Abstract Text: Measurement of Natural Killer (NK) cell function is a valuable tool in the evaluation of Hemophagocytic Lymphohistiocytosis and reduced function forms part of the established diagnostic criteria. Typically, NK cell cytotoxicity is assessed via measurement of the lysis of human K562 (leucoerythroblastic leukemia) cells. Here, K562 cells are incubated with chromium51 to allow uptake into the cells. Following an incubation period with patient Peripheral Blood Mononuclear Cells (PBMCs), lysis of K562 cells is assessed by measuring the supernatant radioactivity. This assay is laborious, requiring the use and storage of radioactive material in the laboratory which is onerous and a potential hazard. Fetal Hemoglobin (HbF) is the dominant hemoglobin expressed in erythrocytes in-utero and in neonates, but expression is typically < 1% after 12 months of age. Human K562 cells are known to express HbF. We hypothesised that NK cell cytotoxicity of K562 cells could be measured by quantifying HbF release into the supernatant, therefore abrogating the need for radioactive material. We performed a series of NK cell function testing by isolating PBMCs through Ficoll-Paque separation and incubating these with K562 cells spiked with chromium and unmarked K562 cells in reducing effector to target ratios (50:1, 25:1, 10:1, 5:1 and 1:1). The traditional chromium51 assay was done in parallel with HbF measurement. HbF in the supernatant was measured by NanoBit® (Promega) bioluminescent detecting antibodies. We found excellent correlation between the two methods, suggesting that bioluminescent measurement of HbF is a suitable surrogate method for measurement of NK cell cytotoxicity.
