EVALUATION OF FLOW CYTOMETRIC METHODS FOR DIAGNOSIS OF CHRONIC GRANULOMATOUS-DISEASE VARIANTS UNDER ROUTINE LABORATORY CONDITIONS

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 dihydrorhodami ne 123 and subsequent flow cytometry. Additionally, neutrophils from t hree patients with myeloperoxidase deficiency were assessed. Cells wer e activated to produce H2O2 by the phorbol ester phorbol-myristate-ace tate (PMA) and by phagocytosis of Escherichia coli bacteria. To evalua te the sensitivity of the method, H2O2-production by neutrophils which was inhibited by different concentrations of diphenyljodonium (DPI) w as measured. The results were compared to those from other methods (NB T-testing, cytochrome c-reduction, and especially chemiluminescence). Normal values and ranges of scatter profile were evaluated in terms of peak channel fluorescence: 97% > 700, ($) over bar x = 840 +/- 59 (S. D.), 97% < 890, for pediatric patients. Normal quantitative values als o resulted from small blood samples of infants (<1 year, n = 6, ($) ov er bar x = 830 +/- 52). For CGD(+) (n = 4) the results were clearly fa r below the normal range. In indicating decreased production of reacti ve oxygen intermediates the method was at least as sensitive as lucige nin enhanced chemiluminescence. Cytochrome b(558)-expression of neutro phils from patients and healthy controls was established by flow cytom etry following staining with the monoclonal antibody 7D5. The normal r ange 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 di sease compared to conventional methods. (C) 1994 Wiley-Liss, Inc.