Neutrophils from 50 pediatric patients with normal phagocyte functions, from 150 healthy adults, from 10
chronic granulomatous disease (CGD)-patients (4 CGD+), and from 18 X-linked carriers for CGD have been tested for their production of H2O2 using staining with
dihydrorhodamine 123 and subsequent flow cytometry. Additionally, neutrophils from three patients with
myeloperoxidase deficiency were assessed. Cells were activated to produce H2O2 by the
phorbol ester phorbol-myristate-acetate (PMA) and by phagocytosis of Escherichia coli bacteria. To evaluate the sensitivity of the method, H2O2-production by neutrophils which was inhibited by different concentrations of diphenyljodonium (DPI) was measured. The results were compared to those from other methods (NBT-testing,
cytochrome c-reduction, and especially chemiluminescence). Normal values and ranges of scatter profile were evaluated in terms of peak channel fluorescence: 97% > 700, x = 840 +/- 59 (S.D.), 97% < 890, for pediatric patients. Normal quantitative values also resulted from small blood samples of infants (< 1 year, n = 6, x = 830 +/- 52). For CGD+ (n = 4) the results were clearly far below the normal range. In indicating decreased production of
reactive oxygen intermediates the method was at least as sensitive as
lucigenin enhanced chemiluminescence.
Cytochrome b558-expression of neutrophils from patients and healthy controls was established by flow cytometry following staining with the
monoclonal antibody 7D5. The normal range was 97% > 485, 97% < 680, peak channel fluorescence. We conclude that flow cytometric routine diagnostics of CGD can easily enhance the reliability of recognition and the yield of information about this disease compared to conventional methods.