Chapter 33 : Functional Assays for the Diagnosis of Chronic Granulomatous Disease

Analysis of H2O2 production by dihydrorhodamine-123 staining. PMN populations of buffer-treated (Basal) and PMA-treated (PMA) samples were gated using FS × SS shown in the contour plots on the left panel for each condition. The middle panel for each condition represents the DHR histogram which is presented on a log scale. The right panel for each condition represents a dot plot of the back-gated region of the DHR histogram to identify the nature of the population analyzed. Stimulation with PMA induces a shift in the FS × SS pattern of the PMN population in all subjects tested: a normal subject (A), a patient with gp91phox CGD (B), an X-linked carrier of CGD (C), and a patient with p47phox CGD (D). DHR+ cells are indicated in red in back-gated FS × SS dot plots.

Histochemical staining of PMN with NBT. (A) PMN from a normal subject under basal condition exhibit only the safranin counterstain of the cytosol and nucleus. (B) PMN from a normal subject stimulated with PMA exhibit blue-black formazan deposits in the cytosol and the safranin counterstain in the nucleus. (C) PMN from a patient with gp91phox CGD stimulated with PMA fail to generate ROS and exhibit only the safranin counterstain of the cytosol and nucleus. (D) PMN from an X-linked carrier of CGD stimulated with PMA exhibit both an NBT− population and an NBT+ population of PMN. In this case, the X-linked carrier was 52% NBT+.

Analysis of PMN ROS generation by luminol-enhanced chemiluminescence. PMN (1 × 105 cells/200 μl HBSS/HEPES with 100 μM luminol) were incubated for 120 min in the presence of PMA (100 ng/ml). Luminescence of each well was determined every 2 min. The data from each subject were performed in triplicate. Blue circles represent the mean response of normal subjects (n = 20); yellow circles, an X-linked carrier of gp91phox CGD; green circles, a patient with p47phox CGD; red circles, a patient with gp91phox CGD.

Immunoblot analysis of phox subunits of NOX2. DFP-treated PMN (5 × 106 cells) were sonicated in 100 μl of sample buffer. For each subject, an aliquot (20 μl or 1 × 106 cell equivalents) of PMN lysate was loaded into a well of the precast BisTris SDS-PAGE gel. In this composite figure, the bands corresponding to each phox subunit from each subject have been aligned. Lane 1 represents PMN from a normal subject and defines the position and the expression of each phox subunit. Lanes 2 and 3 represent CGD patients with missense mutations in gp91phox and illustrate the proportional expression observed with gp91phox and p22phox. Lane 4 represents a CGD patient with a nonsense mutation in gp91phox with undetectable expression of gp91phox but faint expression of p22phox. Lane 5 represents a CGD patient with p22phox deficiency with undetectable expression of both p22phox and gp91phox. Lane 6 represents a CGD patient with undetectable p47phox. Lane 7 represents a CGD patient with undetectable expression of p67phox.