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IS-6
Amotosalen and UVA is more effective in the inactivation of T-Cells than gamma irradiation when assessed by a Limiting Dilution Assay (LDA)
Thursday, May 4th, 12:05 PM - Monitor IS-1

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Transfusion-associated graft-versus-host-disease (TA-GvHD) results in high morbidity and mortality and is caused by contaminating T-cells present in transfused blood products. Gamma irradiation (G-IRR) of platelet components (PC) with a dose of 2500 cGy has, until now, been the accepted methodology to prevent TA-GvHD in high-risk patients. Although G-IRR has been used for decades with presumptive success, diagnosis of GvHD as related to transfusion requires temporal and phenotypical connection to the blood product in use. This leaves open the possibility that some atypical cases of GvHD in blood product recipients may be inappropriately diagnosed and thus may go unrecognized, despite G-IRR of blood products. The effectiveness of the G-IRR methodology has been established based on in vitro limiting dilution assays (LDAs) that quantify the inactivation of T-cells (Figure 1). 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 sensitivity. LDAs usually have a dynamic range of 4-5 log10.
An alternative methodology to prevent TA-GvHD is the photochemical treatment of PC with a psoralen (amotosalen) and UVA light (PCT; psoralen treatment; INTERCEPT® Blood System) (Figure 2). PCT via psoralen/UVA light treatment has been demonstrated to inactivate a broad spectrum of pathogens, including bacteria, parasites, viruses, and spirochetes, as well as donor T-cells, all of which may contaminate PC. PCT has been shown to prevent TA-GvHD in an animal model, to inhibit clonal T-cell proliferation, to prevent allogeneic antigen stimulation in mixed lymphocyte reactions, and to inhibit transcription-mediated cytokine production and early activation antigen expression.1 For more than 10 years, PCT of PC has replaced G-IRR in parts of Europe for prevention of TA-GvHD, and PCT is authorized for prevention of TA-GvHD in the U.S. by the AABB Standards.2
In this study, we evaluated both G-IRR and PCT for inactivation of T-cells in a highly sensitive in vitro LDA assay. By using a higher number of peripheral blood mononuclear cells (PBMCs) isolated by leukapheresis, and an increased number of cells cultured per well (107/well), achieved by using larger wells and optimal culture conditions, we were able to improve the dynamic range of the LDA assay sensitivity (Figure 3).

PBMCs harvested by leukapheresis from individual donors (AllCells, Alameda, CA) were spiked (106/mL) into identical units of human plasma and inactivated using either G-IRR with 2500 cGy, PCT, or were retained as untreated control (Figure 3). For the inactivation assays, PCT- or G-IRR-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. For the precursor frequency determination, live cells isolated from the pool prior to treatments (Figure 4) were spiked in defined numbers in the presence of allostimulators and growth factors and the ability to proliferate was quantitated (Figure 4; Table 2) in 12 wells per dose. The frequency was then calculated as described in Figure 4.
In all cases, proliferation was assessed by tritiated thymidine ([3H]TdR 6.7 Ci/mmol) incorporation into PBMCs, as well as by the observation of bright large clusters that were clearly observed at 40x magnification. This study was performed in plasma to avoid interference of platelets in the cell proliferation assay under high cell input conditions and no compound adsorption device was used to remove residual psoralen or photo-by products resulting from PCT.

Initial experiments showed that 107 PBMCs/well or 106 PBMCs/well resulted in proliferation after G-IRR 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]TdR above background. T-cell precursor frequency was measured for each PBMC donor by incubation of serial dilutions of viable PBMCs (50 - 1 per well; 12 wells per dilution) in the presence of 107 inactivated PBMCs. The experiment was repeated 10 times with PBMCs from different donors.
For G-IRR, [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 106 PBMCs/well were cultured. No T-cell growth was detected by [3H]TdR incorporation when 105 G-IRR 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 (Figure 5; Table 3). The results obtained in this study are applicable to leukocyte inactivation by PCT via psoralen treatment in both plasma and platelet components, as the photochemistry of activation of the psoralen amotosalen is the same in blood components with and without platelets.

The accepted dose of 2500 cGy G-IRR for prevention of TA-GvHD results in more than 4.1 log10 but less than 5.1 log10 T-cell inactivation. G-IRR still allows the proliferation of T-cell clones from some donors. The significance of the remaining live clones still remains to be evaluated for G-IRR treated T-cells in conventional PCs. Treatment of T-cells with PCT via psoralen treatment results in complete inactivation of T-cells (>6.1 log10) with no breakthrough proliferation detected.

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