Pathogen Reduction Technology Treatment Research Articles

The effects of pathogen reduction technology on apheresis platelet concentrates stored in PAS.

The impact of pathogen reduction technology (PRT) such as Mirasol, and the effect of platelet additive solutions (PAS) on the activity and hemostatic profile of transfused apheresis platelets remain largely unknown. The aim of this study was to assess the in vitro hemostatic and metabolic profile of Mirasol treated platelets in PAS during a 7-day storage period. Ten split bags containing apheresis platelets stored in PAS were split into two groups; control platelets (No.=10 units) and PRT-treated platelets (No.=10 units). In vitro evaluation of the platelet components was performed on the 1st, 3rd, 5th, and 7th days of the storage period. Several metabolic parameters including pH, glucose, and lactate levels were evaluated, while assessment of their hemostatic capacity was performed using light transmission aggregometry (LTA) and viscoelastic studies such as rotational thromboelastometry (ROTEM) and thromboelastography (TEG). Last, Annexin V levels were measured though flow cytometry for evaluation of platelet activation. Clot strength, as reflected by the maximum clot firmness (MCF) and the maximum amplitude (MA) parameters of the viscoelastic studies was significantly decreased in the PRT-treated platelets compared to the control platelets (p<0.05). Clot strength based on MCF and MA values was also found to be decreasing over storage time in PRT-treated platelets (p<0.001), while this was not evident in control platelets. Moreover, the comparison between pH, glucose, and lactate levels were indicative of increased metabolic activity in PRT-treated platelets compared to control platelets (p<0.001). Last, Annexin-V was significantly higher in PRT-treated platelets compared to control platelets on the 7th day of the storage period (p<0.001). The results of this study indicate that increased PSL induced by PRT treatment leads to a decreased in vitro platelet hemostatic efficacy and increased metabolic activity. However, the clinical impact of these alternations needs further investigation.

Haemostatic profile of riboflavin-treated apheresis platelet concentrates.

The haemostatic activity of platelet concentrates (PCs) treated with pathogen reduction technology (PRT) remains a subject of debate. Our aim was to investigate the effect of Mirasol PRT on the haemostatic properties of PCs stored in plasma. Untreated and Mirasol-treated platelets stored in plasma and derived from ten split double-dose apheresis PCs were evaluated in vitro on days 1, 3 and 5 post collection for functionality, microparticle procoagulation activity (MPA), endogenous thrombin potential (ETP), and haemostatic profile using rotational thromboelastometry (ROTEM). P-selectin expression was significantly higher in Mirasol-treated platelets compared with untreated counterparts on days 3 and 5 (p=0.003 and p=0.002, respectively). Clot strength, as shown by EXTEM maximum clot firmness (MCF), was significantly lower in the Mirasol-treated platelets at all time points (days 1, 3, 5) than in untreated platelets (p=0.009, p<0.001, p<0.001, respectively). There was a considerable increase in MPA over time (p<0.001) and this was significantly higher in the Mirasol-treated platelets on day 5 (p=0.015). A notable acceleration of decrease in ETP values was observed for Mirasol-treated PCs over time (p<0.001), with significant differences between PRT-treated and untreated PCs on days 3 and 5 (p=0.038 and p=0.019, respectively). Clot strength attenuation was significantly associated with pH reduction (p<0.001, Spearman's rho: 0.84), increased microparticle procoagulant activity (p<0.001, Spearman's rho: -0.75), and with decreased ETP (p<0.032, Spearman's rho: 0.41). Increased platelet activation induced by PRT treatment leads to a decrease in in vitro haemostatic capacity as seen by reduced clot strength and thrombin generation capacity over time. The clinical relevance of this needs to be investigated.

Improvement of Blood Processing and Safety by Automation and Pathogen Reduction Technology

Introduction: The objective of the present study was to describe the experience of the Blood and Tissues Bank of Aragon with the Reveos® Automated Blood Processing System and Mirasol® Pathogen Reduction Technology (PRT) System, comparing retrospectively routine quality data obtained in two different observation periods. Methods: Comparing quality data encompassing 6,525 blood components from the period 2007–2012, when the semi-automated buffy coat method was used in routine, with 6,553 quality data from the period 2014–2019, when the Reveos system and subsequently the Mirasol system were implemented in routine. Results: Moving from buffy coat to Reveos led to decreased discard rates of whole blood units (1.2 to 0.1%), increased hemoglobin content (48.1 ± 7.6 to 55.4 ± 6.6 g/unit), and hematocrit (58.9 ± 6.5% to 60.0 ± 4.9%) in red blood cell concentrates. Platelet concentrates (PCs) in both periods had similar yields (3.5 ×10¹¹). Whereas in the earlier period, PCs resulted from pooling 5 buffy coats, in the second period 25% of PCs were prepared from 4 interim platelet units. The mean level of factor VIII in plasma was significantly higher with Reveos (92.8 vs. 97.3 IU). Mirasol PRT treatment of PCs reduced expiry rates to 1.2% in 2019. One septic transmission was reported with a non-PRT treated PCs, but none with PRT-treated PCs. Conclusion: Automation contributed to standardization, efficiency, and improvement of blood processing. Released resources enabled the effortless implementation of PRT. The combination of both technologies guaranteed the self-sufficiency and improvement of blood safety.

Haemostatic function measured by thromboelastography and metabolic activity of platelets treated with riboflavin and UV light.

Pathogen reduction technology (PRT) may damage platelet (PLT) components. To study this, metabolic activity and haemostatic function of buffy coat (BC) PLT concentrates, with or without riboflavin and UV light PRT treatment, were compared. Twenty-four BC PLT concentrates, leukoreduced and diluted in additive solution, were grouped into 12 type-matched pairs, which were pooled and divided into 12 non-PRT-treated BC PLT concentrates (control units) and 12 riboflavin and UV PRT-treated BC PLT concentrates (test units). Haemostatic function and metabolic parameters were monitored by thrombelastography at days 1, 3, 7 and 14 post collection in both PLT groups. Loss of PLT discoid shape, glucose consumption, lactate production, and decrease in pH were greater in the PRT-treated PLTs than in control PLTs over time (p<0.001). PLT haemostatic function evaluated by clot strength was also significantly weaker in PRT-treated PLTs compared with the excellent clot quality of control PLTs at day 7 (maximum amplitude: 41.27 vs 64.27; p<0.001), and even at day 14 (21.16 vs 60.39; p<0.001) of storage. Pathogen reduction technology treatment accelerates and increases platelet storage lesion, resulting in glucose depletion, lactate accumulation, PLT acidification, and discoid shape loss. The clots produced by control PLTs at day 14 were still remarkably strong, whereas at day 7 PRT-treated PLTs produced weaker clots compared to the control group. Clinical trials investigating the efficacy of PRT-treated PLTs transfused at the end of the storage period (day 7), when the in vitro clot strength is weaker, are needed.

Mirasol pathogen reduction technology treatment of human whole blood does not induce acute lung injury in mice.

In resource-limited settings and in the military theater, fresh human whole blood is commonly transfused, but infectious risks are a concern. Sophisticated molecular testing for potential infectious agents in the whole blood is often unavailable. To address this unmet need, pathogen reduction technology (PRT) has been developed, and it is an effective approach to inactivate a broad range of pathogens found in human blood. However, studies are needed to determine if it is harmful to blood cells and whether these cells could damage the transfused recipient, including the development of acute lung injury/acute respiratory distress syndrome. In this study, we used a commercial PRT system to treat human whole blood that was then transfused into immunodeficient mice, and the development of acute lung injury was determined. In a model of transfusion-related acute lung injury (TRALI), BALB/c SCID mice developed more robust lung injury when challenged with a MHC Class I monoclonal antibody compared to BALB/c wild-type and NOD/SCID mice. Transfusion of control versus Mirasol PRT-treated whole blood (25% blood volume exchange) into BALB/c SCID mice did not produce lung injury at storage day 1. However, mild lung injury at storage days 14 and 21 was observed without significant differences in lung injury measurements between Mirasol PRT-treated and control groups. The mild storage-dependent acute lung injury correlated with trends for increased levels of cell-free hemoglobin that accumulated in both the control and Mirasol PRT-treated groups. Neutrophil extracellular traps were elevated in the plasma of BALB/c SCID mice in the monoclonal antibody TRALI model, but were not different in mice that received exchange transfusions. In conclusion, exchange transfusion of human whole blood into immunodeficient mice produces mild lung injury that is storage-dependent and not related to pathogen reduction treatment.

Introducing Pathogen Reduction Technology in Poland: A Cost-Utility Analysis

Background: Mirasol® pathogen reduction technology (PRT) uses UV light and riboflavin to chemically inactivate pathogens and white blood cells in blood components. In the EU, Mirasol PRT is CE-marked for both plasma and platelet treatment. In Poland, the decision to introduce PRT treatment of the national supply of fresh frozen plasma has spurred interest in evaluating the cost-effectiveness of this strategy. Methods: A decision-analytic model evaluated the incremental costs and benefits of introducing PRT to the existing blood safety protocols in Poland. Results: Addition of PRT treatment of plasma to current screening in Poland is estimated to cost 2.595 million PLN per quality-adjusted life year (QALY) (610,000 EUR/QALY); treating both plasma and platelet components in addition to current safety interventions had a lower cost of 1.480 million PLN/QALY (348,000 EUR/QALY). Conclusions: The results suggest that in Poland the cost per QALY of PRT is high albeit lower than found in previous economic analyses of PRT and nucleic acid testing in North America. Treating both platelets and plasma components is more cost-effective than treating plasma alone. Wide confidence intervals indicate high uncertainty; to improve the precision of the health economic evaluation of PRT, additional hemovigilance data are needed.

Pathogen reduction technologies

BackgroundIdeally, pathogen reduction technology (PRT) should provide proactive protection against emerging pathogens, reduce the need to introduce additional testing, minimize bacterial contamination and potentially replace processing steps such as gamma‐irradiation.Material and MethodsAvailable literature regarding technologies currently in use or under development for the treatment of labile blood components during processing was reviewed.ResultsThe INTERCEPT and Mirasol PRT systems are used widely in blood centres in Europe, Asia and the Middle East for the treatment of both platelets and plasma, whereas clinical trials of platelets treated using the THERAFLEX UV‐Platelets system are underway. In vitro data suggest that PRT treatment leads to some loss of platelet function. However, haemovigilance reports from countries where PRT‐treated platelets and plasma are transfused indicate no change in component usage, and indeed a reduction in transfusion‐related adverse events. Animal models are being used to elucidate mechanisms for these observed reductions. A second‐generation S‐303 system for red cells is also under development, as is a Mirasol PRT system for treatment of whole blood. Preliminary data indicate that these technologies may eventually become available.ConclusionThe challenge for blood services is to understand and rationalize the costs, risks and benefits of PRT compared with removing any tests or processes, whilst aiming to provide the highest level of blood safety. Ongoing post‐marketing surveillance and clinical trials are still required for all PRT systems.

Effects of riboflavin and ultraviolet light treatment on platelet thrombus formation on collagen via integrin αIIbβ3 activation

The adoption of pathogen reduction technology (PRT) is considered for the implementation of safer platelet (PLT) transfusion. However, the effects of PRT treatment on PLT thrombus formation under blood flow have not yet been fully clarified. Leukoreduced PLT concentrates (PCs) obtained by plateletpheresis were treated with riboflavin and ultraviolet light (Mirasol PRT). PC samples were passed through a column filled with collagen-coated beads at a fixed shear rate after 1, 3, and 5 days of storage. The thrombus formation ability was evaluated by measuring collagen column retention rate. The change in the activation state of integrin αIIbβ3 on PLTs during storage was examined by flow cytometry. The retention rate of the PRT-treated PLTs was significantly higher than that of the control PLTs on the day of treatment and decreased with storage but remained higher than those of the control during storage. This modification did not correlate with the total αIIbβ3 or fibrinogen binding on the PLTs but correlated significantly with PAC-1 binding. Mn(2+) -induced αIIbβ3 activation also fully restored the retention rate in the Day 5 PRT-treated PLTs along with the increase in PAC-1 binding. Riboflavin-based PRT treatment of PCs leads to the enhancement of thrombus formation on collagen, which is related to the activation status of αIIbβ3, which does not bind to fibrinogen but binds to PAC-1. The impact of this finding on the hemostatic or even thrombogenic potential in vivo must await clinical evaluation.

Pathogen reduction treatment alters the immunomodulatory capacity of buffy coat–derived platelet concentrates

Storage of platelet concentrates (PCs) after Mirasol pathogen reduction technology (PRT) treatment changes platelet (PLT) surface marker expression and secretion of immunomodulatory factors. Given that PLTs are known to participate in immune function, PRT may alter the way PLTs interact with the immune cells of a recipient upon transfusion. As such, the aim of this study was to assess the effects of PRT treatment on the functional ability of PLTs to interact with peripheral blood mononuclear cells (PBMNCs). Buffy coat-derived PCs were pooled and split to obtain matched pairs. One unit was treated using the Mirasol PRT system, while the control PC remained untreated. After 5 days of storage, either the PLTs or the PLT supernatants from the PCs were cocultured with PBMNCs, with or without lipopolysaccharide (LPS). The immunomodulatory factors secreted into culture medium after coculture were examined. PRT-treated PLTs and PLT supernatant significantly increased the interleukin (IL)-8 concentration, which was manifested only in the presence of LPS. Conversely, PRT-treated PLTs secreted less soluble P-selectin (sCD62P) upon coculture with PBMNCs. PRT-treatment induced differential secretion of IL-8 and sCD62P during coculture, which may be attributed to either bioactive substances present in PLT supernatant or as a result of cell-cell interactions.

Effect of Mirasol pathogen reduction technology system on in vitro quality of MCS+ apheresis platelets

Reducing the risk of pathogen transmission to transfusion recipients is one of the great concerns in transfusion medicine. Important among the measures suggested to minimise pathogen transmission is pathogen reduction technology (PRT) systems. The present study examined the effects of Mirasol PRT system on MCS+ apheresis platelets in vitro quality measures during a seven-day storage period at 22°C. Statistical analysis indicated no significant difference in platelet concentrations between the control and treated platelet concentrates (PCs) during the storage period. Glucose and lactate levels were measured to determine metabolic activities of control and treated platelets. In both control and treated platelets, the amount of glucose consumed and lactate produced increased significantly with storage time, but glucose consumption and lactate production rates were significantly higher in treated platelets compared with control platelets. The mean pH of treated PCs was decreased at all time points relative to control PCs but remained within acceptable limits. The expression of P-selectin was also higher in Mirasol PRT treated platelets throughout the storage period, but differences were not statistically significant on Days 1 and 4. Finally, visual inspection of swirling indicated that Mirasol PRT treatment of platelets is associated with platelet shape change. Overall, our results show that MCS+ apheresis platelets treated with Mirasol PRT can preserve adequate in vitro properties for at least 5days of storage.

Primary hemostatic capacity of whole blood: a comprehensive analysis of pathogen reduction and refrigeration effects over time

Whole blood (WB) has been used in combat since World War I as it is readily available and replaces every element of shed blood. Component therapy has become standard; however, recent military successes with WB resuscitation have revived the debate regarding wider WB use. Characterization of optimal WB storage is needed. We hypothesized that refrigeration preserves WB function and that a pathogen reduction technology (PRT) based on riboflavin and ultraviolet light has no deleterious effect over 21 days of storage. WB units were stored for 21 days either at 4°C or 22°C. Half of each temperature group underwent PRT, yielding four final treatment groups (n = 8 each): CON 4 (WB at 4°C); CON 22 (WB at 22°C); PRT 4 (PRT WB at 4°C); and PRT 22 (PRT WB at 22°C). Testing was at baseline, Days 1-7, 10, 14, and 21. Assays included coagulation factors; platelet activation, aggregation, and adhesion; and thromboelastography (TEG). Prothrombin time (PT) and partial thromboplastin time increased over time; refrigeration attenuated the effects on PT (p ≤ 0.009). Aggregation decreased over time (p ≤ 0.001); losses were attenuated by refrigeration (p ≤ 0.001). Refrigeration preserved TEG parameters (p ≤ 0.001) and PRT 4 samples remained within normal limits throughout the study. Refrigeration in combination with PRT inhibited fibrinolysis (p ≤ 0.001) and microparticle formation (p ≤ 0.031). Cold storage increased shear-induced platelet aggregation and ristocetin-induced platelet agglutination (p ≥ 0.032), as well as GPIb-expressing platelets (p ≤ 0.009). The in vitro hemostatic function of WB is largely unaffected by PRT treatment and better preserved by cold storage over 21 days. Refrigerated PRT WB may be suitable for trauma resuscitation. Clinical studies are warranted.

Pathogen reduction treatment of buffy coat platelet concentrates in additive solution induces proapoptotic signaling

Pathogen reduction technology (PRT) can potentially reduce the risk of transfusion-transmitted infections. However, PRT treatment of platelet (PLT) concentrates also results in reduced PLT quality and increased markers of apoptosis during storage. The aim of this study was to investigate changes to the expression and activation of proteins involved in apoptosis signaling. Samples from riboflavin and ultraviolet light PRT-treated and untreated (control) buffy coat-derived PCs in 70% SSP+ and 30% plasma were taken on Days 1, 5, and 7 of storage. Phosphatidylserine (PS) exposure, expression of Bcl-2 family proteins, cytochrome c release, and cleavage of caspase-3 and caspase-3 substrates were analyzed using flow cytometry and Western blotting. Compared to untreated controls, markers of apoptosis signaling were increased after PRT and subsequent storage. PS exposure on the PLT outer membrane was significantly higher after PRT on Days 5 and 7 of storage (p < 0.05). Expression of proapoptotic Bak and Bax was higher after PRT and subsequent storage. Cytochrome c release and caspase-3 cleavage were also greater and occurred earlier in the PRT-treated PLTs. The cleavage of caspase-3 substrates gelsolin and ROCK I were also increased after PRT, compared to untreated controls. This study demonstrated an increase in proapoptotic signaling during PLT storage, which was exacerbated by PRT. Many of these differences emerged outside the current 5-day storage period. These changes may not currently influence PLT transfusion quality, but will need to be carefully evaluated when considering extending PLT storage beyond 5 days.

Pathogen Reduction Treatment of Whole Blood Inhibits Clot Lysis and Thrombin Generation Is Preserved to 14 Days by Storage At 4°C

Abstract 2272 Introduction:Fresh whole blood (WB) collected from a “walking blood bank” is used by the U.S. military to supplement component therapy when blood component supplies are exhausted. Currently, WB is used for emergency transfusion within 24 hours of collection, before results of pathogen testing are available. A pathogen reduction technology (PRT), which uses riboflavin and ultraviolet light to damage nucleic acids in pathogens, is being considered as a transfusion-transmitted disease (TTD) risk mitigation measure. The effect of this technology on the hemostatic properties of whole blood, particularly on clotting capacity and clot lysis, are poorly understood, and optimal storage conditions are not defined. We previously reported that activated partial thrombin time (aPTT) and prothrombin time (PT) are prolonged by treatment; however, these tests do not always correlate with clinical findings. Thromboelastography is a more robust measure of clot formation and stability over time; we previously found that maximum amplitude (MA), which represents clot strength, did not decrease with PRT treatment and was preserved by storage at 4°C. Here we explore the effects of PRT on other parameters important to clotting capacity and clot lysis, and present the effects of WB storage at 4°C compared to 22°C. Hypothesis:WB treated with PRT demonstrates similar hemostatic function to non-treated WB, and storage at 4°C reduces degradation of blood components essential to clotting capacity and clot lysis compared to 22°C. Methods:Under an IRB-approved protocol, 8 units per treatment group of WB were collected in CPD anticoagulant from healthy donors of normal hemostatic status according to standard blood donor guidelines. Pathogen reduction was performed using riboflavin and ultraviolet light (265–400nm phosphor; Mirasol® System, CaridianBCT) dosed at 80 J/mLRBC. Treatment groups included: control WB stored at 4° C (CON-04); control WB stored at 22° C (CON-22); PRT-treated WB stored at 4° C (PRT-04); and PRT-treated WB stored at 22° C (PRT-22). The hemostatic function of the blood was assessed at baseline, days 1–7, 10, 14, and 21. Factor VIII and fibrinogen were measured from assayed samples (BCS® XP system, Siemens). Thromboelastography (TEG®, Haemoscope Corp.) estimated total thrombin generation by calculating the first derivative of the TEG tracing, the Total Thrombus Generation variable (TTG). TEG was also used to measured lysis (LY30). Data were analyzed as repeated measures, followed by analysis of variance to assess interactions. Significance was set at p<0.05. Results:Treatment with PRT caused an initial drop in fibrinogen (baseline: 244 ± 77.5 mg/dL versus post-treatment: 185 ± 63.2 mg/dL, p≤0.04) and factor VIII (baseline: 96 ± 39% versus day post-treatment: 46 ± 23%, p≤0.001); however, levels stabilized thereafter (p≥0.987 and p≥0.871, respectively; see Fig. 1–2). Baseline fibrinogen levels were similar between groups p≥0.386). PRT-04 was the only group in which both fibrinogen and Factor VIII levels fell below clinical reference ranges (fibrinogen: p’0.039; factor VIII: p≤0.016). TTG was unaffected by PRT and was preserved through day 14 by storage at 4° C (p≥0.979, see Fig. 3), but only through day 10 when stored at 22°C (p≤0.290 at day 10). PRT treatment inhibited clot lysis (LY30) compared to storage at 22°C (p≤0.001), and variability was the lowest in the PRT-04 group (p≤0.001, see Fig. 4). [Display omitted] [Display omitted] Conclusions:Our data demonstrate that pathogen reduction inhibited clot lysis. Decreased clot formation could conceivably account for the smaller degree of lysis; however, we previously found that MA is unaffected, and now demonstrated that thrombin generation was preserved despite a treatment-related decrease in factor VIII levels. While fibrinogen levels were diminished in the PRT-04 group, they were preserved in the PRT-22 group, which also demonstrated the diminished lysis. Cold storage preserved WB clotting capacity compared to storage at room temperature. The clinical significance of these findings has yet to be established; a coagulopathic animal hemorrhage model could determine whether the effects of PRT-induced lysis inhibition and cold storage are beneficial. Disclosures:Mora:CaridianBCT: Research Funding. Pidcoke:CaridianBCT: Research Funding. Valdez-Delgado:CaridianBCT: Research Funding. Fedyk:CaridianBCT: Research Funding. Reddy:CaridianBCT: Employment, Research Funding. Goodrich:CaridianBCT: Employment, Research Funding. Cap:CaridianBCT: Research Funding.

Cell quality of apheresis‐derived platelets treated with riboflavin‐ultraviolet light after resuspension in platelet additive solution

Previously, we evaluated the Mirasol pathogen reduction technology (PRT) system on platelet (PLT) function before resuspension. We now evaluated this system in the presence of PLT additive solution (PAS). Double-dose PLTs (n = 15) were generated using a commercially available apheresis system (Trima, Version 5.2, CaridianBCT) allowing for the resuspension in SSP+ (MacoPharma) immediately after collection. Paired units (n = 30) were PRT treated (M) or remained untreated (C) and analyzed for metabolism (pH, pO(2) , glucose, lactate, adenosine triphosphate [ATP]), swirl, hypotonic shock response (HSR), turbidometric aggregation, CD62P expression, annexin A5 and lactate dehydrogenase (LDH) release, mitochondrial enzymatic reduction activity (MTS), transmembrane mitochondrial potential (Δψ), and surface coverage (SC) during shear-induced adhesion throughout 8 days of storage. As seen previously, PRT treatment of PLT units, containing a mean of 3.9 × 10(11) ± 0.3 × 10(11) PLTs in 397 ± 10 mL with a 32% to 34% plasma carryover, was associated with significantly (p < 0.001) increased cell activation, acidity, and glycolytic flux. PRT treatment appeared to up regulate both oxidative pathway and adhesional properties as evidenced by significantly higher MTS reduction, oxygen consumption, and shear-induced SC on Day 1 (p ≤ 0.016). While no significant differences were found for LDH release and ATP content (except for Day 8), M units were significantly inferior (p ≤ 0.021) for aggregation (TRAP-6); for Δψ and annexin A5 release (by Day 5); and for swirl, HSR, and MTS reduction (by Day 7). PRT treatment in the presence of PAS was comparable to PRT treatment before resuspension preserving ATP content and mitochondrial function.

Generation of neutrophil priming activity by cell‐containing blood components treated with pathogen reduction technology and stored in platelet additive solutions

Storage of cell-containing blood components such as platelet concentrates (PCs) and red blood cells (RBCs) results in generation of biologically active compounds, many of which may be associated with adverse transfusion events. Priming of the neutrophil oxidase activity is a common characteristic of many of the biologically active compounds found in stored blood. We evaluated the priming activity of pathogen reduction technology (PRT)-treated PCs stored in plasma or platelet additive solution (PAS) and PRT-treated RBCs. PCs were collected with Trima or Amicus equipment and were PRT treated with the Mirasol PRT system or the Intercept Blood System. Some units were gamma irradiated. Products were stored in 100% plasma or 35% plasma plus PAS. RBCs were washed and PRT treated before storage. Samples were removed throughout storage and priming of the oxidase activity was measured. Platelets collected on Trima or Amicus equipment and stored in plasma or PAS demonstrated increasing priming activity during 5 to 7 days of storage. Gamma irradiation, but not PRT treatment with either technology, further enhanced this priming activity. Supernatants of RBCs stored for 42 days induced priming in untreated controls, but not in washed or Mirasol PRT-treated test products. Production of oxidase priming activity increased during storage in all blood products. No significant differences were associated with the collection method, storage in PAS, or PRT treatment. The generation of biologically active compounds, which may serve as an etiology for adverse events, appears to be independent of these processes for collection, storage, and pathogen reduction.

Inactivation of human white blood cells in platelet products after pathogen reduction technology treatment in comparison to gamma irradiation

During the transfusion of blood products, the transfer of allogeneic donor white blood cells (WBCs) can mediate adverse reactions in recipients. To minimize reactions, blood components are leukoreduced and/or exposed to gamma irradiation. Nucleic acid-targeted pathogen reduction technologies (PRTs) processes are well suited for WBC inactivation. The Mirasol PRT system (CaridianBCT Biotechnologies) uses riboflavin and ultraviolet light to reduce the active pathogen load and inactivate residual WBCs in blood products used for transfusion. The aim of this study was to compare the effect of PRT treatment to gamma irradiation. WBCs were resuspended in 100% plasma or in plasma containing 65% platelet additive solution (SSP+). A single product was split into three aliquots: control, PRT treatment, or gamma irradiation. After treatment, peripheral blood mononuclear cells were isolated from products, and cell viability and functionality were assessed in limiting dilution assays (LDAs), by CD69 expression, by proliferation, and by cytokine accumulation after lipopolysaccharide addition. PRT treatment and gamma irradiation reduced the ability of the T cells to proliferate by similar degrees as evidenced by a 6 log or greater reduction in the LDA and a lack of response to several stimuli. However, gamma-irradiated T cells were still capable of up regulating surface CD69, inducing a proliferative response in allogeneic responder cells, and producing levels of proinflammatory cytokines similar to those of untreated control cells, responses that PRT-treated T cells were incapable of mediating. These in vitro results demonstrate that PRT treatment is more effective than gamma irradiation at abrogating selected WBC immune functions.

Expression of P-Selectin and Platelet-Neutrophil (PMN) Interactions In Untreated and Mirasol® PRT Treated Platelet Concentrates During Routine Storage.

AbstractAbstract 1123 Introduction:Platelet concentrates develop biologically active compounds during storage which may play a role in adverse events of transfusion. Although many studies have focused on release of soluble pro-inflammatory compounds, changes in cells such as platelets may contribute to the pro-inflammatory effects of transfusion products. We evaluated untreated and Mirasol Pathogen Reduction Technology (PRT) treated platelet concentrates for expression of P-selectin and interactions between platelets and PMNs. Methods:Four, double apheresis platelet products were collected from healthy volunteers with TRIMA system under an IRB approved protocol. One product of each pair was PRT treated with Mirasol PRT and both were stored under standard conditions. At various times, aliquots were sterilely removed from products. Expression of P-selectin on platelets was defined with a specific antibody. Neutrophils, isolated from ABO type specific peripheral blood of volunteers, were incubated with platelet concentrates for 3 min at 37° C, and labeled CD41 was added. PMNs were identified with flow cytometry by forward/side scatter and platelets by fluorescence and the % of PMNs associated with platelets determined. Inhibition of platelet/neutrophil interaction with antibodies to adhesion molecules was also examined. In parallel experiments, activation of neutrophils after incubation with platelets was determined by oxidation of dihydrorhodamine (DHR). Results:During storage, platelet expression of P-selectin increased in both untreated and Mirasol treated platelet concentrates (p<0.05) with greater expression on treated products (p<0.05). On day 0 of storage, PMNs associated with platelets was 9.7±1.1% (mean±SEM, n=4, untreated) and 12.1±1.3% (treated products), and on day 5 results were 16.8±4.3% and 27.1±4.3% respectively (significant, p<0.05). Platelet-PMN interactions were inhibited by ≥90% by antibodies to P-selectin or its PMN adhesion partner PSGL-1, but not Mac1, LFA-1, GPIb or ICAM-1. Platelet-PMN interactions were directly related to the stored platelets, not the effects of stored plasma. Activation of the PMN respiratory burst by oxidation of DHR was not shown in any product type over storage time. Conclusion:During storage, platelets exhibit increased P-selectin expression and the capability to adhere to PMNs. These effects were greater after Mirasol PRT treatment. The cell association depended on the interaction between P-selectin (platelets) and PSGL-1 (PMNs). These changes occurring during storage of platelet concentrates may explain platelet recovery patterns in patients requiring repeated transfusions with untreated and PRT treated products and could play a potential role in adverse events for either product type. Disclosures:Goodrich:CaridianBCT Biotechnologies: Employment, Patents & Royalties, Research Funding. Marschner:CaridianBCT Biotechnologies: Employment, Research Funding.

Annexin V Release and Transmembrane Mitochondrial Potential during Storage of Apheresis-Derived Platelets Treated for Pathogen Reduction

Background: In vitro function of stored platelet (PLT) con-centrates was analyzed after applying two different techniques of pathogen reduction technology (PRT) treatment, which could increase cellular injury during processing and storage. Methods: Nine triple-dose PLT apheresis donations were split into 27 single units designated to riboflavin-UVB (M) or psoralen-UVA (I) treatment or remained untreated (C). Throughout 8 days of storage, samples were analyzed for annexin V release, the mitochondrial transmembrane potential (ΔΨ) and some classical markers of PLT quality (pH, LDH release, hypotonic shock response (HSR)). Results: PLT count and LDH release of all units maintained initial ranges. All units exhibited a decrease in pH and HSR and an increase in annexin V release and ΔΨ disruption. Notably, throughout the entire storage period, annexin V release re-mained lowest in M units. Throughout 7 days of storage, M units remained comparable to C units (p > 0.05), whereas inferior values were observed with I units. Here, differences to C units reached significance by day 1 (pH: p < 0.0001), day 5 (annexin V release: p < 0.014), and day 7 (HSR, ΔΨ: p ≤ 0.003). After PRT treatment, annexin V release and ΔΨ disruption were significantly (p < 0.001) correlated with pH and HSR. Conclusion: During storage, all units showed a de-crease in HSR and an increase in acidity, annexin V release and ΔΨ disruption. While M units remained comparable to C units, I units demonstrated significantly inferior values dur-ing terminal storage. This could have resulted from differences in PRT treatment or simply be due to differences in storage media and should be analyzed for clinical relevance in future investigations.

Mirasol Pathogen Reduction Technology®treatment does not affect acute lung injury in a two-event in vivo model caused by stored blood components

Mirasol Pathogen Reduction Technology (PRT) treatment uses riboflavin and UV light to inactivate pathogens in blood components. Neutrophil [polymorphonuclear cells (PMN)] priming activity accumulates during routine storage of cellular blood components, and this activity has been implicated in transfusion-related acute lung injury (TRALI). We hypothesize that PRT-treatment of blood components affects the priming activity generated during storage of packed RBCs (PRBCs) or platelet concentrates (PCs), which can elicit ALI in vivo. Plasma, PRBCs and PCs were isolated from healthy donor's whole blood or by apheresis. Half of a collected unit was treated with PRT treatment and the remainder was left as an unmodified control. Supernatant was collected during storage of PCs and PRBCs and assayed for PMN priming activity and used as the second event in a two-event in vivo model of TRALI. PRT treatment did not induce priming activity in plasma or affect the priming activity generated during storage of PCs or PRBCs as compared with the unmodified controls. The supernatants from stored, but not fresh, PCs and PRBCs did cause ALI as the second event in a two-event animal model of TRALI, which was unaffected by PRT treatment. We conclude that the PRT treatment does not induce priming activity in plasma nor does it affect the priming activity generated during storage of PCs or PRBCs or their ability to cause ALI as the second event in a two-event in vivo model of TRALI. Moreover, the amount of priming activity in TRIMA-isolated PCs was significantly less than SPECTRA-isolated PCs.

Inactivation of Human Leukocytes in Platelet Products After Pathogen Reduction Technology Treatment in the Presence of Platelet Additive Solution.

Abstract 2116Poster Board II-93During the transfusion of blood or blood products, a recipient can receive a large number of allogeneic leukocytes. This can lead to leukocyte-mediated adverse reactions in the recipient and include donor anti-recipient responses such as the life-threatening transfusion-associated graft versus host disease (TA-GVHD) and cytokine production; or recipient anti-donor responses that are induced by direct presentation of foreign antigen by donor leukocytes or indirectly after processing of the donor cells by recipient antigen-presenting cells. To avoid or minimize leukocyte mediated reactions, the leukocytes present in blood products are inactivated or depleted prior to administration. Nucleic acid targeted pathogen reduction processes (PRT) are well suited for leukocyte inactivation. The Mirasol® PRT System uses riboflavin (Vitamin B2) and ultraviolet (UV) light to reduce the active pathogen load and inactivate residual leukocytes in blood products used for transfusion. To make the PRT System more widely applicable, the effect of treating leukocytes in the presence of platelet additive solution (PAS) was tested. Human peripheral blood mononuclear cells (PBMNC) were purified by Ficoll-Hypaque discontinuous centrifugation and placed in 350 ml of storage solution consisting of 65% PAS (SSP+) and 35% plasma. An untreated control sample was removed before addition of 35 ml of riboflavin (500 μM) and exposure to UV light (9.1 J/ml). PBMNC were recovered after treatment and tested for their ability to proliferate in response to polyclonal stimulators such as phytohemagglutinin, and anti-CD3/CD28 or to allogeneic stimulator cells in a mixed lymphocyte culture (MLC). Treatment was found to inhibit proliferation as well as T cell activation as measured by the upregulation of CD69 expression when incubated with phorbol 12-myristate 13-acetate. Treated PBMNC were unable to produce inflammatory or TH1/TH2 cytokines when stimulated with lipopolysaccharide for 24 hours or anti-CD3/CD28 for 72 hours. Levels of cytokines that are released in the absence of activation, such as IL-6, IL-8 and IL1β, were reduced below levels of detection of the assay after PRT-treatment. Quantitation of the degree of inactivation using limiting dilution assays showed that 5.2 log inactivation could be achieved at the specified energy doses. These treatment conditions resulted in acceptable platelet cell quality over 8 days in storage. In summary, PRT treatment was able to functionally inactivate leukocytes in the presence of PAS to the levels seen with gamma-irradiation without adversely affecting the quality of the platelets. Disclosures:Fast:CaridianBCT Biotechnologies: Research Funding. Marschner:CaridianBCT Biotechnologies: Employment. Goodrich:CaridianBCT Biotechnologies: Employment.