NK cell activity in PNH. (A) NK cell granule exocytosis in PNH. NK cells were purified from blood samples from healthy controls (HCs) or PNH patients using indirect selection reagents from Miltenyi. NK cell degranulation was assayed using a modification of a standard method to allow detection of GPI⁺ and GPI-deficient NK cells using fluorescent aerolysin (FLAER) (supplemental Text).⁸  Briefly, purified NK cells were cocultured with K562 target cells (for 4 hours). Cocultures were then stained with anti-CD56 antibody to identify NK cells, FLAER to distinguish GPI-deficient and GPI⁺ cells, and anti-CD107 to identify degranulated NK cells. This analysis is from 1 PNH patient and 1 HC (gating on the purified CD56⁺ NK cells) with the data from the cohort shown in panel B. We also compared the mean fluorescence intensity of CD107 staining in paired GPI-deficient and GPI⁺ cells within each patient. This demonstrated a significant increase in CD107 display on the GPI-deficient NK cells (supplemental Figure 1). (B) Summary of NK cell degranulation activity from 15 PNH patients and 8 HCs. The box plot shows the percentage of GPI-deficient (GPI^neg) and GPI⁺ NK cells that have degranulated in response to target cells. The range (whiskers), median (horizontal line), and interquartile range (box) are shown. The percentage of CD107⁺ NK cells was not statistically significant (NS) between any 2 groups according to the Mann-Whitney test. Comparison of the percentage of CD107⁺ NK cells within individual patients showed no significant differences between matched GPI⁺ and GPI^neg NK cells (supplemental Figure 1). (C) Number of total NK cells in 39 PNH patients (cells per microliter). Patient values ranged from 3 to 725 cells per microliter (mean, 100 cells/μL). A European reference range (77-427 cells/μL) is shown by the dotted lines. The patients with NK cell counts within the normal range were unremarkable in terms of gender, age, or treatment (cyclosporin or eculizumab). Furthermore, there was no correlation between the NK cell counts and the absolute numbers of other cell types (supplemental Figure 2). Peripheral blood samples used in this work were collected after informed consent in accordance with the Declaration of Helsinki.