Irradiation is not sufficient to eradicate residual immortalized erythroid cells in in vitro‐generated red blood cell products

Abstract Background The generation of immortalized erythroid progenitor cell lines capable of producing enough red blood cells (RBCs) for blood transfusion typically requires the overexpression of oncogenes in stem cells or progenitor cells to permanently proliferate immature cells. It is essential that any live oncogene‐expressing cells are eliminated from the final RBC products for clinical use. Study Design and Methods It is believed that safety issues may be resolved by using a leukoreduction filter or by irradiating the final products, as is conventionally done in blood banks; however, this has never been proven to be effective. Therefore, to investigate whether immortalized erythroblasts can be completely removed using γ‐ray irradiation, we irradiated the erythroblast cell line, HiDEP, and the erythroleukemic cell line, K562 that overexpress HPV16 E6/E7. We then analyzed the extent of cell death using flow cytometry and polymerase chain reaction (PCR). The cells were also subjected to leukoreduction filters. Results Using γ‐ray irradiation at 25 Gy, 90.4% of HiDEP cells, 91.6% of K562‐HPV16 E6/E7 cells, and 93.5% of non‐transduced K562 cells were dead. In addition, 5.58 × 10 7 HiDEP cells were passed through a leukoreduction filter, and 38 intact cells were harvested, revealing a filter removal efficiency of 99.9999%. However, both intact cells and oncogene DNA were still detected. Discussion Irradiation cannot induce total cell death of oncogene‐expressing erythroblasts and leukocyte filter efficiency is not 100%. Therefore, our findings imply that for clinical applications, safer methods should be developed to completely remove residual nucleated cells from cell line‐derived RBC products.