. In vitro PLT function was assessed by blood gas and metabolite analyses, flow cytometry–based assays (CD62P, JC-1, annexin V, PAC-1), hypotonic shock response, and static adhesion to fibrinogen-coated wells. RESULTS: In our experimental conditions, riboflavin/ UVB-treated PCs showed the most pronounced differences compared to untreated and amotosalen/UVAtreated PCs. The riboflavin/UVB treatment led to a significant increase of anaerobic glycolysis rate despite functional mitochondria, a significant increase of CD62P on Day 2, and a decrease of JC-1 aggregates and increase of annexin V on Day 7. The expression of active GPIIbIIIa (PAC-1) and the adhesion to fibrinogen was significantly increased from Day 2 of storage in riboflavin/ UVB-treated PCs. Importantly, we showed that these lesions were caused by the UVB radiation alone, independently of the presence of riboflavin. CONCLUSION: The amotosalen/UVA-treated PCs confirmed previously published results with a slight increase of PSLs compared to untreated PCs. Riboflavin/ UVB-treated PCs present significant in vitro PSLs compared to untreated PCs. These lesions are caused by the UVB radiation alone and probably involve the generation of reactive oxygen species. The impact of these observations on clinical use must be investigated.


athogen inactivation technologies (PITs) have been implemented in blood bank to improve the safety of blood components with regard to the emergence of new pathogens and the risk of bacterial contamination.1,2 Current PITs use ultraviolet (UV) light illumination with or without a photosensitizer to target nucleic acids. The INTERCEPT Blood System (Cerus, Concord, CA) utilizes amotosalen-HCl, a psoralen, which cross-links nucleic acids upon UVA radiation (320400 nm)3,4 and generates reactive oxygen species (ROS).5 The Mirasol technology (TerumoBCT, Lakewood, CO) uses riboflavin as a photosensitizer that generates ROS upon UVA-UVB radiation (270-360 nm). Its excited state oxidizes preferentially purine bases (mainly guanine residues) by direct electron transfer.6 INTERCEPT7-20 and Mirasol21,22 treatments are efficient against a broad range of viruses, bacteria, and parasites, as well as residual white blood cells (WBCs). Clinical ABBREVIATIONS: HSR 5 hypotonic shock response; PC(s) 5 platelet concentrate(s); PE 5 phosphatidylethanolamine; PIT(s) 5 pathogen inactivation technology(-ies); PSL(s) 5 platelet storage lesion(s); ROS 5 reactive oxygen species; TCA 5 tricarboxylic acid. From the Laboratoire de Recherche sur les Produits Sanguins Epalinges, Transfusion Interr egionale CRS, Epalinges, Switzerland. Address reprint requests to: Niels Lion, PhD, Transfusion Interr egionale CRS, Laboratoire de Recherche sur les Produits Sanguins, Route de la Corniche 2, 1066 Epalinges, Switzerland; e-mail: [email protected] MA and GS are supported by a grant from the research committee of “Transfusion SRC Switzerland.” *These authors contributed equally to this work. Received for publication September 4, 2014; revision received February 24, 2015; and accepted February 28, 2015. doi:10.1111/trf.13123 C 2015 AABB V

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trials on INTERCEPT reported a lower corrected count increment (CCI) in treated PCs.23-30 Hemovigilance programs tend to indicate a lack of adverse events, no differences in platelet (PLT) transfusion requirement, and a significantly lower incidence of acute transfusion reactions with INTERCEPT-treated PLTs compared to untreated PLTs31-33 or gamma-irradiated PLTs.30 The clinical trial on Mirasol (MIRACLE trial) reported a slightly lower CCI with treated PCs with no concomitant increase in blood product consumption.34 However, it is still to be determined whether the lower CCI observed in Mirasoltreated PLTs translates into an increased risk of bleeding.34 PLT storage lesions (PSLs) observed in PCs seem to be increased after the use of PITs. In vitro studies reported metabolic changes, impaired mitochondrial function, accelerated passive activation, and altered agonistinduced PLT aggregation in INTERCEPT-treated compared to untreated PLTs.23,35-41 Mirasol-treated PLTs exhibit an increased expression of P-selectin (CD62P), higher lactate production, and increased glucose and oxygen consumption, as well as lower ATP over storage time.21,42-55 The Mirasol PIT leads to hyperactive PLTs resulting in a reduced degranulation capacity upon acute stimulation.56 Both amotosalen and riboflavin are photosensitizers able to directly oxidize other molecules and generate ROS thereby potentially triggering other damages to the cells. Recently, we characterized the oxidations induced by amotosalen/UVA and riboflavin/UVB treatments on model peptides, suggesting that such modifications could also occur at the protein level.57 Proteomic studies revealed that a limited number of PLT proteins were altered after PITs.39,58-63 The INTERCEPT treatment seems to affect proteins involved in PLT activation and aggregation pathways, whereas Mirasol impacts mainly actin polymerization, cytoskeleton organization, and PLT shape change.58 Although in vitro functional studies and proteomic studies on PCs allowed getting insights into the pathways affected after the use of PITs, many questions are still remaining. An important concern is the role of the UV radiation alone on PSLs. Thus far, the influence of UVA on PLT function has been poorly investigated. Our group has shown that the UVA alone does not contribute to the changes in aggregation response observed in INTERCEPT-treated PLTs compared to untreated PLTs.41 The effect of UVB on PLTs is better documented as UVB radiation alone has been used to prevent alloimmunization and refractoriness to PLT transfusions caused by contaminating WBCs in PCs.64-68 These studies have shown significant differences in PLT aggregation for a UVB dose equal or superior to 3 J/cm2 in response to collagen, ADP, and arachidonic acid activation.64-68 In a two-event mouse model of transfusion, Gelderman and colleagues69 observed an increased PLT activation, 2220 TRANSFUSION Volume 55, September 2015

reduced PLT count, disruption of mitochondrial function, reduced in vivo recovery in circulation, and accumulation of PLTs in the lung at a UVB dose of 2.4 J/ cm2 with an opened illumination setup. However, they have recently shown that the commercial Mirasolinduced PLT activation reduced in vivo recovery of treated PLTs but did not lead to PLT spontaneous aggregation and PLT lung accumulation or acute lung injury in their model.70 Finally, the UVC radiation has been shown to activate GPIIbIIIa via reduction of the disulfide bonds regulating integrin conformation.71 In this study, we evaluated the in vitro properties of PLTs treated either with amotosalen/UVA (commercial INTERCEPT system) or riboflavin/UVB (in-house built illumination device), all units being prepared with the same additive solution (AS). In addition, we evaluated the contribution of the UVB alone in the PSLs observed upon riboflavin/UVB treatment.

MATERIALS AND METHODS Preparation of PCs Whole blood was obtained from regular donors who gave their consent for the use of their blood component in research. Whole blood units (450 6 50 mL 1 63 mL CPD) were collected in a quadruple bag system (NGR6428B, Fenwal, Lake Zurich, IL) and kept at 22 C overnight. After whole blood centrifugation at 3500 3 g for 14 minutes, red blood cells (RBCs) and plasma were expressed on an automated blood component extractor (Optipress II, Fenwal). Five ABO-matched buffy coats (60 mL, hematocrit 0.4, from donors without nonsteroidal antiinflammatory drugs) were pooled and leukoreduced with a buffy coat processing system (Orbisac, TerumoBCT). All the PCs were prepared with the same AS, that is, Intersol (77 mmol/L NaCl, 30 mmol/L sodium acetate, 10 mmol/L sodium citrate, 26 mmol/L phosphate).

Treatment of PCs On Day 1 postcollection, three ABO-matched buffy coat– derived PCs (each coming from five donors) were pooled in a 2-L bag (Plasmix, Grifols, Barcelona, Spain), agitated for 5 minutes, and equally split into three identical PCs. Two bags were prepared for the inactivation, while the control bag was simply diluted with Intersol. Two series of experiments were run with a minimum of three biologic replicates (n  3), each representing 15 donors: untreated and amotosalen/UVA- and riboflavin/UVB-treated PCs were compared in the first series of experiments whereas untreated and riboflavin/UVB- and UVB-treated PCs were compared in the second series. The characteristics of the PCs are summarized in Table 1. Volume losses in PCs throughout storage result from the sample collection to perform the different tests.


TABLE 1. Characteristics of PLT concentrates* Variable Volume (mL) Untreated (n 5 6) Amotosalen/UVA (n 5 4) Riboflavin/UVB (n 5 3) UVB (n 5 3) PLT dose (31011) Untreated (n 5 6) Amotosalen/UVA (n 5 4) Riboflavin/UVB (n 5 3) UVB (n 5 3)


Day 2

Day 5

Day 7


341 6 22 348 6 10 349 6 8 341 6 19

329 6 30 310 6 35 325 6 10 323 6 34

327 6 15 287 6 15 313 6 7 292 6 35


4.09 6 0.02 4.39 6 0.01 3.93 6 0.01 3.55 6 0.01

3.69 6 0.02 3.54 6 0.02 3.71 6 0.01 3.42 6 0.01

3.75 6 0.01 3.38 6 0.02 3.58 6 0.01 3.18 6 0.01

* Values are shown as mean 6 SD. All units had a plasma content of 39%, an AS content of 61%, a RBC count of fewer than 4 3 109/L, and a WBC count of fewer than 1.0 3 106/unit. n is the number of biologic replicates entering the study.

Control unit A quantity of 17.5 mL of Intersol was added to the control PC to compensate for the addition of photosensitizer. The PC was transferred into a storage bag (1300 mL, PL2410 plastic) and stored under agitation at 22 C.

Amotosalen/UVA unit The amotosalen/UVA unit was processed with the INTERCEPT kit for large volume (INT2203B, Cerus). A quantity of 17.5 mL of 3 mmol/L amotosalen-HCl solution was added to the PC, and the mixture was further illuminated with the INTERCEPT illuminator (INT100, Cerus) at 3.9 J/ cm2. After illumination, the amotosalen/UVA-treated PC was transferred to the CAD container and kept under agitation for 14 hours. The amotosalen/UVA unit was then transferred to a storage bag (1300 mL, PL2410 plastic) and stored under agitation at 22 C.

storage bag (1300 mL, PL2410 plastic) and stored under agitation at 22 C.

UVB unit The UVB unit was processed similarly to the riboflavin/ UVB unit, except that the 17.5 mL of riboflavin solution was replaced with 17.5 mL of Intersol. After illumination with the UVB setup described above, the PLTs were transferred to a storage bag (1300 mL, PL2410 plastic) and stored under agitation at 22 C.

Sampling and storage of PCs Day 0 was defined as day of collection. After treatment, all PCs were stored in standard blood banking conditions up to Day 7. Samples were collected and tested on Days 2, 5, and 7 of storage.

PLT metabolism Riboflavin/UVB unit The riboflavin/UVB unit was processed using an inhouse–built UV illumination setup.57 In a dark cabinet, two sets of four fluorescent lamps (Philips UVB broadband TL 20W/12, 280-350 nm, Elevite, Spreitenbach, Switzerland) were disposed above and below an agitating plate. The temperature inside the illumination chamber was air-cooled to maintain the PC between 23 and 26 C. The dose delivered was measured with a radiometer (UVX-31, UVP, Cambridge, UK). In this configuration, a dose of 5 J/cm2 was dispensed in 20 minutes. Directly after splitting the PCs, 17.5 mL of 1 mmol/L riboflavin was diluted in Intersol and added to the riboflavin/UVB unit (final concentration of 50 6 3 mmol/L), which was further illuminated in the illumination bag of the INTERCEPT kit. One milliliter of PCs was sampled before and after illumination to measure the conversion of riboflavin by highperformance liquid chromatography.57,72 According to the published data, a riboflavin conversion of approximately 21.1% is reached with the standard Mirasol PIT.72 In our experiments we reached 21.9 6 1.8% of riboflavin conversion. After illumination, the PLTs were transferred to a

Blood gas analysis including pH value, pO2, pCO2, bicarbonate, and lactate were performed immediately after sampling on an automated blood gas analyzer (ABL800 FLEX, Radiometer, Neuilly-Plaisance, France). One milliliter of PCs was centrifuged at 1000 3 g for 10 minutes and the supernatant was analyzed for glucose and lactate dehydrogenase (LDH). Glucose and LDH were measured with the hexokinase assay (Roche Diagnostics, Basel, Switzerland) and lactate to pyruvate conversion (IFCC 37 C, Roche Diagnostics) methods, respectively (Cobas 8000, Roche Diagnostics).

Flow cytometry–based, hypotonic shock response, and adhesion assays Samples were analyzed on a flow cytometer (FACSCalibur, BD Biosciences, Franklin Lakes, NJ) using computer software (CellQuest Pro, BD Biosciences). The flow cytometer was calibrated with a bead kit (CaliBRITE 3, BD Biosciences). PE-Cy mouse anti-human CD62P (Cat. 551142), FITC–annexin V (Cat. 556420), and FITC Pac-1 (Cat. 340507) were provided by BD Biosciences. MitoProbe JC-1 (M34152) was purchased from Invitrogen (Zug, Volume 55, September 2015 TRANSFUSION 2221


Fig. 1. Blood gas and metabolic analyses of amotosalen/UVA-, riboflavin/UVB-, and UVB-treated PLTs compared to untreated PLTs throughout 7 days of storage. The (A) pH, (B) bicarbonate, (C) pCO2, (D) pO2, (E) glucose, (F) lactate, and (G) LDH release were assayed on Days 2, 5, and 7 in the untreated and treated PCs (n  3, with n the number of biologic replicates). *p < 0.05, **p < 0.01, and ***p < 0.001 for a significant difference of the treated unit compared to the untreated unit.

Switzerland). The hypotonic shock response (HSR) and the static adhesion assay to fibrinogen were realized as described in our recent publication.41 2222 TRANSFUSION Volume 55, September 2015

Statistical analysis Data were expressed as the mean 6 standard deviation (SD). Comparisons were made using analysis of variance


with repeated-measures and post hoc comparisons (R software, version 2.15.1, windows/base/old/2.15.1/). Differences were considered significant when p values were not greater than 0.05.

RESULTS Product characteristics The PCs were conformed to the specifications of the INTERCEPT treatment (Table 1). On Day 7, the averaged volume of the amotosalen/UVA and UVB units were slightly lower than the other conditions due to over sampling but the PLT dose was still in the recommended range for PCs.

PLT metabolism The PLT metabolism throughout the storage period is presented in Fig. 1. In all PCs, the anaerobic glycolysis occurring in PLTs was highlighted by a consumption of glucose, an increase in lactate production, and a decrease in pH. The bicarbonate concentration, a buffering agent present in the storage medium to compensate the acidification, was also decreased throughout storage. The oxygen consumption in the PCs was relatively stable until Day 5 and decreased (i.e., increase of pO2 in the PC) up to Day 7. The CO2 produced during the mitochondrial respiration (tricarboxylic acid [TCA] cycle) and within the buffering reaction of the bicarbonate tends to decrease throughout the storage period diminishing the pCO2. Finally, the PLT lysis was significantly increased at the end of the storage period, except for the riboflavin/UVB-treated PLTs for which the difference in released LDH was not significant. The metabolism of amotosalen/UVA-treated PLTs was similar to the one of untreated units throughout storage except for a significantly lower bicarbonate level after Day 5 of storage. On the contrary, riboflavin/UVB-treated units showed a significant increase of anaerobic glycolysis rate compared to the untreated unit with a glucose consumption rate of 1.58 and 1.01 mmol/L/day and a lactate production rate of 3.09 and 1.71 mmol/L/day, respectively, until Day 5. The glycolysis in riboflavin/UVB units was increased up to Day 5 of storage until all the glucose present in the PLT storage medium was depleted. The pH and bicarbonate levels in riboflavin/UVB units were significantly lower than in the untreated counterparts. The pH value of the riboflavin/UVB unit on Day 7 was higher than on Day 5 probably because of the anaerobic glycolysis slowing down and bicarbonate buffering. The pCO2 in the riboflavin/UVB unit was significantly lower than in the untreated unit at the end of the storage period. When comparing the riboflavin/UVB and UVB treatments we found similar results indicating that the UVB radiation alone was responsible for the metabolic changes observed in riboflavin/UVB-treated PLTs. The PLT lysis

TABLE 2. Flow cytometry–based assays* Variable

Day 2

CD62P expression (%) Untreated Amotosalen/UVA Riboflavin/UVB UVB JC-1 aggregates (%)† Untreated Amotosalen/UVA Riboflavin/UVB UVB Annexin V expression (%) Untreated Amotosalen/UVA Riboflavin/UVB UVB

Day 7

32.7 6 5.0r,uv 34.0 6 4.7r,uv 49.8 6 2.8u,a,uv 44.3 6 2.9u,a,r

48.9 6 8.1r,uv 52.5 6 4.3r,uv 79.2 6 2.1u,a 79.0 6 4.1u,a

90.9 6 4.3 92.9 6 2.7 89.6 6 3.6 90.2 6 1.7

89.7 6 1.6r,uv 88.7 6 0.9r,uv 50.1 6 6.0u,a,uv 41.5 6 5.1u,a,r

1.0 6 0.4a,r,uv 1.8 6 0.7u 1.7 6 0.4u 1.8 6 0.4u

4.7 6 2.2a,r,uv 8.6 6 2.2u,r,uv 24.6 6 4.0u,a 24.8 6 2.2u,a

* Values are shown as mean 6 SD. u,a,r,uvsignificant (p < 0.05) compared to untreated, amotosalen/UVA, riboflavin/UVB, and UVB, respectively. † JC-1 aggregates correspond to FL21/FL11 PLTs.

throughout storage was similar in all units except for the amotosalen/UVA-treated units that presented a significantly increased lysis at all time points compared to the other units.

Surface expression and preapoptotic or preactivation markers The results of the flow cytometry–based assays are reported in Table 2. Passive PLT activation (CD62P expression) occurred throughout storage in all PCs. The amotosalen/UVA-treated PLTs showed a profile of activation similar to the untreated units. However, PLTs either treated with riboflavin/UVB or UVB alone exhibited a significant increase of CD62P expression from Day 2 of storage compared to the untreated unit. The percentage of JC-1 aggregates represents the percentage of PLTs with functional mitochondrial transmembrane potential. Amotosalen/UVA-treated PLTs showed a similar percentage of aggregates compared to the untreated units whereas riboflavin/UVB- and UVB-treated PLTs exhibited a significantly decreased percentage of JC1 aggregates on Day 7 compared to the untreated units. Annexin V binds phosphatidylserine and phosphatidylethanolamine (PE) at the surface of PLTs as a result of membrane shape changes. It is an indicator of preapoptotic events (phosphatidylserine exposure) as well as PLT activation events (PE exposure). All the treated units were significantly different compared to the untreated unit throughout storage. The differences of riboflavin/UVBand UVB-treated PLTs were more pronounced at the end of the storage period.

HSR The HSR is an indicator of the PLT membrane integrity and metabolism, which has been shown to be well Volume 55, September 2015 TRANSFUSION 2223


Fig. 2. HSR, surface expression of active GPIIbIIIa, and static adhesion to fibrinogen-coated wells of amotosalen/UVA-, riboflavin/UVB-, and UVB-treated PLTs compared to untreated PLTs on Days 2 and 7 of storage. (A) HSR measured with an aggregometer. (B) Expression of active GPIIbIIIa at the surface of PLTs detected by flow cytometry. (C) PLT adhesion to fibrinogen under static flow determined by colorimetric assay (n  3, with n the number of biologic replicates). *p < 0.05 and ***p < 0.001 for a significant difference of the treated unit compared to the untreated unit at the same storage time. *p < 0.05 for a significant difference between two storage time.

correlated with in vivo PLT survival and recovery.73 Riboflavin/UVB- and UVB-treated PLTs showed a significant decrease of HSR values throughout storage (Fig. 2A). These two conditions exhibited a significantly lower HSR value compared to untreated and amotosalen/UVA units from Day 2 of storage. The decrease of HSR in riboflavin/ UVB-treated units was mainly due to the UVB radiation. 2224 TRANSFUSION Volume 55, September 2015

PLT adhesion to fibrinogen and expression of active GPIIbIIIa (PAC-1) on PLT surface PLTs treated with the amotosalen/UVA system showed a significant increase of active GPIIbIIIa expression (Fig. 2B) and adhesion to fibrinogen (Fig. 2C) both from Day 2 and from Day 7 of storage. Riboflavin/UVB- and UVB-treated PLTs exhibited a similar trend with a significant increase


of active GPIIbIIIa expression and PLT adhesion to fibrinogen compared to the untreated unit from Day 2 of storage. The capacity of riboflavin/UVB- and UVB-treated PLTs to adhere to fibrinogen-coated plates decreased at the end of the storage period.

DISCUSSION The results from this study reveal that the riboflavin/UVB treatment significantly impacts the metabolism of PLTs as evidenced by higher glucose consumption and lactate production rates. This phenomenon has been observed by other groups in Mirasol-treated PLTs.21,36,42-56 It has been hypothesized that the increased anaerobic glycolysis rate in riboflavin/UVB-treated PLTs was either due 1) to an impaired mitochondrial functionality leading to a decrease of oxidative phosphorylation, 2) to an alteration of the PLT storage medium characteristics (pH, oxygen content, plasma protein content), or 3) to changes in ATP consumption rate.46 During storage at 22 C PLTs use the anaerobic pathway of glycolysis for energy production only to a minor extent (approx. 15%), whereas the main part of the required energy (85%) is generated through the oxidative pathway of the TCA cycle.74,75 The increase of glycolysis rate in riboflavin/UVB-treated PLTs led to glucose depletion in the storage medium after Day 5 of storage. The subsequent diminution of lactate production and increase of pH suggest a switch off of the anaerobic glycolysis. As the LDH levels in the medium remain similar to the controls, it means that the oxidative pathway is sufficient for PLT survival. In agreement with other reports, our results showed that the oxidative pathway is maintained in riboflavin/UVB-treated PLTs.46,76 The oxygen consumption74 in riboflavin/UVB-treated units is stable until Day 5 of storage as for the untreated units. After then, a decrease of oxygen consumption is noticed in all units. As the oxygen serves as a substrate for the oxidative phosphorylation to produce ATP and H2O, it suggests that the mitochondria are still functional after the riboflavin/UVB treatment or at least at early storage times. The CO2 is produced both in mitochondria (TCA cycle) and as a result of bicarbonate buffering (acid-base equilibrium) subsequently to a medium acidification. The decrease of pCO2 observed throughout the storage in all units would, however, suggest a linear decline of the oxidative pathway. The lower level of pCO2 on Day 7 in riboflavin/UVB-treated PLTs suggests a possible alteration of the mitochondria function (as evidenced by a decrease of JC-1 aggregates) and/ or a modification of the bicarbonate buffering (due to the glycolysis switch off).44 The influence of the extracellular medium composition is undoubtedly a key variable in the regulation of PLT metabolism. In this study, the Intersol was used as AS in all the units whereas the standard Mirasol system

recommends the use of SSP1 solution that contains magnesium and potassium. These two ions have been shown to suppress PLT metabolism and activation.45 Galan and colleagues50 showed that SSP1 provides a better preservation of adhesive and cohesive function than Intersol. It is likely that the use of Intersol instead of SSP1 in the riboflavin/UVB units contributed to the exacerbated increase of PLT metabolism we observed compared to other reports on Mirasol-treated PLTs. Recently, Paglia and coworkers77 published a large-scale metabolomic study of stored PLTs supporting a model of successive metabolic shifts throughout the storage period, in contrast to the traditional concept of a linear decay over storage time. The change in composition of the extracellular medium (secretion of metabolites, pH decrease due to the increased glycolysis) of PLTs was most likely the root cause of the metabolic shift they have detected at 4 days of storage.77 Unlike those of Paglia and coworkers, our data tend to support the model of a linear decay throughout storage as illustrated by the constant anaerobic glycolysis rate and pCO2 decrease in untreated PCs. A change of ATP consumption rate is another hypothesis for the increased glycolytic flux in riboflavin/ UVB-treated PLTs. Increased expression of P-selectin and lower ATP content were reported in riboflavin/UVBtreated PLTs.21,36,42-56 We also found an increased expression of CD62P, active GPIIbIIIa at the surface of the PLTs, and an increased adhesion to fibrinogen indicating that the riboflavin/UVB-treated PLTs were somehow activated upon treatment. The membrane integrity of PLTs was also altered as shown by a lower HSR. In agreement with our observations, Zeddies and colleagues56 demonstrated that Mirasol leads to an hyperactive PLT phenotype, probably caused by continuous basal degranulation throughout storage. As the ATP pool in PLTs is located both in the alpha granules and in the cytoplasm, the continuous degranulation and the energy required for achieving this process would result in an increased ATP demand after PITs. To sustain this demand in ATP, the PLTs would therefore accelerate their metabolism and preferentially use the anaerobic glycolysis as long as enough glucose is available in the storage medium. Interestingly, Schubert and coworkers61 showed that the Mirasol treatment (5.3 J/cm2) potentiates the phosphorylation of VASP consistent with an alteration of actin dynamics.61 In another publication,78 they demonstrated the increased phosphorylation of p38MAPK upon riboflavin/UVB treatment, which is a protein involved in granule secretion and clot retraction.79 Altogether, these data suggest that the Mirasol treatment impacts important protein kinases involved in signal transduction, probably via the generation of ROS. In addition, the Mirasol treatment has been associated with an accumulation of PLT-derived cytokines during Volume 55, September 2015 TRANSFUSION 2225


storage, likely caused by PLT destruction and/or PLT degranulation or cytokine de novo synthesis.80,81 The same has been observed in amotosalen/UVA-treated PLTs.82 However, opposite results were published by Tauszig and coworkers83 who observed a low release of cytokine from the alpha granule compared to the high level of activation in Mirasol-treated PLTs and suggested an effect of the PIT on de novo cytokine synthesis. Unlike previous reports on Mirasol-treated PLTs,46,84 we observed that riboflavin/UVB-treated PLTs present increased signs of shape modification (preapoptotic and preactivation events) increasing annexin V binding on Day 7 of storage. Considering that no increase in cell lysis was observed, significant decrease in JC-1 aggregates probably indicate an abnormal oxidative phosphorylation resulting in a decreased polarization of the mitochondrial membrane. An imbalance of ATP might be at the origin of this behavior. The effect of the amotosalen/UVA treatment has already been extensively characterized.35-41 Although amotosalen/UVA-treated PLTs exhibit a slightly higher passive activation, increase of active GPIIbIIIa expression and adhesion to fibrinogen, lower HSR, and increase of annexin V binding, their metabolism seems to remain very similar to the one of untreated PLTs. Nevertheless, we systematically observed an increased LDH release straight after the amotosalen/UVA treatment and throughout storage. This might be due to a physical stress caused by the incubation with the compound adsorbing device (to remove the residual amotosalen in PCs) during the INTERCEPT process. We then focused our attention on the riboflavin/ UVB treatment, which presented the most pronounced differences and we questioned the contribution of the UVB radiation alone. We demonstrated that the lesions observed in riboflavin/UVB-treated PLTs were due to the UVB radiation, independently of the presence of riboflavin. The effect of the UVB radiation on PLT aggregation has been documented in several studies.6468 It seems that there is a threshold dose at approximately 3 J/cm2 above which the PLTs present significant differences in aggregation.64-68 Unlike these studies that were limited to the study of aggregation and activation markers, here we followed the PLT metabolism in addition to a panel of surface expression and preapoptotic and preactivation markers, as well as the membrane integrity and adhesive properties of PLTs throughout storage. In support of our data, Gelderman and colleagues69 also observed an increased PLT activation, reduced PLT count, disruption of mitochondrial function, reduced in vivo recovery in circulation, and accumulation of PLTs in the lung in a two-event mouse model of transfusion upon UVB irradiation (2.4 J/cm2) with an in-house–built open illumination setup. Later, they performed a similar study using the Mirasol PIT 2226 TRANSFUSION Volume 55, September 2015

(riboflavin 1 UVA/UVB dose at 5 J/cm2, use of an illumination and storage bag) and showed that Mirasoltreated PLTs were damaged less than those exposed to open air, which may lead to a lower illumination dose delivered to the PLTs and less ROS production.70 UVB has been shown to activate important protein kinases, such as the protein kinase C that is involved in aggregation signaling.85,86 The presence of oxygen radicals upon UVB radiation is probably the cause of the activation of protein kinases and alteration of signal transduction in the PLTs. In addition to our observations on PLT function, the recent study of Bakkour and colleagues87 showed an equal extent of PLT mitochondrial DNA damages upon riboflavin/UVB and UVB alone treatments suggesting a secondary role of the riboflavin regarding the DNA modification.

Study limitations The first objective of this study was to fundamentally understand the lesions occurring in PLTs treated with amotosalen/UVA or riboflavin/UVB. The pronounced differences observed upon riboflavin/UVB treatment led us to investigate more in depth the role of the UVB alone. It is important to point out that our conclusions on the riboflavin/UVB treatment cannot directly be extended to the commercial Mirasol technology as we used different UV lamps, illumination and storage bag, and AS. Nevertheless, we calibrated our illumination setup in function of the extent of riboflavin conversion in the PCs based on the data published on the Mirasol system.72 To reach a similar conversion of riboflavin, a dose of approximately 5 J/cm2 was then delivered to the PC in about 20 minutes, which is a longer illumination time compared to the Mirasol PIT (5-10 min). In addition, we chose to process all our PCs the same way: 1) suspension in 39% plasma and 61% Intersol while the recommended AS for the Mirasol is SSP1 and 2) illumination and storage in the bags derived from the INTERCEPT kit. This experimental design allowed us to compare the influence of the two treatments in similar conditions. To conclude, this study clearly supports the negative impact of UVB radiation on in vitro PLT function upon riboflavin/UVB treatment. It would be of interest to get more insights into the generation of ROS upon UVB radiation and to evaluate whether UVB-treated PLTs could recover their functionality in vivo. Finally, large-scale mass spectrometry-based metabolomic studies would also be a useful tool to deeply characterize PIT-induced lesions in PLTs. ACKNOWLEDGMENTS The authors thank the research committee of “Transfusion SRC Switzerland” for financial support and Mr Guillaume Riat for his help in building the illumination setup.


CONFLICT OF INTEREST NL received a conference honorarium on two occasions from Cerus, the provider of the INTERCEPT Blood System. JDT received an honorarium from TerumoBCT (European customer panel). The other authors have disclosed no conflict of interest.

14. Brecher ME, Hay S, Corash L, et al. Evaluation of bacterial inactivation in prestorage pooled, leukoreduced, whole blood-derived platelet concentrates suspended in plasma prepared with photochemical treatment. Transfusion 2007; 47:1896-901. 15. Van Voorhis WC, Barrett LK, Eastman RT, et al. Trypanosoma cruzi inactivation in human platelet concentrates and

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transfusion-related acute lung injury in a two-event animal model. Transfusion 2013;53:722-31. 87. Bakkour S, Chafets DM, Wen L, et al. Development of a mitochondrial DNA real-time polymerase chain reaction assay for quality control of pathogen reduction with riboflavin and ultraviolet light. Vox Sang 2014;107:351-9.

UVB-treated platelet concentrates.

Platelet inactivation technologies (PITs) have been shown to increase platelet storage lesions (PSLs). This study investigates amotosalen/ultraviolet ...
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