(P-TS-119) T Cell Inactivation Efficacy: Comparison of Amotosalen-UVA Pathogen Inactivation and Gamma Irradiation Using a Validated EdU Incorporation Assay to Detect T-Cell Proliferation

Background/Case Studies: Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare but fatal complication caused by donor T cell proliferation in immunocompromised blood transfusion recipients. This study compares the efficacy of Amotosalen-UVA pathogen reduction treatment (AMO-UVA PRT) and gamma irradiation in inactivating donor T cells in platelet concentrates. A validated, non-radioactive EdU incorporation assay was used to assess T cell proliferation. Unlike traditional methods that rely on radioactive isotopes (H³ thymidine assay), the EdU assay utilizes 5-Ethynyl-2’-deoxyuridine (EdU) -a thymidine analog containing an alkyne group - which is incorporated into newly synthesized DNA enabling single-cell detection of proliferating T cells. A fluorescent dye with an azide group is then added and, in the presence of a copper catalyst, an azide-alkyne cycloaddition forms a stable covalent bond between the EdU and fluorophore, labelling proliferating cells and enabling flow cytometry detection.

Study

Design/Methods: Three-arm pool and split studies were conducted using apheresis-derived platelet concentrates (100% plasma) spiked with 1 × 10⁶ CD3⁺ T cells/mL. The arms were treated with AMO-UVA PRT using either INT100 Illuminator (UVA 320-400 nm), the recent CE marked INT200 Illuminator (340-350nm) , or irradiated with a dose of 2500 cGy gamma rays. Treated units were stimulated with CD3/CD28 Dynabeads, PHA-L, and rhIL-2. Parallel cultures were prepared with no stimulation to serve as unstimulated controls. Donor T cell dilutions were seeded in 96- or 12-well plates, depending on the cell number, with each dilution replicated in 10 wells containing allogeneic stimulator cells. Plates were cultured for 10 days before EdU was added. Cells were harvested the next day for CD3 surface staining and EdU incorporation assay. T cell proliferation was quantified using an Attune^TM NxT Acoustic Focusing Cytometer by selecting for CD3+ T cell leukocytes that were labeled with EdU. Twelve replicates using independent T cell donors were performed.

Results/Findings: AMO-UVA PRT treatment using both INT100 and INT200 Illuminators resulted in a log reduction of >5.7 ± 0.4 log T cells/mL. Similarly, following treatment with 2,500 cGy dose of gamma irradiation, >5.7 ± 0.4 log T cells/mL were inactivated. No proliferation of T cells was detected following either AMO-UVA PRT treatment or gamma irradiation.

Conclusions: Using the validated EdU incorporation assay to detect T cell proliferation, this study demonstrated that both INT100 and INT200 AMO-UVA PRT can effectively inactivate contaminating T cells, with a log reduction of at least 5.7±0.4 log T cells/mL. This inactivation is robust and consistent with that achieved with gamma irradiation.