American Society of Hematology

Fig. 1.

$Fig. 1. Analysis of apoptotic lymphocytes isolated from WAS patients. The levels of apoptosis were measured using TUNEL immediately after isolation or after incubation in vitro. PBL were not stimulated. (A) WAS lymphocytes had increased side scatter and decreased forward scatter, characteristics of apoptosis, after 4 days of incubation in vitro relative to normal lymphocytes. (B) Representative data acquired after TUNEL indicate that a higher fraction of WAS lymphocytes were undergoing apoptosis relative to normal, healthy donor lymphocytes after 4 days of incubation in vitro. The fraction of TUNEL-positive cells is indicated in the upper right corner. (C) WAS lymphocytes underwent apoptosis at a greater frequency than normal lymphocytes. Levels of apoptosis were measured immediately after isolation of PBL from WAS patients or from normal individuals using the TUNEL assay. N, the mean value for a group of 8 different normal controls (3 children and 5 adults). W, the mean value for a group of 5 different WAS patients. 1 through 5, the mean values for each individual WAS patient. Patient no. 5 was analyzed twice in 2 independent experiments (with 6 and 3 replicates); all other patients were analyzed once in single experiments (with 6 replicates). An unbalanced repeated measures analysis of variance (ANOVA) showed that the values for patients no. 2, 3, 4, and 5 differed significantly from the mean normal value (P ≤ .0001). The mean value for the group of 5 WAS patients (W) also differed significantly from the mean normal (P = .0123). n, the number of different samples; *, statistical significance. (D) WAS lymphocytes were more susceptible to apoptosis than normal lymphocytes after in vitro incubations of 24, 48, and 96 hours. Considerable variability in the apoptotic susceptibility of PBL from different WAS patients was apparent. Mean levels of apoptosis are reported in Table 1. Patient no. 1 was analyzed once in a single experiment, patient no. 2 was analyzed 3 times in 3 independent experiments, and patients no. 3, 4, and 5 were analyzed twice in 2 independent experiments. Results from 8 different normal controls (3 children and 5 adults) are shown. The number of repeated measures made in each experiment was varied, ranging from 2 to 6 (generally 4), depending on the number of PBL isolated from the blood samples. An unbalanced repeated measures ANOVA showed that differences between the groups, WAS and normal, are significant at 24, 48, and 96 hours (P = .000181, .00283, and .000190, respectively). *Statistical significance.$

Analysis of apoptotic lymphocytes isolated from WAS patients. The levels of apoptosis were measured using TUNEL immediately after isolation or after incubation in vitro. PBL were not stimulated. (A) WAS lymphocytes had increased side scatter and decreased forward scatter, characteristics of apoptosis, after 4 days of incubation in vitro relative to normal lymphocytes. (B) Representative data acquired after TUNEL indicate that a higher fraction of WAS lymphocytes were undergoing apoptosis relative to normal, healthy donor lymphocytes after 4 days of incubation in vitro. The fraction of TUNEL-positive cells is indicated in the upper right corner. (C) WAS lymphocytes underwent apoptosis at a greater frequency than normal lymphocytes. Levels of apoptosis were measured immediately after isolation of PBL from WAS patients or from normal individuals using the TUNEL assay. N, the mean value for a group of 8 different normal controls (3 children and 5 adults). W, the mean value for a group of 5 different WAS patients. 1 through 5, the mean values for each individual WAS patient. Patient no. 5 was analyzed twice in 2 independent experiments (with 6 and 3 replicates); all other patients were analyzed once in single experiments (with 6 replicates). An unbalanced repeated measures analysis of variance (ANOVA) showed that the values for patients no. 2, 3, 4, and 5 differed significantly from the mean normal value (P ≤ .0001). The mean value for the group of 5 WAS patients (W) also differed significantly from the mean normal (P = .0123). n, the number of different samples; *, statistical significance. (D) WAS lymphocytes were more susceptible to apoptosis than normal lymphocytes after in vitro incubations of 24, 48, and 96 hours. Considerable variability in the apoptotic susceptibility of PBL from different WAS patients was apparent. Mean levels of apoptosis are reported in Table 1. Patient no. 1 was analyzed once in a single experiment, patient no. 2 was analyzed 3 times in 3 independent experiments, and patients no. 3, 4, and 5 were analyzed twice in 2 independent experiments. Results from 8 different normal controls (3 children and 5 adults) are shown. The number of repeated measures made in each experiment was varied, ranging from 2 to 6 (generally 4), depending on the number of PBL isolated from the blood samples. An unbalanced repeated measures ANOVA showed that differences between the groups, WAS and normal, are significant at 24, 48, and 96 hours (P = .000181, .00283, and .000190, respectively). *Statistical significance.

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