Evaluation of a consensus protocol for the analysis of NK-cell and CTL degranulation. (A-B) Degranulation of resting NK cells analyzed with protocol 1. (A) FACS plots illustrating the induction of CD107a expression in CD3⁻CD56⁺ NK cells using PBMCs from a healthy donor and a patient with FHL5 after incubation with medium or with NK-sensitive K562 target cells. (B) Results from the same sample analyzed in 3 different laboratories (▴, Genoa; □, Freiburg; and ●, Stockholm). (C-D) Degranulation of IL-2–stimulated NK cells. (C) NK-cell degranulation assay using PBMCs that had been stimulated for 48 hours with PHA and IL-2. (D) Results from the same sample analyzed in 3 different laboratories. (E-F) Degranulation of T-cell blasts. (E) FACS plots illustrating the induction of CD107a expression in 48h PHA/IL-2–stimulated CD8⁺ T cells from a healthy donor and from a patient with FHL5 after incubation with medium alone or medium plus anti-CD3/anti-CD28 beads. (F) Overlay of stimulated (white) and unstimulated (shaded gray) samples. Numbers inside quadrants represent ΔCD107a (A,C) and ΔMFI of CD107a (E-F). ΔCD107a indicates the difference in the percentage of cells expressing CD107a before stimulation subtracted from the percentage of cells expressing CD107a after stimulation. ΔMFI indicates the respective difference in MFI.