The diagnosis of CGD is usually suggested by the unusual clinical histories outlined earlier or by a family history of CGD. The NBT slide test on fresh blood is the classic diagnostic test. A typical result is shown in Fig. 1. Fig. 1A shows the normal positive staining of a group of seven neutrophils and one monocyte. Fig. 1B shows the complete absence of NBT staining in a patient with X91° CGD, the classic X-linked form of the disease. Fig. 1C shows the mixed population of NBT-positive and NBT-negative cells observed in that patient’s mother, reflecting random X chromosome inactivation. Because nearly 100% of the normal cells in this test are positive, the carrier state in X-linked CGD can be detected when as few as 5% of the cells are NBT-negative. This test also permits detection of diffuse populations of weakly positive cells such as those seen in X91− CGD, which are characterized by a partial deficiency of flavocytochrome b. Because X-linked CGD can arise by new mutations in the maternal germline, one does not always see NBT-negative cells in the mother. Flow cytometric assays of oxidase activity, such as those based on the conversion of dihydroxyrhoda mine (DHR) 123 to rhodamine 123, can also provide both quantitative measurements of oxidant generation and the cell-by-cell distribution of activity (see Fig. 1D–G). The DHR 123 assay for oxidase activity is now available in many referral centers and reference laboratories. In addition to X91− CGD neutrophils, weak staining in the NBT test or a small but measurable level of DHR fluorescence can be seen in A47° cells (see Fig. 1H) because of a small amount of residual oxidant production. Regardless of diagnostic assay used, it is important to have these tests performed on appropriately handled blood samples and by experienced laboratories to avoid inconclusive or false-normal results.
Fig1. ANALYSIS OF NEUTROPHIL NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE OXIDASE ACTIVITY FOR THE DIAGNOSIS OF CHRONIC GRANULOMATOUS DISEASE. (A–C) Nitroblue tetrazolium (NBT) slide test. Peripheral blood neutrophils and monocytes from a drop of fresh whole blood were made adherent to glass slides and stimulated with phorbol myristate acetate. (A) Normal neutrophils and monocytes, all of which are NBT-positive. (B) Neutrophils and monocytes from an X-linked chronic granulomatous disease (CGD) patient, which are all NBT-negative. (C) A mixture of NBT-positive and NBT-negative neutrophils from the X-linked carrier mother of the patient in (B). (D–G) DHR 123 flow cytometry test. Nonfluorescent DHR 123 is taken up by neutrophils, which become fluorescent after reaction with reactive oxygen species produced in the respiratory burst. (D) Normal neutrophils. (E) Neutrophils from an X-linked CGD patient, which do not fluoresce after stimulation. (F) A mixture of nonfluorescent and fluorescent neutrophils from an X-linked CGD carrier. (G) Neutrophils from a p47phox-deficient patient, which show weak fluorescence after stimulation. DHR, Dihydrorhodamine; PMA, phorbol myristate acetate.
Genetic testing is useful to solidify the diagnosis and for genetic counseling and is commercially available for the five genetic subgroups involving NADPH oxidase subunits (see Table 1). The analysis of mutations in NCF1 can require specialized polymerase chain reaction (PCR) testing due to the presence of adjacent pseudogenes. Laboratories specializing in neutrophil biochemistry can also perform immunoblot analysis of neutrophil extracts, flavocytochrome b spectroscopy, or functional analysis of membrane and cytosol fractions in the cell-free oxidase assay.
Table1. Classification of Chronic Granulomatous Disease
Testing for the McLeod red cell phenotype should be done in patients diagnosed with X-linked CGD who harbor large CYBB deletions that potentially involve the locus encoding the Kell erythrocyte antigen. Loss of Kell causes a mild hemolytic anemia. More importantly, there can be serious problems with development of hemolytic antibodies if these patients are transfused with Kell positive blood.
