▪Background: Transfusion Associated Graft vs. Host Disease (TA GVHD) results in high morbidity and mortality, caused by contaminating T-cells present in transfused blood products. Gamma irradiation (GI) of platelet components (PC) with a dose of 2500 cGy is the approved methodology to prevent TA GVHD in high-risk patients. That methodology has been established based on in vitro Limiting Dilution Assays (LDA) that quantify the inactivation of T-cells. The limited natural abundance of T-cells in blood products and the number of T-cells that can be reproducibly cultured in single wells have defined LDA assay sensitivity. LDA assays usually have a dynamic range of 4-5 log10. Even though that methodology has been used for decades with presumable success, cases of GVHD recognized as transfusion associated, have to be connected temporally and phenotypically to the blood product in use, in order to be characterized as such. That leaves open the possibility that some atypical cases of GVHD in blood product recipients may be miss-assigned and go unrecognized.An alternative methodology to prevent TA GVHD is the photochemical treatment of PC with amotosalen/UVA (PCT; INTERCEPTTM Blood System). PCT has replaced GI in Europe for pathoen inactivated PC for more than 10 years. In this study, we evaluated both GI and PCT on inactivation of T-cells using a highly sensitive approach that includes the use of higher numbers of PBMCs isolated by leukapheresis, and an increased number of cells cultured in a single well (107/well) by using larger wells and optimizing culture conditions.Methods: PBMCs harvested by leukapheresis from individual donors (AllCells, Alameda, CA) were spiked (106/mL) into identical units of human plasma and inactivated using either GI with 2500 cGy, PCT, or were retained as untreated control. For the LDA assay, PCT- or GI-treated cells isolated post treatment, were incubated in individual wells for 14 days in the presence of pooled allostimulator cells from 10 unrelated donors (5X106 treated with 7500 cGy) and growth stimulating factors (PHA and IL-2) under standard culture conditions. Proliferation was assessed by tritiated thymidine ([3H]TdR6.7 Ci/mmol) incorporation into PBMC, as well as by the observation of bright large clusters that were clearly observed at 40x magnification.Results: Initial experiments showed that 107PBMCs/well or 106PBMCs/well resulted in proliferation after GI at 2500 cGy, while 105 PBMCs/well did not. On the other hand, 107 PCT treated-PBMCs/well were found neither to proliferate, nor to incorporate [3H]TdRabove background. T-cell precursor frequency was measured for each donor by incubation of serial dilutions of viable PBMCs (50 - 1 /well; 12 wells per dilution) in the presence of 107 inactivated PBMCs. The experiment was repeated 10 times with PBMCs from different donors.For GI, [3H]TdR incorporation above background indicative of T-cell growth, as well as T-cell proliferating colonies were observed in 4 of 10 experiments when 106PBMCs/ well were cultured. No T-cell growth was detected by [3H]TdR incorporation when 105 GI PBMCs/well were cultured, while proliferating colonies were observed in 1 of 10 experiments. No T-cell proliferation was detected by either criterion, when 107 PCT-treated PBMCs/well were cultured.Conclusions: The accepted dose of 2500 cGy gamma irradiation for prevention of TA GVHD results in more than 4.2log10 but less than 6.2 log10 T-cell inactivation. However, it still allows the proliferation of T-cell clones from some donors. Treatment of T-cells with amotosalen/UVA results in complete inactivation of T-cells (>6.2 log10) with no break though proliferation detected.The data in this study have not been reviewed by the FDA. DisclosuresMerkel:Cerus Corporation: Other: fee for service with Cerus Corporation. Giclas:Cerus Corporation: Other: fee for service with Cerus Corporation. Gibula:Cerus Corporation: Other: fee for service with Cerus Corporation. Andersen:Cerus Corporation: Other: fee for service with Cerus Corporation. Knight:Cerus Corporation: Other: fee for service with Cerus Corporation. Lin:Cerus corp: Employment. Castro:Cerus Corporation: Employment. Green:Cerus Corporation: Employment. Stassinopoulos:Cerus Corporation: Employment.