Granulocytes from GSD1b patients display apoptotic features. (A) Freshly purified neutrophils from a patient with GSD1b (n = 5) and an age-matched control (n = 10) were stained by Annexin-V–fluorescein isothiocyanate (FITC) and PI, and analyzed by FACScan. Representative dot plots are shown. Values indicate the proportion of Annexin-V⁺ cells. (B) The summarized data of all measurements performed in fresh cells and cells cultured overnight in the absence or presence of exogenous G-CSF (100 ng/mL; n = 5). Bars represent mean ± SD of Annexin-V⁺ cells in percentage of total number of neutrophils. (C) Overall caspase proteolytic activity in cell lysates prepared from 0.5 × 10⁶ fresh healthy control neutrophils and neutrophils cultured overnight (∼ 70% apoptotic cells as measured by Annexin-V–FITC staining and morphologic examination; n = 5) as well as from freshly isolated neutrophils of 3 GSD1b patients was measured by cleavage of the fluorogenic caspase substrate DEVD-AMC. To check the specificity of the caspase proteolytic activity, the general caspase inhibitor zVAD-fmk (10 μM final concentration; Alexis Biochemicals) was added to the assay mixture. The maximum speed (maximal slope) of AMC release was used as a measure of caspase activity. Results are presented as mean ± SD maximal slope. *P < .01 (by Student t test) versus control fresh neutrophils. RFU indicates relative fluorescence units. (D) Fresh neutrophils from a healthy control and from patients with GSD1b were stained with antibody specific for Bax (green), counterstained with PI (1 μg/mL; red) to visualize nuclear morphology, and analyzed by confocal microscopy. Note that in the control image a punctate-dispersed distribution of Bax and polysegmented nucleus with normal chromatin are present. In contrast, both patients' images (nos. 3 and 5) reveal Bax clustering and typical apoptotic nuclei with condensed chromatin (for further comparison with normal neutrophils, see also Maianski et al¹⁷ ). Representative images from 5 controls and 5 GSD1b patients are shown. Bar is 5 μm. (E) Whole leukocyte preparation from patients with GSD1b (1.5-2 × 10⁵ cells) was obtained after erythrocyte lysis. Cytospins stained with Giemsa solution were analyzed by light microscopy. Engulfment of apoptotic material (left panel) and intact cellular remnant (arrow, right panel) in monocytes are shown. Original magnification, × 400.