ALBERT

Description: Key Points Pathogen-inactivated platelets were noninferior in preventing bleeding only in intention-to-treat analysis. In contrast to animal models, alloimmunization could not be prevented when using pathogen-inactivated platelets.

Description: Abstract 1179 Background Previous studies by our laboratory have demonstrated changes in apheresis collected, PLT concentrates during storage that are enhanced by Mirasol®PRT treatment. These include increased expression of P-selectin on PLTs and increased formation of neutrophil (PMN)/PLT aggregates. To explore the effect of these changes in a microvascular environment, we measured PLT adherence in a microfluidic chamber coated with collagen. Methods Three, double apheresis platelet products were collected from healthy volunteers with TRIMA system under an IRB approved protocol. One product of each pair was treated with Mirasol PRT and both were stored under standard conditions. Aliquots were sterilely removed from products during storage. To 1000 μl of fresh, ABO type specific, citrate anticoagulated whole blood was added 60 μl of apheresis platelet concentrate with or without PRT treatment on day 1 or 7 of storage and incubated for 5 min at 37C. The sample was recalcified with 20 mM CaCl2 and then pulled through the inlet of a microfluidic chamber at a flow sheer rate of 100 s−1. The flow chamber was composed of two parts, a glass slide patterned with collagen and a series of four polydimethylsiloxane microfluidic chambers per inlet. Stored PLTs were labeled with fluorescently labeled anti-CD41 to distinguish from PLTs of the whole blood. The chamber was mounted on an inverted fluorescence microscope, and video microscopic images sampled on observation field every 7 sec over 7 min. The number of fluorescent cells (stored PLTs) per field and the total area covered by all PLTs were determined at 7 min. Results The area, expressed as % of total field, covered by all of the PLTs in the sample was not different for whole blood alone (29 ± 3); Day 1 untreated (23 ± 9); Day 1 PRT treated (26 ± 8); Day 7 untreated (21 ± 5); and Day 7 treated (25 ± 9) PLTs. In contrast, there was an increase in the number of treated apheresis PLTs at Day 1 compared to untreated apheresis platelets binding to the chamber (2.26 ± 0.61 untreated, vs. 5.45 ± 1.45 treated, numbers are mean ± SEM for numbers of cells adhered normalized to the PLT count in the product, significant, p=0.0229). There was also an increase in binding of treated compared to untreated PLTs at Day 7 (2.43 ± 0.47 untreated, vs. 4.42 ± 1.35 treated). However, the results for untreated PLTs on Day 1 and 7 and treated PLTs on study days were not different. The increased binding of Mirasol PRT treated PLTs parallels the small decrease in recovery seen in clinical studies of patients receiving treated PLT concentrates compared to untreated products. Conclusion PRT treatment increases adherence of PLTs early in storage but does not appear to progress further. The changes may have implications for clinical responses to platelet transfusions. Evaluation of adherence in the microfluidic chamber provide a model which simulate the microvascular environment. Disclosures: Ambruso: Terumo BCT: Research Funding. Seewald:Terumo BCT: Research Funding. Marschner:Terumo BCT: Employment. Goodrich:Terumo BCT: Employment.

Description: Abstract 718 Introduction: The presence of donor white blood cells (WBC) in transfused blood products can induce alloimmunization, and reducing or eliminating this response may prove to be of clinical benefit. The use of a pathogen reduction method based on UV light illumination in the presence of riboflavin has been shown to induce changes in WBCs that result in a failure to bind to, or induce proliferation of allogeneic PBMCs in vitro. In addition, a study in rats has shown a reduction in alloimmunization in vivo using this treatment. Transfusion of cells illuminated with UV light at other doses without riboflavin has been shown to induce some degree of tolerance with a reduced antibody response to subsequent allogeneic transfusions. We sought to assess both the degree of alloimmunization in mice given pathogen reduced versus untreated allogeneic platelets, as well as determine if cells from mice given pathogen reduced platelets exhibited signs of tolerance ex vivo. Methods: Peripheral blood was collected from C57Bl/6 and Balb/cJ mice into CPDA-1, and platelet rich plasma (PRP) was prepared by gentle centrifugation. WBCs were isolated from the remainder of the blood and were added back to a portion of the PRP to generate either WBC-enriched or WBC-poor PRP. These products were either left untreated or pathogen reduced using the Mirasol pathogen reduction technology system, which uses a combination of riboflavin and UV illumination. These products were transfused via tail vein injection into Balb/cJ mice. Two weeks after transfusion the treated mice were sacrificed, and peripheral blood and spleens were collected. Serum levels of circulating alloantibodies were measured by flow cytometry. Splenocytes were cultured for 48 hours in the presence or absence of C57Bl/6 splenocytes, and levels of secreted cytokines were measured in culture supernatants using multiplexing techniques. Groups were compared using one-way ANOVA with Tukey's multiple comparison post-test, α=0.05. Results: Mice given allogeneic PRP transfusions had significantly elevated levels of alloantibodies compared with non-transfused control mice, whereas mice given syngeneic PRP or pathogen reduced PRP did not. Mice given either the WBC-enriched PRP or WBC-poor PRP generated alloantibodies, though higher levels of antibodies were observed with WBC-enriched PRP. Levels of IFN-γ, TNF-α, IL-10 and GM-CSF were significantly higher following secondary allogeneic challenge of cells from mice given untreated allogeneic PRP compared with those given no transfusion or syngeneic PRP, but not with those given pathogen reduced PRP. Levels of IL-1β, IL-4, IL-5, IL-6, IL-12(p70), and IL-13 were significantly reduced following secondary allogeneic challenge of cells from mice given pathogen reduced allogeneic PRP compared with those given no transfusion or syngeneic PRP. Conclusions: Treatment of allogeneic PRP with riboflavin and UV light prior to transfusion blocks alloimmunization in mice. Furthermore, secondary cytokine responses to allogeneic cells ex vivo are reduced, in some cases bellow the levels observed in cells from mice without prior exposure, suggesting induction of tolerance. Disclosures: Marschner: CaridianBCT Biotechnologies: Employment. Goodrich:CaridianBCT Biotechnologies: Employment. Norris:CaridianBCT Biotechnologies: Consultancy, Research Funding.

Description: Abstract 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

Description: Abstract 2288 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 response or pro-inflammatory effects of transfusion products. In previous studies we have shown that pathogen reduction with Mirasol PRT of apheresis PLT concentrates induced changes in PLTs which increased during storage including expression of P-selectin and the capacity to form PLT-PMN aggregates. These changes were also associated with increased adherence of stored PLTs in a microfluidic chamber. In the current study we evaluated P-selectin and PLT-PMN aggregate formation in whole blood after treatment with Mirasol PRT. Methods Ten units of whole blood from healthy donors were drawn into CPD under an IRB approved protocol. Five units where treated with Mirasol PRT and five units remained untreated. Aliquots were taken before and after treatment (or at same times for untreated units) on Day 0 and 24–27 hours after draw on Day 1 after storage at 22–24°C. Platelet rich plasma (PRP) was produced by standard technique and PLTs isolated on a Sepharose 2B column. PLTs were incubated with APC-CD62P and FITC-CD41a for comparison with P-selectin expressed as percent positive increase over isotype control. PMNs were isolated from 50 ml of heparinized peripheral blood drawn from healthy, ABO-matched donors. After a 5 min pre-incubation at 37°C in a shaking water bath, untreated, treated PLTs, and PMNs were incubated separately or together for 3 min then quenched at 4°C. FITC-CD41a and FITC-IgG1 isotype for platelet-PMN aggregate quantitation or PE-CD11b for CD11b expression on neutrophils were added. Platelet-PMN aggregates was expressed as percent of all PMN events above CD41a isotype control. CD11b expression was determined for PMNs after exposure to PLTs by flow cytometry. Results P-selectin positive cells did not change significantly over time in samples from untreated products (Table 1). After Mirasol PRT treatment, cells positive for P-selectin increased on Day 0 and 1 (p

Description: Background: Across sub-Saharan Africa, blood supplies are threatened by numerous pathogens. In some locations, Plasmodium parasitemia prevalence in donor blood is nearly 50%. Donor testing for malaria in these areas is not effective and the risk of transfusion-transmitted malaria (TTM) is high. The Mirasol® PRT System for Whole Blood (WB) is a medical device intended for extracorporeal pathogen reduction of WB. The current clinical study evaluated the ability of Mirasol-treated WB to reduce the incidence of TTM. Study Design/Methods: This was a prospective, randomized, double-blind, controlled, single-center study in Ghana, which is hyperendemic for malaria. The study had 90% power to demonstrate a 90% reduction in TTM. Hospitalized patients requiring WB transfusions were randomly allocated to receive ≤ 2 transfusions of standard (untreated) or Mirasol-treated WB. The primary endpoint was the incidence of TTM as measured by quantitative polymerase chain reaction and Plasmodium alleleic sequence homology between transfused and patient WB during 28 days of follow-up. Patient safety was assessed by monitoring treatment-emergent adverse events (TEAEs) and transfusion reactions.Clinical outcomes related to hemoglobin increments, hemostatic parameters, and clinical chemistries were monitored for 28 days post-transfusion. Results: Overall, 226 subjects (113 Mirasol, 113 Untreated) were enrolled; 223 subjects were included in the safety analysis. Sixty-five (65) subjects were non-parasitemic at pre-transfusion (28 Mirasol, 37 Untreated) and received at least 1 parasitemic WB transfusion. Of 16 cases of suspected TTM (3 Mirasol, 13 Untreated) with 2 consecutive days of parasitemia, 9 were confirmed by alleleic homology (1 Mirasol, 8 Untreated). Incidence of TTM was significantly reduced in patients receiving treated products. Hemoglobin (mean [standard deviation]) was similar between groups at baseline (6.71 g/dL; p = 1.0), and Day 1 following 1 transfusion (8.53 [2.0] vs 8.49 [1.5] g/dL; p = 0.93) or 2 transfusions (7.09 [1.5] vs 7.38 [1.6] g/dL; p = 0.33). Ninety-two subjects (48 Mirasol, 44 Untreated) reported 145 TEAEs (75 Mirasol, 70 Untreated). Transfusion reactions were observed in 8.1% and 13.4% of subjects receiving Mirasol-treated and untreated WB, respectively. Table. Incidence of TTM Mirasol n (%); 95% CI Untreated n (%); 95% CI P-Value 2 Consecutive days of ParasitemiaN = 65 3 (10.7); 2.3, 28.2 n = 28 13 (35.1); 20.2, 52.2n = 37 〈 0.05 2 Consecutive days of Parasitemia and 〉2 Allele match by PCRN = 65 1 (3.6); 0.1, 18.3n = 28 8 (21.6); 9.8, 38.2n = 37 〈 0.05 ITT PopulationN = 223 1 (0.9); 0.0, 4.9n = 111 8 (7.1); 3.1, 13.6n = 112 〈 0.05 Abbreviation: CI = confidence interval, ITT = intent-to-treat, PCR = polymerase chain reaction. Conclusions: The primary endpoint of the study was met. Mirasol treatment of WB clinically and statistically reduced TTM infections in the study population. This was the first human clinical study demonstrating that a PRT system can reduce transmission of a bloodborne pathogen. No safety issues were related to the device or device-treated WB. Transfusion reactions did not differ between patients receiving Mirasol-treated or untreated WB. Hemoglobin increments and transfusion outcome parameters in transfused patients did not differ between the treatment groups. Disclosures Allain: Terumo BCT: Consultancy. Owusu-Ofori:Terumo BCT: Other: Clinical Study Sub-Investigator. Marschner:Terumo BCT: Employment. Goodrich:Terumo BCT: Employment. Owusu-Ofori:Terumo BCT: Other: Clinical Study Investigator.

Description: The transfusion of blood products can result in transmission of pathogens and immunological consequences such as transfusion associated graft-versus-host disease and alloimmunization. Previous studies have shown that exposure of platelet concentrates to riboflavin and light (Mirasol PRT treatment) causes irreparable modification of nucleic acids that results in inactivation of a wide range of pathogens as well as inhibition of the immunological responses mediated by the WBC present in platelet concentrates. Initial studies investigated the effects of Mirasol PRT treatment of RBC concentrates on WBC function. Human peripheral blood mononuclear cells (PBMNC) were purified by Ficoll-Hypaque discontinuous centrifugation from control or test non-leukoreduced RBC units exposed to riboflavin and varying doses of light. These PBMNC were tested for their ability to be activated in response to PMA, to proliferate in response to PHA or allogeneic stimulator cells, and to stimulate proliferative responses of allogeneic responder cells. While lower light doses were sufficient to inhibit activation and proliferation, higher energies (30J/ml RBC) were required for inhibition of antigen presentation. These Mirasol treatment conditions did not induce crossmatch incompatibility, methemoglobin (MetHb) levels stayed within an acceptable range (2.6% + 1.5) and hemolysis was below 1% during storage. Interestingly, MetHb levels decreased to background levels during storage at 4°C, suggesting that the activity of the enzyme NADH methemoglobin reductase was not compromised by the exposure to riboflavin and light. These treatment conditions also reduced levels of Gram positive and negative bacteria to the limits of detection and reduced infectivity of both enveloped and non-eveloped viruses by 〉 4 logs on average in packed, washed RBC units. In summary, Mirasol PRT is able to functionally inactivate WBCs and pathogens in washed RBC products without adversely affecting the quality of the RBCs. This novel pathogen reduction therapy for RBC products complements the Mirasol PRT treatment for platelets and offers inactivation of pathogens in cellular blood components using a single technology.