Evaluating 6- and 18-hour stimulation durations for natural killer cell degranulation (CD107a assay) to optimize workflow efficiency in a clinical immunology laboratory

BackgroundNatural killer (NK) cell degranulation is a key immune defense mechanism where exposure to tumor or virus-infected cells triggers the fusion of cytoplasmic granules containing apoptotic proteins, perforin, and granzyme with the cell membrane. This process transiently expresses CD107a on the NK cell surface, and measuring CD107a is a standard method to assess NK cell activity. MethodsWe compared two stimulation protocols differing only in duration (6-hour vs. 18-hour) using K562 target cells to induce NK cell degranulation. Isolated PBMCs without stimulation served as controls to assess spontaneous degranulation. Anti-CD107a-PE antibody was present throughout stimulation in both test and control samples. After stimulation, cells were stained with anti-CD45, anti-CD3, and anti-CD56 and analyzed by flow cytometry. ResultsFor 6 of 7 healthy controls, results from both methods fell within 2 standard deviations. Notably, longer (18-hour) stimulation resulted in lower CD107a expression than the 6-hour assay. Interlaboratory comparisons of two samples showed no significant difference (p>0.05). In a suspected hemophagocytic lymphohistiocytosis (HLH) case, two labs reported similarly reduced CD107a expression (9% and 7%). Inter-day variability was observed in a donor across both time points. The 6-hour assay showed higher sensitivity and specificity than the 18-hour assay. A resting period before ex vivo PBMC assays was found necessary. ConclusionStimulation periods beyond 6 hours are unsuitable for clinical NK degranulation assays. Screening for HLH should include multiple stimulants to improve assay reliability.