Abstract
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.
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