Subjects
The diagnosis of AIN in children was based on the presence of chronic neutropenia and positivity for antineutrophil antibodies in sera according to the criteria published in the Nelson Textbook (21). Antineutrophil antibody was detected using an indirect granulocyte immunofluorescence test, as previously described (4). Table Ipresents the characteristics of 25 patients with AIN enrolled in this study: 13 patients for the analysis of the frequency of total Tregs and activated Tregs (median age: 17 months, range: 7–69 months) and 17 patients for the analysis of the TCR-Vβ repertoire (median age: 20 months, range: 11–57 months). Five patients were included in both analyses. AIN in childhood shows spontaneous resolution within a few years without the need for treatment. Hence, these TCR repertoire assays were performed in the middle of the neutropenic period. Parts of the assays in some patients were performed at the end of the spontaneous recovery from neutropenia. Treg and TCR repertoire assays were longitudinally performed in three patients with AIN. The results of the analyses in these patients did not show any fluctuations. Therefore, the TCR repertoire assay was compared between patients with AIN and age-matched control subjects. Eighteen (median age: 17.5 months, range: 4–78 months) and 22 (median age: 20 months, range: 8–49 months) heathy children without neutropenia were also examined as age-matched control subjects for respective analyses.
Informed consent was obtained from the guardians of patients and control subjects, and approval for these studies was obtained from the institutional review board.