Riboflavin and ultraviolet light for pathogen reduction of murine cytomegalovirus in blood products

Abstract Abstract 2116 Poster Board II-93 During 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.

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Filtration Leukoreduction Followed by Pathogen-Reduction (Mirasol Treatment) Prevents Alloimmune Platelet Refractoriness in a Dog Platelet Transfusion Model

Blood ◽

10.1182/blood.v120.21.271.271 ◽

2012 ◽

Vol 120 (21) ◽

pp. 271-271 ◽

Cited By ~ 2

Author(s):

Sherrill J. Slichter ◽

Esther Pellham ◽

S. Lawrence Bailey ◽

Todd Christoffel

Keyword(s):

Research Funding ◽

Platelet Transfusion ◽

High Rate ◽

Combined Approach ◽

Blood Products ◽

Success Rates ◽

Dog Model ◽

Pathogen Reduction ◽

Antigen Presenting ◽

Rate Of Success

Abstract Abstract 271 Background: The largest transfusion (tx) trial to evaluate methods of preventing platelet (plt) alloimmunization (TRAP Trial; NEJM 1997;337:1861) demonstrated residual alloimmunization rates of 17% to 21% in AML patients (pts) undergoing induction chemotherapy despite receiving either filter-leukoreduced (F-LR) or UV-B irradiated (UV-BI) blood products, respectively. Our pre-clinical dog plt tx studies, the basis for testing UV-BI in the TRAP Trial, demonstrated this model was able to predict pt results; i.e., prevention of alloimmunization was 45% in the dog but 79% in pts. The greater effectiveness in pts was probably because they had chemotherapy-induced immunosuppression compared to the immunocompetent dogs. Our current dog plt tx studies have focused on evaluating F-LR to remove antigen-presenting WBCs (APCs) or pathogen-reduction (PRT) (Mirasol treatment) to inactivate APCs. Methods: For pts, plts are obtained using either apheresis procedures or as plt concentrates prepared from whole blood (WB). To re-duplicate these types of plts in our dog model, we prepared plt-rich-plasma (PRP) from WB which would be equivalent to non-leukoreduced apheresis plts. The PRP was then either unmodified, F-LR, PRT, or the treatments were combined. Because the success rates were very poor with the single treatments of PRP (see table), the WB studies evaluated only combined F-LR and PRT treatments. In clinical practice, the treated WB would then be used to prepare a plt concentrate. The WB studies assessed either PRT of the WB followed by F-LR of PRP made from the WB or, conversely, F-LR of the WB using a plt-sparing filter (Terumo Immuflex WB-SP) followed by PRT of the WB and then preparation of PRP. After completion of all treatments, PRP from each study was centrifuged to prepare a plt concentrate, the plts were radiolabeled with 51Cr, injected into a recipient, and samples were drawn from the recipient to determine recovery and survival of the donor's (dnr's) plts. Dnr and recipient pairs were selected to be DLA-DRB incompatible and crossmatch-negative. Eight weekly dnr plt txs were given to the same recipient or until the recipient became refractory to the dnr's plts defined as ≤5% of the dnr's plts still circulating in the recipient at 24-hours post-tx following 2 sequential txs. Results: The table shows the percent of recipients who accepted 8 weeks of dnr plts and the total number of dnr plts and WBC injected. Using either filter, there was equal reduction in WBCs to 105/tx. Acceptance of unmodified dnr plts was 1/7 recipients (14%), PRT 1/8 recipients (13%), PL1-B filter 1/5 recipients (20%), and PLS-5A filter 4/6 recipients (66%). None of these differences were statistically significant. In contrast, combining F-LR of the PRP followed by PRT of the PRP was effective in 21/22 recipients (95%), regardless of the filter used. WB studies showed dnr plts were accepted by 2/5 recipients (40%) when WB was first treated with PRT followed by F-LR of the PRP made from the WB. Conversely, if the WB was first F-LR followed by PRT of the WB, 5/6 (83%) accepted dnr plts; more of these studies are in progress. Data are given as average ±1 S.D. Conclusions: F-LR of PRP or WB followed by PRT of the same PRP or WB is highly-effective in preventing alloimmune plt refractoriness in our dog plt tx model. These data suggest that most of the APCs must be removed by filtration before PRT can eliminate the activity of any residual APCs. Based on the high rate of success of this combined approach in our immunocompetent dog model, similar results should be achieved in pts even those who are not immunocompetent as were the AML pts receiving chemotherapy in the TRAP Trial. Disclosures: Slichter: Terumo BCT: Research Funding.

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Riboflavin-ultraviolet light pathogen reduction treatment does not impact the immunogenicity of murine red blood cells

Transfusion ◽

10.1111/trf.13432 ◽

2015 ◽

Vol 56 (4) ◽

pp. 863-872 ◽

Cited By ~ 8

Author(s):

Christopher A. Tormey ◽

Manjula Santhanakrishnan ◽

Nicole H. Smith ◽

Jingchun Liu ◽

Susanne Marschner ◽

...

Keyword(s):

Red Blood Cells ◽

Ultraviolet Light ◽

Blood Cells ◽

Reduction Treatment ◽

Pathogen Reduction

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Irradiation eradication and pathogen reduction. Ceasing cesium irradiation of blood products

Bone Marrow Transplantation ◽

10.1038/bmt.2009.124 ◽

2009 ◽

Vol 44 (4) ◽

pp. 205-211 ◽

Cited By ~ 25

Author(s):

P D Mintz ◽

G Wehrli

Keyword(s):

Blood Products ◽

Pathogen Reduction

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Separation, Identification and Quantification of Riboflavin and its Photoproducts in Blood Products using HPLC with Fluorescence Detection: A Method to Support Pathogen Reduction Technology

Photochemistry and Photobiology ◽

10.1562/2004-04-14-tsn-139 ◽

2004 ◽

Author(s):

Christopher C. Hardwick ◽

Troy R. Herivel ◽

Shiloh C. Hernandez ◽

Patrick H. Ruane ◽

Raymond P. Goodrich

Keyword(s):

Fluorescence Detection ◽

Blood Products ◽

Pathogen Reduction ◽

Identification And Quantification

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Lymphocyte Survival and Activation in Stored Platelet Concentrates After Gamma-Irradiation or Pathogen Reduction Technology Treatment.

Blood ◽

10.1182/blood.v114.22.2113.2113 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2113-2113

Author(s):

Jolanta J Wozniak ◽

Agnieszka Krzywdzinska ◽

Elzbieta Lachert ◽

Karolina Janik ◽

Jolanta Antoniewicz-Papis ◽

...

Keyword(s):

Gamma Irradiation ◽

Conflicts Of Interest ◽

Lymphocyte Activation ◽

Equal Weight ◽

Ratio Test ◽

Blood Products ◽

Platelet Concentrates ◽

Becton Dickinson ◽

Lymphocyte Survival ◽

Pathogen Reduction

Abstract Abstract 2113 Poster Board II-90 The presence of white blood cells, particularly lymphocytes, in blood products has been shown to contribute to the development of a variety of adverse events including both donor anti-recipient and recipient anti-donor responses. Therefore leukoreduction of blood products or inactivation of leukocytes is necessary. We are currently using gamma-irradiation (Radiator Gammacell 3000 Elan) to inactivate leukocytes in blood components. This study shows the comparison of irradiation and PRT treatment (Mirasol*®) on lymphocyte survival and inactivation in non-leukoreduced platelet concentrates (PCs). PRT treatment is a pathogen reduction technology that targets nucleic acids after exposure to riboflavin and UV-light. We analyzed 7 untreated (C), 7 PRT treated (M) and 7 irradiated PCs (RD) in our study. Non-leukoreduced buffy coats (mean volume 65 ml) were obtained from the Regional Blood Center, Warsaw. PCs were prepared by pooling 15 buffy coats (ABO identical) suspended in 3 plasma units, and dividing the pool into 3 equal-weight units in bags made of the same material. Following addition of 35 ml of riboflavin solution, M units were illuminated. The same volume of saline solution, was added to the C group and RD units. All PCs were then stored at 22°C with agitation for 5 days. Samples were removed on days 1, 3 and 6 for analysis. The lymphocyte survival rate was determined by 7AAD (7-amino-actinomycin D staining of dead cells, Becton Dickinson) and their activation by anti-CD69-APC staining (Becton Dickinson). Samples were also stained with anti-CD45-PE antibodies to identify and gate on lymphocytes. Samples were analyzed on the Becton Dickinson Cytometer FACSCanto I. No increase in the number of dead cells was observed during 6 days of storage in the C group. After 3 days of storage however, in the M group the percentage of dead cells was significant higher than in C and RD groups (Student t-test, p=0.004 and p=0.03, respectively). After 6 days, the percentage of dead cells in the M samples was 72% vs. 30% following irradiation. The percentage of 7AAD-positive cells was significantly higher compare to C samples, both in M (p=0,001) and in RD samples (p=0,004). The percentage of dead lymphocytes was also observed to be statistically higher in M samples than in RD (p= 0,001). Analysis of lymphocyte activation was performed on live (7AAD-negative, CD45-positive) cells only. CD69 expression ranged between 20% and 40% in all tested samples (C, M, RD) during 6 days of storage. On days 1 and 3 of storage, Mirasol treatment significantly reduced lymphocyte activation as shown by the ratio (test/control) of %CD69-positive cells (p=0,004 and p=0,001, respectively) and mean fluorescence expression intensity of CD69. Interestingly, after 6 days of storage, RD samples showed significant higher lymphocyte activation then C and M samples (p=0,03 and p=0,02, respectively). In summary, a significant increase of dead lymphocytes after 6 days of storage was observed in PRT-treated PCs. This increase was two-fold higher than in gamma-irradiated PCs. At the same time a decrease in lymphocyte activation during 6 days of storage was observed in PRT-treated PCs. Overall, the use of PRT achieves better leukocyte inactivation than gamma-irradiation. Disclosures: No relevant conflicts of interest to declare.

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