There are three subsets of circulating human lymphocytes: B-cells, T-cells, and natural killer (NK) cells. Natural killer cells are most easily identified on a peripheral smear by their characteristic morphology of large granular lymphocytes. They comprise approximately 10–20% of the mononuclear cell fraction in normal peripheral blood. Natural killer cells can be isolated from the peripheral blood by first separating mononuclear cells (removing red cells and neutrophils) from the whole blood by removing interface cells after centrifugation over a ficoll gradient. Although further enrichment steps over other density gradients (percoll) have been described, techniques are now readily available for NK cell purification without the use of flow cytometry. Probably, the best method to isolate resting NK cells with high purity is by immunomagnetic beads. This can be performed by positive selection using an antigen expressed on the NK cells or, our preferred method, which is to isolate the NK cells by negative selection (removing B-cells, T-cells, and monocytes). Activated NK cells can also be isolated by their adherence to plastic after 24-hours of incubation with

IL-2 using the adherent lymphokine-activated killer (ALAK) cell method (1–4). Historically, NK cells were first identified by their ability to lyse tumor cells without prior immunization or activation. Now, the NK cells and their subsets can be identified precisely by the use of monoclonal antibodies. By definition, NK cells lack the T-cell receptor (TCR) complex, they are CD32 and do not rearrange TCR genes. The neural cell adhesion molecule (NCAM, also called CD56), first identified in the brain tissue, is probably the best human surface antigen to identify all NK cells (5). Several other antibodies can be used to identify important subsets. CD16 (FcRgIII) is the Fc receptor present on CD56+ dim NK cells, which identifies the most mature cell of this lineage (Fig. 1). In contrast, CD56+ bright NK cells are the best-characterized unique subset circulating in the peripheral blood (6). CD56+ bright NK cells lack CD16 (FcRgIII), are lowly cytotoxic (7), highly proliferative (7, 8), constitutively express the intermediate affinity IL-2 receptor (9), express c-kit (the receptor for stem cell factor or c-kit ligand)(10, 11), and may be a precursor of mature NK cells. However, definitive proof that CD56+ bright NK cells can further mature into CD56+ dim NK cells is lacking.