The impact of anticoagulant used for peripheral blood collection on NK cell yield, function, and proliferation

Natural killer (NK) cells are under investigation for cellular immunotherapy of cancer, infectious diseases, and autoimmunity. The collection of autologous or allogeneic starting material for the expansion of NK cells - whether whole blood, cord blood, or leukocyte apheresis - requires the use of anticoagulants. Heparin and acid citrate dextrose A (ACD-A) are most commonly used for collection, but their impact on NK cells has not been clearly described. As functional impairment has been described for irreversible calcium chelation with EDTA, we hypothesized that prolonged calcium chelation by ACD-A might also have an adverse impact on NK cell health, particularly with prolonged exposure. To test this hypothesis, peripheral blood was collected as paired samples in heparin and in ACD-A, stored for 24 h at room temperature, and then tested for viable NK cell yield and proliferative capacity for 2 weeks under ex vivo expansion. Both conditions yielded similar viability and T/NK cell content after initial isolation. However, compared to ACD-A-treated blood, heparin-treated blood provided a mean 23 % higher initial yield of NK cells (p = 0.03) and a non-significant trend toward greater fold expansion, which resulted in 60 % higher overall yield after expansion (p = 0.048). These data suggest that both ACD-A and heparin can be used for NK cell collection, but that heparin may be the preferred anticoagulant if there is an expected delay in the product processing such as a pause for overnight testing or shipping. Process development for cellular therapeutics should assess the impact of anticoagulant used for cell collection.