Assessment of the soluble proteins HMGB1, CD40L and CD62P during various platelet preparation processes and the storage of platelet concentrates: The BEST collaborative study.

BACKGROUND: Structural and biochemical changes in stored platelets are influenced by collection and processing methods. This international study investigates the effects of platelet (PLT) processing and storage conditions on HMGB1, sCD40L, and sCD62P protein levels in platelet concentrate supernatants (PCs). STUDY DESIGN/METHODS: PC supernatants (n = 3748) were collected by each international centre using identical centrifugation methods (n = 9) and tested centrally using the ELISA/Luminex platform. Apheresis versus the buffy coat (BC-PC) method, plasma storage versus PAS and RT storage versus cold (4°C) were investigated. We focused on PC preparation collecting samples during early (RT: day 1-3; cold: day 1-5) and late (RT: day 4-7; cold: day 7-10) storage time points. RESULTS: HMGB1, sCD40L, and sCD62P concentrations were similar during early storage periods, regardless of storage solution (BC-PC plasma and BC-PC PAS-E) or temperature. During storage and without PAS, sCD40L and CD62P in BC-PC supernatants increased significantly (+33% and +41%, respectively) depending on storage temperature (22 vs. 4°C). However, without PAS-E, levels decreased significantly (-31% and -20%, respectively), depending on storage temperature (22 vs. 4°C). Contrastingly, the processing method appeared to have greater impact on HMGB1 release versus storage duration. These data highlight increases in these parameters during storage and differences between preparation methods and storage temperatures. CONCLUSIONS: The HMGB1 release mechanism/intracellular pathways appear to differ from sCD62P and sCD40L. The extent to which these differences affect patient outcomes, particularly post-transfusion platelet increment and adverse events, warrants further investigation in clinical trials with various therapeutic indications.

Minimal impact of anticoagulant on in vitro whole blood quality throughout a 35-day cold-storage regardless of leukoreduction timing.

BACKGROUND: There is increasing interest in leukoreduced whole blood (WB) as a transfusion product for trauma patients. In some jurisdictions, few leukoreduced filters are approved or appropriate for WB leukoreduction and quality information is therefore limited. This study assessed the impact of filtration timing of WB collected in CPDA-1 versus CPD on in vitro quality. STUDY DESIGN AND METHODS: WB was collected in CPDA-1 or CPD and leukoreduction filtered either after 3-8 h (early) or 18-24 h (late) from stop bleed time. In vitro quality was assessed after filtration and throughout 5 weeks of storage at 4°C. Cell count and hemoglobin levels were determined by hematology analyzer, platelet activation and responsiveness to ADP by surface expression of P-selectin by flow cytometry, hemolysis by HemoCue, and metabolic parameters by blood gas analyzer. Hemostatic properties were assessed by rotational thromboelastometry. Plasma protein activities and clotting times were determined by automated coagulation. RESULTS: Although there were some data points which showed statistically significant differences associated with anticoagulant choices or the filtration timing, no general trend in inferiority/performance could be discerned. After 35 days' storage, only clotting time, alpha angle and factor II in the early filtration arm comparing anticoagulants and prothrombin time and factor II in the CPDA-1 study arm comparing filtration timing showed a significant difference. CONCLUSION: In vitro WB quality seems to be independent on the choice of anticoagulant and filtration timing supporting WB hold-times to up to 24 h, increasing operational flexibility for transfusion services.

Preparing small-dose red cell concentrates (RCCs) for neonatal and pediatric transfusions: Impact of RCC volume, storage, and irradiation.

BACKGROUND: Preparing small-dose red cell concentrates (RCCs) is a common practice for pediatric and neonatal transfusions. However, there is a lack of quality monitoring data to indicate that both the preparation and storage of small-dose RCCs does not alter in vitro red cell quality. The present study seeks to provide data to support this practice. MATERIALS AND METHODS: To evaluate quality of stored small aliquots, six ABO/Rh matched leukoreduced citrate phosphate-dextrose/saline-adenine-glucose-mannitol (LR CPD/SAGM) RCCs were pooled and split into 30 ml aliquots, 80 ml aliquots, and a standard 290 ml unit, with testing performed for up to 43 days post-collection. To evaluate the impact of irradiation on small-dose RCC preparation, a total of 48 independent LR CPD/SAGM RCCs were used (non-irradiated: n = 24; irradiated: n = 24). Aliquoting with/without irradiation was performed within 7 days of collection and baseline testing was performed within 24 h of aliquot production. RESULTS: Limited variability in hemolysis, mean cell volume, and extracellular potassium concentrations were seen between the different aliquot sizes throughout the 43-day storage period. Aliquot production did not accentuate damage based on any of these tested parameters in both the non-irradiated and irradiated subsets. A significant increase was seen in the potassium concentrations in the irradiated parent and aliquot samples relative to their non-irradiated counterparts. CONCLUSIONS: Non-irradiated small-aliquot dose RCCs meet in vitro quality criteria required for safe transfusion throughout the 42-day storage period. The same can be said for aliquots derived from irradiated units and tested within 24 h of aliquot production.

Assessment of bacterial growth in leukoreduced cold-stored whole blood supports overnight hold at room temperature prior to filtration: A pilot study.

BACKGROUND AND OBJECTIVES: Whole blood (WB) transfusion has regained attention to treat trauma patients. We reported no significant changes in in vitro quality through 21 days of cold storage for leukoreduced WB (LCWB) when time to filtration was extended from 8 to 24 h from collection. This study evaluated the impact of extended WB-hold at room temperature (RT) prior to leukoreduction on proliferation of transfusion-relevant bacteria. MATERIALS AND METHODS: WB units were spiked with suspensions of Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Listeria monocytogenes prepared in saline solution (SS) or trypticase soy broth (TSB) to a concentration of ~0.2 CFU/ml (N = 6). Spiked units were held at RT for 18-24 h before leukoreduction and cold-stored for 21 days. Bacterial growth was determined on days 2, 7, 14 and 21. In vitro quality of WB inoculated with unspiked diluents was assessed. RESULTS: K. pneumoniae and S. pyogenes proliferated in WB prior to leukoreduction reaching concentrations ≤102 CFU/ml. These bacteria, however, did not proliferate during the subsequent cold storage. S. aureus did not survive in WB while L. monocytogenes reached a concentration of ~102 CFU/ml by day 21. LCWB in vitro quality was not affected by SS or TSB. CONCLUSION: Extended WB-hold prior to leukoreduction allowed proliferation of bacteria able to resist immune clearance, although they did not grow to clinically significant levels. While L. monocytogenes proliferated in LCWB, clinically relevant concentrations were not reached by day 21. These data suggest that transfusing LCWB may not pose a significant bacterial contamination safety risk to transfusion patients.

Pathogen reduction of whole blood: Supplementing fibrinogen partly corrects clot formation in a massive transfusion model.

BACKGROUND: The use of whole blood (WB) to treat trauma patients is becoming more common. Similar to the treatment of individual components, pathogen inactivation (PI) technologies are available to treat WB. The impact of PI on WB function is not well understood. This study investigated the impact of PI of WB with riboflavin/ultraviolet (UV) light on its hemostatic function by modeling transfusion scenarios for trauma patients and assessing transfusion efficacy by rotational thromboelastometry (ROTEM). As fibrinogen is affected by PI of WB, the effect of fibrinogen supplementation commonly used in trauma patients was also analyzed in this model. STUDY DESIGN AND METHODS: Trauma transfusion scenarios were simulated by mixing untreated WB or WB treated with the Mirasol PI technology (riboflavin/UV) in different ratios with hemodiluted blood, and the thromboelasticity was monitored by ROTEM. The impact of supplementation with the fibrinogen concentrate RiaSTAP was investigated in this model. RESULTS: Pathogen-inactivated WB (PI-WB) showed decreased activity in the hemostatic profile compared to the untreated control. Hemodiluted blood at a hematocrit (hct) of 20%, which was reconstituted with PI-WB or untreated WB, exhibited increased alpha values, maximum clot firmness, and clot formation time. Simulating transfusion scenarios by blood replacement with PI-WB resulted in a significant difference in ROTEM parameters between reconstituted PI-treated and -untreated WB (p ≥ .05). The effect of PI treatment waned when PI-WB was enriched with fibrinogen. CONCLUSION: ROTEM investigations suggest that PI treatment has a negative impact on WB clot formation unless fibrinogen supplementation is used.

Releasates of riboflavin/UV-treated platelets: Microvesicles suppress cytokine-mediated endothelial cell migration/proliferation.

BACKGROUND: Accelerated development of the platelet (PLT) storage lesion upon pathogen inactivation (PI) is associated with the release of proteins from granules and platelet microvesicles (PMVs). Whether PI treatments alter the interaction between PLT factors and the vessel endothelium is of interest in understanding the risk profile of these technologies. STUDY DESIGN AND METHODS: In a pool-and-split study, one platelet concentrate (PC) was treated with riboflavin/UV (RF/UV) light, while the other one was kept as an untreated control. Releasates and PMV-depleted releasates were prepared by differential centrifugation steps on days 0, 1, 5, and 7 of storage. Cytokine/chemokine release following PI treatment was analyzed by an antibody array, and results were verified by the enzyme-linked immunosorbent assay. PMVs were enumerated by CD41 labeling and flow cytometry. Wound scratch assays were performed using cultured Ea.hy926 cells exposed to the differently prepared releasates. Effects of releasates on the phosphorylation levels of kinases ERK and p38 expressed by endothelial cells were analyzed by immunoblot. RESULTS: Cytokine/chemokine assays identified a 2-fold increase in epidermal growth factor released from PCs treated with RF/UV light compared with control. PMV count increased ~100-fold following PI treatment. Unmodified releasates and PMV-depleted releasates displayed different contributions to the kinetics of endothelial cell wound closure. This observation was associated with an increased ERK versus unaltered p38 activation in the endothelial cells. CONCLUSION: This study identified an inhibitory impact of PMVs on endothelial cell migration/proliferation upon stimulation by released cytokines and PMVs from PLTs treated with RF/UV light for endothelial cell wound closure.

Reconstituted cryopreserved platelets synthesize proteins during short-term storage and packaging a defined subset into microvesicles.

BACKGROUND: Cryopreservation of platelets (PLTs) could allow extension of their shelf-life to years, compared to days for liquid stored platelets. Due to their greater hemostatic effect, reconstituted cryopreserved platelets (cryo-PLTs) would be able to support bleeding emergencies. Since protein synthesis has been linked to PLT functions, such as clot formation and immune responses, the translational capacity of reconstituted cryo-PLTs was assessed upon thawing and short-term storage. METHODS/MATERIALS: Platelets were frozen at -80°C with 5-6% DMSO. Upon thawing, they were reconstituted in plasma and then aliquoted (12 ml) into mini-bags and assessed over 24 h of storage at RT. One series served as control; the second and third series were spiked with either 300 µM puromycin (Pm) or 227 nM biotin-labeled Pm. Samples were tested for in vitro quality and PLT microvesicle enumeration by flow cytometry. Protein synthesis in cryo-PLTs was assessed using a modified method based on puromycin-associated nascent chain proteomics. RESULTS: In vitro parameters of reconstituted and subsequently stored platelets were consistent with previously published results. Mass-spectrometry analyses identified that 22 proteins were synthesized in PLTs and 13 of those were observed in platelet microvesicles (PMVs). CONCLUSION: Cryo-PLTs can synthesize proteins upon reconstitution and storage. Discovery of a subset of these proteins in the PMV suggests a role in vesicle encapsulation, possibly in a selective manner. This observation provides novel insights into the capacity for protein synthesis in cryo-PLTs and the potential regulation of protein packaging into PMV.

Cold-stored leukoreduced whole blood: Extending the time between donation and filtration has minimal impact on in vitro quality.

BACKGROUND: Leukoreduced whole blood (LR-WB) has received renewed attention as alternative to component-based transfusion in trauma. According to the manufacturer's instructions, leukoreduction should be carried out within 8 h after collection. This study assessed impact of (1) WB collection bag, (2) LR filtration, and (3) timing of filtration on in vitro quality. STUDY DESIGN AND METHODS: WB collected into different vendor bags was held at room temperature for <8 h or >16 h but <24 h prior to LR. In vitro quality was assessed before and after filtration, and throughout 3 weeks of storage at 4°C. Cell count and hemoglobin levels were determined by hematology analyzer, platelet activation, and responsiveness to ADP by surface expression of P-selectin by flow cytometry, hemolysis by HemoCue, and metabolic parameters by blood gas analyzer. Hemostatic properties were assessed by rotational thromboelastometry. Plasma protein activities and clotting times were determined by automated coagulation analyzer or quantitative immunoblotting. RESULTS: Bag type had no impact on WB in vitro quality. LR by filtration had some impact, but is aligned with data in the literature. The time between donation and filtration resulted in some statistically significant differences in metabolic activity, platelet yield, platelet activation, and factor protein activity initially; however, these differences in in vitro quality attributes decreased throughout 21-day cold storage. CONCLUSION: WB hold time showed only a minor impact on WB in vitro quality, so it may be possible for blood processing facilities to explore extended hold times prior to filtration in order to provide greater operational flexibility.

Improved in vitro quality of stored red blood cells upon oxygen reduction prior to riboflavin/UV light treatment of whole blood.

BACKGROUND: The application of riboflavin/UV-based pathogen inactivation (PI) to whole blood (WB) is currently limited by its negative impact on red blood cell (RBC) quality. The generation of reactive oxidative species in RBC products contributes to increased hemolysis. This study evaluated the impact of deoxygenation of WB prior to riboflavin/UV light treatment versus deoxygenation of RBC concentrates after PI treatment by monitoring RBC in vitro quality parameters. STUDY DESIGN AND METHODS: Six ABO-matched WB units were pooled and split. Within three pairs, one unit was treated with riboflavin/UV light while the other was kept as an untreated control prior to manufacture into red cell concentrates (RCCs). The first pair (Cntr; Cntr-PI) served as the normoxic controls. Deoxygenation was performed at the RCC level for the second pair (RCCdeox; PI-RCCdeox), and at the WB level of the third pair (WBdeox; WBdeox-PI). In vitro qualities of the respective RBC units were assessed throughout storage. RESULTS: The data for the Cntr and Cntr-PI units were comparable to previous reports. The PI-RCCdeox units exhibited worse in vitro quality for most parameters tested compared to Cntr-PI and WBdeox-PI units throughout storage. Hemolysis and microvesicle release was significantly (p < 0.05) higher on Days 21 and 42 in Cntr-PI units compared to WBdeox-PI units. CONCLUSION: WB deoxygenation may help to decrease the accelerated deterioration in RCC in vitro quality caused by treatment with riboflavin/UV light. Treatment of WB under reduced oxygen levels needs to be assessed for PI effectiveness.

Proteomic analysis of red blood cells from donors exhibiting high hemolysis demonstrates a reduction in membrane-associated proteins involved in the oxidative response.

BACKGROUND: The development of hemolysis during ex vivo hypothermic storage is multifaceted. Standardization of collection and production processes is used to minimize variability in biologics manufacturing and to maximize product quality. However, the influence of various donor characteristics on product quality is often difficult to evaluate and to control. Using a proteomic approach, we aimed to decipher relevant donor characteristics that may predict red blood cell (RBC) quality during storage. STUDY DESIGN AND METHODS: Ten healthy volunteer donors exhibiting repeated high hemolysis at outdate (>0.8%; RBCHH ) and 10 age- and sex-matched control donors (RBCCtrl ) were studied. Common quality variables were measured on Days 5, 14, 21, 28, and 42 of storage. Protein profiles of hemoglobin-depleted membrane fractions from RBCHH and RBCCtrl donors were analyzed using a quantitative proteomics approach based on iTRAQ (isobaric tags for relative and absolute quantitation). RESULTS: Time-dependent lesion development was apparent in both donor populations. RBCHH exhibited reduced 2,3-bisphosphoglycerate levels (p < 0.001) and morphologic score (p < 0.001), but displayed elevated hemolysis level (p < 0.001), RBC-derived microvesicle formation (p < 0.001), and mean corpuscular fragility (p < 0.001) compared to RBCCtrl , indicating notable differences at the membrane between the two donor populations. Proteomic findings revealed a significant reduction in the level of proteins involved in oxidative response pathways at early time points in RBCHH compared to that of RBCCtrl . CONCLUSION: The recruitment of these candidate proteins might be part of a response mechanism altered in RBCHH donors and therefore may be useful as a donor screening tool.

p38 mitogen-activated protein kinase regulates mitochondrial function and microvesicle release in riboflavin- and ultraviolet light-treated apheresis platelet concentrates.

BACKGROUND: Biochemical analyses of mechanisms triggered in platelets (PLTs) upon pathogen inactivation (PI) are crucial to further understand the impact of PI on PLT functionality and, subsequently, quality. STUDY DESIGN AND METHODS: PLT concentrates (PCs) were split into four small illumination bags: 1) untreated control, 2) treated with riboflavin and ultraviolet light (RF/UV), and spiked with 3) solvent control dimethyl sulfoxide and 4) p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 before RF/UV treatment. Flow cytometry was used to monitor PLT mitochondrial potential (ΔΨm ); generation of intracellular reactive oxygen species (ROS); and release of microvesicles (MVs), mitochondria (MT), and MVs containing MT (MVs/MT). Quantitative polymerase chain reaction (qPCR) was used to quantify extracellular mitochondrial DNA (mtDNA). Translocation of selected mitochondrial proteins was analyzed in subcellular fractions by immunoblot. RESULTS: RF/UV treatment triggered an increased mitochondrial translocation of both Bax and Bid (p < 0.05, Day 7) and cytochrome c release (p < 0.01, Day 7), loss of ΔΨm (p < 0.05, Day 5 and Day 7), and ROS generation (p < 0.01, Day 5 and Day 7) in PCs compared to the untreated control during storage. These PI-triggered changes were inhibited by SB203580 (p < 0.05). The release of MVs, MT, and MVs/MT was increased upon the RF/UV treatment during storage (p < 0.05) and, with the exception of MT, the release was decreased by the inhibitor (p < 0.05). qPCR analysis showed that RF/UV does not trigger mtDNA release during storage. CONCLUSION: These findings further our understanding of mechanisms in PLTs initiated by the RF/UV treatment, demonstrating that this treatment induces p38 MAPK-dependent mitochondrial signaling and MV release in apheresis PCs.

Pathogen inactivation treatment of plasma and platelet concentrates and their predicted functionality in massive transfusion protocols.

BACKGROUND: Trauma transfusion packages for hemorrhage control consist of red blood cells, plasma, and platelets at a set ratio. Although pathogen reduction improves the transfusion safety of platelet and plasma units, there is an associated reduction in quality. This study aimed to investigate the impact of riboflavin/ultraviolet light-treated plasma or platelets in transfusion trauma packages composed of red blood cell, plasma, and platelet units in a ratio of 1:1:1 in vitro by modeling transfusion scenarios for trauma patients and assessing function by rotational thromboelastometry. STUDY DESIGN AND METHODS: Pathogen-reduced or untreated plasma and buffy coat platelet concentrate units produced in plasma were used in different combinations with red blood cells in trauma transfusion packages. After reconstitution of these packages with hemodiluted blood, the hemostatic functionality was analyzed by rotational thromboelastometry. RESULTS: Hemostatic profiles of pathogen-inactivated buffy coat platelet concentrate and plasma indicated decreased activity compared with their respective controls. Reconstitution of hemodiluted blood (hematocrit = 20%) with packages that contained treated or nontreated components resulted in increased alpha and maximum clot firmness and enhanced clot-formation time. Simulating transfusion scenarios based on 30% blood replacement with a transfusion trauma package resulted in a nonsignificant difference in rotational thromboelastometry parameters between packages containing treated and nontreated blood components (p ≥ 0.05). Effects of pathogen inactivation treatment were evident when the trauma package percentage was 50% or greater and contained both pathogen inactivation-treated plasma and buffy coat platelet concentrate. CONCLUSION: Rotational thromboelastometry investigations suggest that there is relatively little impact of pathogen inactivation treatment on whole blood clot formation unless large amounts of treated components are used.

Pathogen inactivation by riboflavin and ultraviolet light illumination accelerates the red blood cell storage lesion and promotes eryptosis.

BACKGROUND: Pathogen reduction treatment using riboflavin and ultraviolet light illumination (Mirasol) effectively reduces the risk of transfusion-transmitted infections. This treatment is currently licensed for only platelets and plasma products, while its application to whole blood (WB) to generate pathogen-inactivated red blood cells (RBCs) is under development. RBC storage lesion, constituting numerous morphologic and biochemical changes, influences RBC quality and limits shelf life. Stored RBCs further show enhanced susceptibility to RBC programmed cell death (eryptosis) characterized by increased cytosolic Ca2+ -provoked membrane phosphatidylserine (PS) externalization. STUDY DESIGN AND METHODS: Using a "pool-and-split" approach, we examined multiple variables of RBC storage lesion and eryptosis in RBC units, derived from Mirasol-treated or untreated WB, after 4 to 42 days of storage, under blood bank conditions. RESULTS: In comparison to untreated RBC units, Mirasol treatment significantly altered membrane microvesiculation, supernatant hemoglobin, osmotic fragility, and intracellular adenosine triphosphate levels but did not influence membrane CD47 expression and 2,3-diphosphoglycerate levels. Mirasol-treated RBCs showed significantly higher PS exposure after 42, but not after not more than 21, days of storage, which was accompanied by enhanced cytosolic Ca2+ activity, ceramide abundance, and oxidative stress, but not p38 kinase activation. Mirasol treatment significantly augmented PS exposure, Ca2+ entry, and protein kinase C activation after energy depletion, a pathophysiologic cell stressor. Mirasol-treated RBCs were, however, more resistant to cell shrinkage. CONCLUSIONS: Prolonged storage of Mirasol-treated RBCs significantly increases the proportion of eryptotic RBCs, while even short-term storage enhances the susceptibility of RBCs to stress-induced eryptosis, which could reduce posttransfusion RBC recovery in patients.

Altered timing of riboflavin and ultraviolet light pathogen inactivation improves platelet in vitro quality.

BACKGROUND: The platelet (PLT) storage lesion is in part caused by the collection and/or production process. Pathogen inactivation (PI) further accelerates its development leading to a reduced in vitro PLT functionality and hence quality. Although the treatment of PLT concentrates (PCs) with riboflavin and ultraviolet light PI should occur within 22 hours of collection, in this study the impact of treatment timing on in vitro PLT quality was investigated. STUDY DESIGN AND METHODS: Apheresis PCs were PI treated on the day of production or on Days 1, 3, or 4 of storage or left untreated as control. A panel of in vitro variables was used to monitor quality throughout 7-day storage, including metabolism, PLT activation, and release of microparticles. Changes in phosphorylation profiles of proteins in the lysate and levels of PLT factor 4, thrombospondin, and epidermal growth factor (EGF) in the releasate were analyzed by immunoblots or enzyme-linked immunosorbent assay. RESULTS: By Day 7 of storage, units illuminated on Day 4 showed a smaller impact of the PI process than units treated on the day of production or one day after on PLT quality such as PLT activation; metabolic activity; microvesicle and EGF release; and phosphorylation of p38, ERK, and HSP27. PCs treated on Day 3 of storage displayed an intermediate effect. CONCLUSION: The timing of PI treatment of PCs influences in vitro PLT quality. Based on these results, timing recommendations should be reconsidered. If PI is applied, inventory management in blood banks might improve with a more flexible collection and treatment regime.

Riboflavin and ultraviolet illumination affects selected platelet mRNA transcript amounts differently.

BACKGROUND: Pathogen inactivation (PI) techniques use ultraviolet (UV) illumination with or without a photosensitizer to destroy pathogen RNA and DNA. Although lacking a nucleus and innate DNA transcription, platelets (PLTs) contain RNA and can synthesize proteins. The impact of PI on PLT protein synthesis and function is unknown; altered synthesis may affect overall PLT quality. In this study we determine to what extent PLT RNA is affected by PI. STUDY DESIGN AND METHODS: In a pool-and-split design, paired apheresis PLT concentrates were treated with riboflavin and UV illumination or were left untreated. PLT total RNA and mRNA amounts specific for glycoproteins (GP)IIIa, GPIIb, and GPIb; α-granule proteins PLT factor (PF)4; osteonectin and thrombospondin (TSP); and housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using absorbance and quantitative polymerase chain reaction. RESULTS: After treatment, amounts of all analyzed mRNAs were significantly reduced (p < 0.05), but to different degrees. For GAPDH and PF4, transcripts appeared less susceptible to the treatment, with 70% remaining 1 hour after UV illumination. For GPIIIa and TSP, less than 15% remained after treatment. There was a correlation (R(2) = 0.85) between transcript length and amount of mRNA remaining 1 hour after treatment. Total RNA demonstrated a life span equal to the PLT life span of 10 to 11 days. CONCLUSION: This is the first report of the impact of riboflavin and UV illumination on PLT mRNA. Results suggest that all mRNA present in PLTs is affected by the treatment although the degree of the effect varies among transcripts.

Extended storage and glucose exhaustion are associated with apoptotic changes in platelets stored in additive solution.

BACKGROUND: The storage of platelets (PLTs) in additive solution (AS) may facilitate improved PLT quality and possibly extension of the PLT shelf life. A minimum amount of plasma is required when PLTs are stored in AS, as a source of glucose. The aim of this study was to assess the effect of reducing the plasma carryover to 20% on PLT quality when stored in SSP+ for an extended period. STUDY DESIGN AND METHODS: Using a pool-and-split design, buffy coat-derived PLTs were stored in either 30% plasma/SSP+ or 20% plasma/SSP+. In vitro analyses were carried out to Day 10. Metabolites and markers of PLT activation and apoptosis were measured using a blood gas analyzer and flow cytometry. PLT apoptotic protein expression was investigated by Western blotting. RESULTS: Glucose exhaustion occurred in the 20% plasma group between Day 7 and Day 10. The surface expression of P-selectin and PAC-1 was comparable on Day 10 in both groups, suggesting that the PLTs were not activated. However, the exposure of phosphatidylserine and the number of phosphatidylserine-positive microparticles were significantly higher in the 20% group on Day 10. The expression of the proapoptotic proteins Bak, Bax, and cleaved caspase-3 were higher in the 20% plasma group by Day 7 of storage, compared to the 30% plasma group. CONCLUSION: Exhaustion of glucose was associated with a proapoptotic phenotype. Results such as these should be considered before extending the PLT shelf life beyond 7 days, particularly when stored in ASs lacking glucose with low plasma carryover.

Dengue virus persists and replicates during storage of platelet and red blood cell units.

BACKGROUND: Dengue virus (DENV) is a transfusion-transmissible arbovirus that threatens blood donor systems with approximately 200 million high-titer asymptomatic infections occurring annually. Here we investigated the viability of DENV during storage of donor-derived platelet (PLT) and red blood cell (RBC) units. While purified PLTs have been shown to generate viable DENV, RBCs are replication incompetent. Combined with different storage criteria, distinct virus persistence profiles were anticipated in PLT and RBC units. STUDY DESIGN AND METHODS: Mimicking the virus titer of asymptomatic donors, purified DENV was spiked (10(5) -10(6) infectious units/mL) into PLT or RBC units produced and stored according to blood bank operating procedures. DENV was measured by infectious plaque-forming assays and by quantitative reverse transcription-polymerase chain reaction. RESULTS: In both PLT (7 days, 20-24°C) and RBC (42 days, 1-6°C) units, infectious DENV persisted throughout storage despite logarithmic decay. In buffer alone, DENV infectivity was insignificant by Day 1 at 20 to 24°C or 14 days at 1 to 6°C. Infectious virus production was identified in stored PLT units using a translation inhibitor and supported by virus genome replication. Surprisingly, DENV was also produced in RBC units, implying the involvement of cells other than RBCs. CONCLUSION: Both virus propagation and effects independent of cell function mitigate the intrinsic lability of DENV. Nevertheless, the overall rapid storage decay suggests that aged PLT and RBC units may be safer. These data raise awareness to the possible persistence of other conceivably more robust RNA viruses during the storage of cellular blood products.

Effect of texture of platelet bags on bacterial and platelet adhesion.

BACKGROUND: Missed detection of Staphylococcus epidermidis contamination in platelet (PLT) storage bags by the standard 24-hour-postcollection BacT/ALERT screening test has been documented. A slow growth rate and the strong tendency of this bacterium to adhere to surfaces can contribute to missed detection of the pathogen. STUDY DESIGN AND METHODS: Topography of two different PLT storage bag surfaces, textured (rough) and smooth surfaces of Terumo 80440 bags (designated A15), was studied. Adhesion of biofilm-positive and -negative S. epidermidis strains on these surfaces was evaluated under static conditions. Quality of stored PLTs in A15 bags under blood bank conditions was compared for two different bag orientations (rough vs. smooth surface down) on Days 2, 5, and 7 of storage. PLT adhesion on the surfaces was evaluated after 7 days of storage. RESULTS: Bacterial adhesion and biofilm formation were significantly higher on the rough surfaces of A15 bags compared to the smooth surfaces. After 7 days of storage in A15 bags, PLTs showed similar metabolite levels, pH, and response capacity in the bags with different orientation and more PLT adhesion and aggregation was observed on rough surfaces. CONCLUSION: Higher bacterial adhesion on rough surfaces can contribute to missed detection of bacterial strains that tend to adhere on surfaces. PLT adhesion and aggregation on rough surfaces can affect the quality and safety of PLTs by promoting more bacterial adhesion and biofilm formation on surfaces.

p38MAPK is involved in apoptosis development in apheresis platelet concentrates after riboflavin and ultraviolet light treatment.

BACKGROUND: Pathogen inactivation (PI) accelerates the platelet (PLT) storage lesion, including apoptotic-like changes. Proteomic studies have shown that phosphorylation levels of several kinases increase in PLTs after riboflavin and UV light (RF-PI) treatment. Inhibition of p38MAPK improved in vitro PLT quality, but the biochemical basis of this kinase's contribution to PLT damage requires further analysis. STUDY DESIGN AND METHODS: In a pool-and-split design, apheresis PLT concentrates were either treated or kept untreated with or without selected kinase inhibitors. Samples were analyzed throughout 7 days of storage, monitoring in vitro quality variables including phosphatidylserine exposure, degranulation, and glucose metabolism. Changes in the protein expression of Bax, Bak, and Bcl-xL and the activities of caspase-3 and -9 were determined by immunoblot analysis and flow cytometry, respectively. RESULTS: The expression levels of the proapoptotic proteins Bax and Bak, but not the antiapoptotic protein Bcl-xL, were significantly increased after the RF-PI treatment. This trend was reversed in the presence of p38MAPK inhibitor SB203580. As a result of increasing proapoptotic protein levels, caspase-3 and -9 activities were significantly increased in RF-PI treatment during storage compared with control (p < 0.05). Similarly, p38MAPK inhibition significantly reduced these caspase activities compared with vehicle control after RF-PI treatment (p < 0.05). CONCLUSION: These findings revealed that p38MAPK is involved in signaling leading to apoptosis triggered by RF-PI. Elucidation of the biochemical processes influenced by PI is a necessary step in the development of strategies to improve the PLT quality and ameliorate the negative effects of PI treatment.