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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.

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.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.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.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.

Platelets are a key component of massive transfusion in treating actively bleeding patients. While optimized for prophylactic transfusions, the effectiveness of the current standard room temperature stored platelets (RPs) in treating actively bleeding patients is not clear. Cold-stored platelets (CPs) have been shown to have superior hemostatic functions and the potential to extend shelf life. In this study, we explored the effect of using CPs versus RPs in an in vitro transfusion model based on the massive transfusion protocol.RPs or CPs were combined with RBCs and plasma in a 1:1:1 volume ratio to make transfusion packages. Whole blood was collected and then either diluted to 20% hematocrit or mixed with tPA (8.8 μg/ml). By volume, 70% of transfusion package was mixed with 30% whole blood to simulate massive transfusions and analyzed by rotational thromboelastometry. Transfusion package supernatant was analyzed for PAI-1 activity as well.Both transfusion packages restored the clot characteristics of hemodiluted or hyperfibrinolytic whole blood. Specifically, only transfusion packages made with CPs significantly reduced the maximum clot lysis of hyperfibrinolytic whole blood. PAI-1 activity in CPs transfusion packages were also significantly higher.Transfusion packages containing cold-stored platelets may be able to restore the blood hemostatic profile of bleeding patients. In addition, transfusion packages made from CPs may provide additional benefit of resisting hyperfibrinolysis in bleeding patients. In trauma where post-transfusion platelet recovery is less of a concern, CPs are a viable option to restore hemostasis.

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.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.K. pneumoniae and S. pyogenes proliferated in WB prior to leukoreduction reaching concentrations ≤10Extended 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.

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

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 16 h but

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.

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.

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.

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

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.

Background Pathogen inactivation (PI) technologies are currently licensed for use with platelet (PLT) and plasma components. Treatment of whole blood (WB) would be of benefit to the blood banking community by saving time and costs compared to individual component treatment. However, no paired, pool-and-split study directly assessing the impact of WB PI on the subsequently produced components has yet been reported. Study Design and Methods In a “pool-and-split” study, WB either was treated with riboflavin and ultraviolet (UV) light or was kept untreated as control. The buffy coat (BC) method produced plasma, PLT, and red blood cell (RBC) components. PLT units arising from the untreated WB study arm were treated with riboflavin and UV light on day of production and compared to PLT concentrates (PCs) produced from the treated WB units. A panel of common in vitro variables for the three types of components was used to monitor quality throughout their respective storage periods. Results PCs derived from the WB PI treatment were of significantly better quality than treated PLT components for most variables. RBCs produced from the WB treatment deteriorated earlier during storage than untreated units. Plasma components showed a 3% to 44% loss in activity for several clotting factors. Conclusion Treatment of WB with riboflavin and UV before production of components by the BC method shows a negative impact on all three blood components. PLT units produced from PI-treated WB exhibited less damage compared to PLT component treatment.

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.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.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.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.

Background Pathogen reduction technologies (PRTs) significantly reduce the risk of transmission of infectious agents in platelet (PLT) concentrates; however, in vitro studies reveal a negative impact on PLT quality after PRT treatment including effects on PLT aggregation, integrin αIIbβ3 conformation, and actin dynamics. Clinically, the interval between transfusions is shortened. Study Design and Methods Seeking to understand the biochemical mechanisms underlying these observed effects, we analyzed signal transduction in PLT concentrates after riboflavin and ultraviolet light (UV; Mirasol) treatment and subsequent storage focusing on the phosphorylation levels of selected protein kinases. Results Among identified candidates, p38MAPK increased fourfold in phosphorylation after PRT. Incubation of PLT concentrates with a p38MAPK-specific inhibitor before PRT significantly improved numerous PLT quality measures. Phosphorylation levels of the p38MAPK substrates AKT, VASP, and HSP27 also decreased with inhibitor treatment. Phospho-HSP27 decrease in the presence of the inhibitor correlated with a reduction in PLT activation determined by surface expression of P-selectin. Conclusion These findings support a model of one dominant underlying molecular signaling mechanism that is impacted by the riboflavin and UV (Mirasol) PRT process resulting in alterations in PLT quality. The identification of such a target should assist in the development of strategies to ameliorate this negative aspect of an otherwise beneficial and important safety development for transfusion medicine.

Background and Objectives Staphylococcus epidermidis forms surface-attached aggregates (biofilms) in platelet concentrates (PCs), which are linked to missed detection during PC screening. This study was aimed at evaluating the efficacy of riboflavin-UV treatment to inactivate S. epidermidis biofilms in buffy coat (BC) PCs. Materials and Methods Biofilm and non-biofilm cells from S. epidermidis ST-10002 and S. epidermidis AZ-66 were individually inoculated into whole blood (WB) units (~106 colony-forming units (CFU)/ml) (N = 4–5). One spiked and three unspiked WB units were processed to produce a BC-PC pool. Riboflavin was added to the pool which was then split into two bags: one for UV treatment and the second was untreated. Bacterial counts were determined before and after treatment. In vitro PC quality was assessed by flow cytometry and dynamic light scattering. Results Bacterial counts were reduced during BC-PC production from ~106 CFU/ml in WB to 103–104 CFU/ml in PCs (P 0·05). Platelet activation was enhanced in PCs produced with WB inoculated with biofilms compared to non-biofilm cells (P < 0·05). Conclusion Riboflavin-UV treatment was similarly efficacious in PCs produced from WB inoculated with S. epidermidis biofilm or non-biofilm cells. Levels of biofilm-derived S. epidermidis ≥103 CFU/ml were not completely inactivated; however, further testing is necessary with lower (real-life) bacterial levels.

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.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.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.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.

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.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 (ΔΨRF/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 ΔΨThese 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.

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.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.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.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.

Quality management of blood products is essential for blood banking. It is influenced by both processing and donor characteristics and assured by monitoring routine in vitro parameters to defined product specifications. However, these measures correlate poorly with the in vivo behavior of transfused platelets and cannot be used to select optimal donors. Since radiolabeled platelet recovery and survival studies are expensive and time consuming, there is an ongoing search for simpler measures that predict platelet transfusion outcomes. We performed a pilot study using semi-qualitative proteomics to assess changes in the platelet protein profile of donors with either acceptable or unacceptable in vivo radiolabeled autologous platelet recovery and survival measurements. Proteins changing during a 9-day storage period included cytoskeletal elements talin, vinculin and moesin as well as signal transduction proteins 14-3-3, RhoGDI and Rap1. Two of nine donations exhibited a decrease in these proteins and poor in vivo platelet recovery and survival whereas the remaining donors showed acceptable platelet recovery and survival and expected protein profiles. Analyses revealed a significant correlation between protein levels of Rap1 and RhoGDI during storage and platelet recovery and survival. This study provides for the first time preliminary data showing evidence of the utility of protein profiling to predict platelet transfusion quality. This article is part of a Special Issue entitled: Integrated omics.

Background and Objectives There is no automated, accurate assay for the enumeration of residual red blood cells (rRBCs) in non-RBC components for transfusion, despite the potential risk of allo-immunization when mismatched components are transfused. Materials and Methods The automated ADVIA 120 cerebrospinal fluid (CSF) assay, which is approved to count RBCs and WBCs in CSF samples, was optimized and tested to measure rRBC in platelet concentrate (PC) and plasma components. Results Sample dilution, incubation time and reagent volume were optimized for use with non-RBC blood products. The assay was linear (R2 = 0·99), even at low rRBCs counts. Intra- and inter-assay variation gave coefficients of variance (CV) between 2·2 and 9·4% and 2·6 and 14·9%, respectively, depending on rRBC levels. Good correlation (r = 0·995) was found between the automated assay and manual counting, which is considered the gold standard. Using the automated assay, the range of rRBCs (count/unit) in buffy-coat platelet concentrate (PCs) was 27–5505 × 106 and in apheresis PCs was 1–361 × 106. Conclusion The ADVIA CSF assay is a sensitive, precise and accurate means to assess rRBC counts in non-RBC components.

BACKGROUND: The in vitro quality of small-volume platelet (PLT) aliquots for pediatric transfusions was assessed to determine the best practice approach. STUDY DESIGN AND METHODS: Small volumes (50 mL) of single apheresis PLT components (APCs), collected on either CaridianBCT Trima or Haemonetics MCS+ instruments, were aliquoted on Days 2, 3, 4, and 5 postcollection into Fenwal PL1240 or 4R2014 bags or 60-mL polypropylene syringes. Samples were tested for in vitro quality at their recommended expiry times (4 hr for 4R2014 bags and syringes or Day 5 for PL1240 bags). Assays included pH, CD62P expression, and metabolic measures. RESULTS: CD62P expression increased throughout storage in all containers. Among the small-volume containers, pH, pCO2, lactate, and bicarbonate varied considerably. Regardless of the day of aliquoting, pCO2 was significantly higher and pO2 was significantly lower in gas-impermeable syringes than other containers. No bacterial growth was detected in any sample. CONCLUSION: The quality of APCs aliquoted into small-volume containers meets regulatory requirements and is generally equivalent to that of full-volume APCs at expiry.

BACKGROUND: A quality monitoring program (QMP) for platelet concentrates (PCs) was implemented at Canadian Blood Services (CBS) to improve standards and to better understand platelet (PLT) products by supplementing routine quality control (QC). STUDY DESIGN AND METHODS: Annual surveys of PCs from CBS production sites were conducted, with four completed to date (QMP Cycles 1-4) spanning two different PC production methods: PLT-rich plasma (PRP) and buffy coat (BC). Randomly selected PCs were sent to a central laboratory and tested 1 day after expiry. An expanded panel of tests including CD62P expression by flow cytometry, mean PLT volume, PLT count and morphology, extent of shape change, and PLT metabolic parameters, were applied. RESULTS: QMP data on the implementation of the BC production method across CBS indicated that BC PCs have less variable in vitro quality measures than PRP PCs. For the QC parameters pH and PLT count per unit, the range of mean values from each site for QMP 3 and 4 fell well within the range defined by regulatory standards, a first step in defining quality benchmarks for PCs. Of the extended panel of quality parameters, CD62P expression was the most sensitive indicator of change and identified an issue with the implementation of the BC PC production method at one site, which was subsequently remedied. CONCLUSION: A QMP was found to be useful to monitor production processes across sites and highlights best practice approaches while deepening understanding of the quality of PLT products at CBS.

BACKGROUND: Pathogen reduction technologies (PRTs) were developed to improve the safety of platelet concentrates (PCs) for transfusion purposes; however, several studies report a negative impact on the in vitro and in vivo platelet (PLT) quality. Therefore, analyses of the underlying molecular processes triggered by PRT treatments are necessary to understand their effects on PLT function. STUDY DESIGN AND METHODS: In two separate two-arm studies PCs prepared in plasma for storage either by the leukoreduced buffy coat (BC-PCs) or by the leukoreduced apheresis (AP-PCs) method were treated with or without riboflavin and ultraviolet (UV) light (Mirasol; 6.24 J/mL; 265-375 nm). Samples were drawn after treatment and after 1, 4, and 6 days of storage with subsequent analyses performed using in vitro measurements for PLT quality monitoring. Semiquantitative proteomic studies identified proteins that changed in band intensities in response to treatment or storage. Protein validation and subsequent biochemical studies were carried out by immunoblot analyses. RESULTS: The proteomic results identified changes mainly of proteins associated with the structure and regulation of the cytoskeleton. Focusing on the vasodilator-stimulated phosphoprotein (VASP) in AP-PCs revealed a storage-dependent, but treatment-independent, delocalization and a strong treatment-dependent phosphorylation at Ser-239 that was also present, but to a much lesser degree in BC-PCs. This modification correlated exponentially with PLT activation as determined by P-selectin expression. CONCLUSION: Treatment of PCs with Mirasol leads to the amplification of VASP Ser-239 phosphorylation, which is linked to actin dynamics and regulation of integrin αIIbβ3 activation. This change offers one explanation for Mirasol's impact on PLT in vitro quality measures. The Ser-239 phosphorylation level of VASP might be a useful protein marker for riboflavin and UV light–mediated PLT compromise.

Summary Introduction: The determination of quality parameters is a necessity for monitoring the efficacy of platelet concentrates. During consolidated quality control studies, there may be a large number of samples to be analyzed at the same time. This common workflow setup triggered the question whether there is an influence of the number of samples to be analyzed on the accuracy of the test results. Methods: Two different sample volumes of platelet concentrates, 1 ml and 50 ml, were analyzed for a set of standard in vitro parameters including pCO2, pO2, pH, glucose, and lactate as well as platelet activation via CD62P expression and responsiveness to adinosine diphosphate in an extent-of-shape-change assay. To assess apoptotic mechanisms triggered by the hold time, changes in the phosphatidylserine exposure were monitored. Results: In total, eleven time points were assessed over a 3-h period as well as an overnight point for assay evaluation. Except for pCO2 and pO2, all in vitro parameters analyzed were unaffected by a sample hold time of up to 3-h. Conclusion: Sampling for pO2 determination should be carried out in small volumes and assessed within 30 min of collection to obtain reliable and comparable results.

BACKGROUND: With buffy coat (BC) processing of whole blood (WB) donations, the preparation of plasma occurs within 24 hours rather than 8 hours of collection. The effect of this change on coagulation factor function in plasma and cryoprecipitate was evaluated during the validation of this production method and with routine production. STUDY DESIGN AND METHODS: Plasma frozen after an overnight hold of WB was prepared via BC or whole blood filtration (WBF) methods and quality control (QC) variables were measured. Additionally, plasma prepared with the BC method was compared to plasma produced using the platelet-rich plasma (PRP) method with an extended plasma factor analysis. Selected plasma factor levels were also measured in both cryoprecipitate and cryosupernatant plasma prepared using the WBF method from plasma frozen on the day of collection or after an overnight hold of WB. RESULTS: When comparing BC plasma to PRP plasma, coagulation factors (F)II, VII, VIII, IX, X, and XI had somewhat lower levels, and fibrinogen and antithrombin levels were elevated. As expected the most sensitive to the prolongation of production time was FVIII with 72 and 78% of the activity of PRP plasma and cryoprecipitate, respectively. However, both still met QC standards. Similarly, products made in routine production show acceptable levels of FVIII. CONCLUSION: Plasma and cryoprecipitate products, prepared using methods in which the plasma is frozen close to 24 hours after collection, meet current quality standards. The longer WB storage time has been implemented into general use in Canada.

BACKGROUND: In high-volume processing environments, manual breakage of in-line closures can result in repetitive strain injury (RSI). Furthermore, these closures may be incorrectly opened causing shear-induced hemolysis. To overcome the variability of in-line closure use and minimize RSI, Fresenius Kabi developed a new in-line closure, the CompoFlow, with mechanical openers. STUDY DESIGN AND METHODS: The consistency of the performance of the CompoFlow closure device was assessed, as was its effect on component quality. A total of 188 RBC units using CompoFlow blood bag systems and 43 using the standard bag systems were produced using the buffy coat manufacturing method. Twenty-six CompoFlow platelet (PLT) concentrates and 10 control concentrates were prepared from pools of four buffy coats. RBCs were assessed on Days 1, 21, and 42 for cellular variables and hemolysis. PLTs were assessed on Days 1, 3, and 7 for morphology, CD62P expression, glucose, lactate, and pH. A total of 308 closures were excised after processing and the apertures were measured using digital image analysis. RESULTS: The use of the CompoFlow device significantly improved the mean extraction time with 0.46 ± 0.11 sec/mL for the CompoFlow units and 0.52 ± 0.13 sec/mL for the control units. The CompoFlow closures showed a highly reproducible aperture after opening (coefficient of variation, 15%) and the device always remained opened. PLT and RBC products showed acceptable storage variables with no differences between CompoFlow and control. CONCLUSIONS: The CompoFlow closure devices improved the level of process control and processing time of blood component production with no negative effects on product quality.

Background In Europe, buffy-coat processing allows for the use of platelet additive solutions (PAS). These solutions, however, have long been questioned for their lack of glucose, a potentially essential nutrient for platelet storage. Using a novel, practical, two-part system for incorporation of glucose into an additive solution (PAS-G), this study compares platelet storage in plasma to storage in PAS-G. Study Design and Methods A paired study design of platelet concentrates (PC) were prepared from leucoreduced pools of eight buffy coats (BCP) split into two equal pools, with suspension in autologous plasma, or PAS-G. On days 2, 5, 7 and 9 of storage, samples were tested using standard in vitro platelet parameters. Data were analysed by paired Student's t-tests. Results During storage, PCs in PAS-G maintain a quality profile that is strikingly similar to PCs stored in plasma in terms of platelet activation (CD62) morphology score, swirl, glucose metabolism and pH. However, PCs in PAS-G perform lower (P

BACKGROUND: Buffy coat (BC) production of platelets (PLTs) has been successfully used in Europe for more than two decades. Currently, Canadian Blood Services is implementing the BC method. This article summarizes results of the validation testing performed to qualify the process of PLT production from whole blood and compares the quality of PLTs produced in routine production by either the PLT-rich plasma method (PRPPCs) or the BC method (BC-PCs). STUDY DESIGN AND METHODS: Validation data included variables used for routine quality control (QC; pH, PLT count, volume, sterility, residual white blood cell count) as well as nonroutine testing of PLTs for PLT activation, metabolic changes during storage, and PLT responsiveness to hypotonic shock and the extent of shape change induced by adenosine 5′-diphosphate. BC-PCs were tested on Days 1 and 6. QC of production runs included the same routine tests performed on Day 6. RESULTS: PLTs produced by the BC method during validation and pilot implementation met all Canadian Standards Association standards with respect to yield, volume, pH, and leukoreduction. Additional validation testing indicated a moderate level of PLT storage lesion development. In comparison to PRP-PCs, in vitro variables of BC-PCs, either pH in this study, or other markers compared to the literature were better, suggesting that BC-PCs have less evidence of productionrelated damage and improved PLT quality during storage. CONCLUSIONS: PLT concentrates produced from whole blood by the BC method after an overnight hold have laboratory variables suggestive of a higher quality than those concentrates produced by the PRP method. C anadian Blood Services undertook to change its method of component production from whole blood from the North American standard platelet-rich plasma (PRP) method to the buffy coat (BC) method of platelet (PLT) production. Developed by investigators in the Netherlands and Sweden in the mid-1970s, 1 the BC production method reverses the sequence of centrifugation steps compared to PRP. A hard spin is used initially to separate whole blood into three components: plasma, red blood cells (RBCs), and a BC layer. Using semiautomated extraction, the most common configuration uses a so-called top-and-bottom collection set in which plasma and RBCs are transferred to storage containers and the BC is left in the donation bag. This BC contains PLTs, white blood cells (WBCs), plasma, and some RBCs. ABO-matched BCs are pooled together with 1 plasma unit from one of the donors or 1 unit of PLT additive solution using a sterile docking device. The pooled BC is then given a soft spin, and the PRP is extracted with or without leukofiltration to produce a pooled PLT concentrate. Although both methods produce a PLT product, the products may have somewhat different in vitro characteristics. There is noticeable quality improvement in laboratory markers of BC-produced PLTs

PURPOSE Pathogen inactivation technologies (PITs) were introduced into blood banking to further improve the safety of blood products. However, the UV light used in PITs to terminate pathogen growth might alter the functionality of the cells in the blood product as well as the protein profile of the blood components. This study employed proteomic approaches to assess changes in the platelet proteome and translatome. EXPERIMENTAL DESIGN Apheresis-derived platelet concentrates treated with riboflavin/UV light or untreated controls were analyzed throughout blood bank storage by quantitative proteomics using iTRAQ and puromycin-associated nascent chain (PUNCH) proteomics. RESULTS Quantitative proteomic analysis identified 408 individual proteins including 26 unique proteins that changed in the treated arm during storage. Proteomic results were confirmed using immunoblot analyses and results suggested a translational control of the protein expression profile. PUNCH proteomic analysis of day 7 samples from illuminated units identified 52 unique platelet proteins that incorporated puromycin, including proteins involved in the cytoskeleton, metabolism, and signaling. CONCLUSION AND CLINICAL RELEVANCE This study demonstrates for the first time that platelets can synthesize proteins despite the riboflavin and UV treatment and suggests that platelets may possess a mechanism to protect their mRNA from damage by the PI 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

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.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.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).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.

Pathogen inactivation (PI) technologies are currently licensed for use with platelet (PLT) and plasma components. Treatment of whole blood (WB) would be of benefit to the blood banking community by saving time and costs compared to individual component treatment. However, no paired, pool-and-split study directly assessing the impact of WB PI on the subsequently produced components has yet been reported.In a "pool-and-split" study, WB either was treated with riboflavin and ultraviolet (UV) light or was kept untreated as control. The buffy coat (BC) method produced plasma, PLT, and red blood cell (RBC) components. PLT units arising from the untreated WB study arm were treated with riboflavin and UV light on day of production and compared to PLT concentrates (PCs) produced from the treated WB units. A panel of common in vitro variables for the three types of components was used to monitor quality throughout their respective storage periods.PCs derived from the WB PI treatment were of significantly better quality than treated PLT components for most variables. RBCs produced from the WB treatment deteriorated earlier during storage than untreated units. Plasma components showed a 3% to 44% loss in activity for several clotting factors.Treatment of WB with riboflavin and UV before production of components by the BC method shows a negative impact on all three blood components. PLT units produced from PI-treated WB exhibited less damage compared to PLT component treatment.

Pathogen reduction technologies (PRTs) significantly reduce the risk of transmission of infectious agents in platelet (PLT) concentrates; however, in vitro studies reveal a negative impact on PLT quality after PRT treatment including effects on PLT aggregation, integrin αIIbβ3 conformation, and actin dynamics. Clinically, the interval between transfusions is shortened.Seeking to understand the biochemical mechanisms underlying these observed effects, we analyzed signal transduction in PLT concentrates after riboflavin and ultraviolet light (UV; Mirasol) treatment and subsequent storage focusing on the phosphorylation levels of selected protein kinases.Among identified candidates, p38MAPK increased fourfold in phosphorylation after PRT. Incubation of PLT concentrates with a p38MAPK-specific inhibitor before PRT significantly improved numerous PLT quality measures. Phosphorylation levels of the p38MAPK substrates AKT, VASP, and HSP27 also decreased with inhibitor treatment. Phospho-HSP27 decrease in the presence of the inhibitor correlated with a reduction in PLT activation determined by surface expression of P-selectin.These findings support a model of one dominant underlying molecular signaling mechanism that is impacted by the riboflavin and UV (Mirasol) PRT process resulting in alterations in PLT quality. The identification of such a target should assist in the development of strategies to ameliorate this negative aspect of an otherwise beneficial and important safety development for transfusion medicine.

The in vitro quality of small-volume platelet (PLT) aliquots for pediatric transfusions was assessed to determine the best practice approach.Small volumes (50 mL) of single apheresis PLT components (APCs), collected on either CaridianBCT Trima or Haemonetics MCS+ instruments, were aliquoted on Days 2, 3, 4, and 5 postcollection into Fenwal PL1240 or 4R2014 bags or 60-mL polypropylene syringes. Samples were tested for in vitro quality at their recommended expiry times (4 hr for 4R2014 bags and syringes or Day 5 for PL1240 bags). Assays included pH, CD62P expression, and metabolic measures.CD62P expression increased throughout storage in all containers. Among the small-volume containers, pH, pCO(2) , lactate, and bicarbonate varied considerably. Regardless of the day of aliquoting, pCO(2) was significantly higher and pO(2) was significantly lower in gas-impermeable syringes than other containers. No bacterial growth was detected in any sample.The quality of APCs aliquoted into small-volume containers meets regulatory requirements and is generally equivalent to that of full-volume APCs at expiry.

A quality monitoring program (QMP) for platelet concentrates (PCs) was implemented at Canadian Blood Services (CBS) to improve standards and to better understand platelet (PLT) products by supplementing routine quality control (QC).Annual surveys of PCs from CBS production sites were conducted, with four completed to date (QMP Cycles 1-4) spanning two different PC production methods: PLT-rich plasma (PRP) and buffy coat (BC). Randomly selected PCs were sent to a central laboratory and tested 1 day after expiry. An expanded panel of tests including CD62P expression by flow cytometry, mean PLT volume, PLT count and morphology, extent of shape change, and PLT metabolic parameters, were applied.QMP data on the implementation of the BC production method across CBS indicated that BC PCs have less variable in vitro quality measures than PRP PCs. For the QC parameters pH and PLT count per unit, the range of mean values from each site for QMP 3 and 4 fell well within the range defined by regulatory standards, a first step in defining quality benchmarks for PCs. Of the extended panel of quality parameters, CD62P expression was the most sensitive indicator of change and identified an issue with the implementation of the BC PC production method at one site, which was subsequently remedied.A QMP was found to be useful to monitor production processes across sites and highlights best practice approaches while deepening understanding of the quality of PLT products at CBS.

Pathogen reduction technologies (PRTs) were developed to improve the safety of platelet concentrates (PCs) for transfusion purposes; however, several studies report a negative impact on the in vitro and in vivo platelet (PLT) quality. Therefore, analyses of the underlying molecular processes triggered by PRT treatments are necessary to understand their effects on PLT function.In two separate two-arm studies PCs prepared in plasma for storage either by the leukoreduced buffy coat (BC-PCs) or by the leukoreduced apheresis (AP-PCs) method were treated with or without riboflavin and ultraviolet (UV) light (Mirasol; 6.24 J/mL; 265-375 nm). Samples were drawn after treatment and after 1, 4, and 6 days of storage with subsequent analyses performed using in vitro measurements for PLT quality monitoring. Semiquantitative proteomic studies identified proteins that changed in band intensities in response to treatment or storage. Protein validation and subsequent biochemical studies were carried out by immunoblot analyses.The proteomic results identified changes mainly of proteins associated with the structure and regulation of the cytoskeleton. Focusing on the vasodilator-stimulated phosphoprotein (VASP) in AP-PCs revealed a storage-dependent, but treatment-independent, delocalization and a strong treatment-dependent phosphorylation at Ser-239 that was also present, but to a much lesser degree in BC-PCs. This modification correlated exponentially with PLT activation as determined by P-selectin expression.Treatment of PCs with Mirasol leads to the amplification of VASP Ser-239 phosphorylation, which is linked to actin dynamics and regulation of integrin α(IIb) β(3) activation. This change offers one explanation for Mirasol's impact on PLT in vitro quality measures. The Ser-239 phosphorylation level of VASP might be a useful protein marker for riboflavin and UV light-mediated PLT compromise.

BACKGROUND: Buffy-coat processing allows for the use of platelet additive solutions (PASs). PASs reduce plasma-associated transfusion reactions and conserve plasma for transfusion or fractionation. Platelet (PLT) storage in plasma was compared to storage in three commercially available PASs compared to assess their influence on in vitro laboratory variables. STUDY DESIGN AND METHODS: Platelet concentrates (PCs) were prepared from leukoreduced pools of four buffy coats (BCPs) suspended in autologous plasma or one of PASs (Composol, Fresenius-Kabi; T-Sol, Baxter Corp.; or SSP+, MacoPharma). On Days 1, 2, 3, 5, and 7 of storage, samples were tested for PLT concentration, mean PLT volume (MPV), CD62P, morphology, pO2, pCO2, glucose, lactate and total protein concentration, pH, extent of shape change (ESC), and hypotonic shock response (HSR). Data were analyzed by analysis of variance (ANOVA) with repeated measures and t tests. RESULTS: PLT recoveries from BCPs were higher (p