Genotype-dependent variability in flow cytometric evaluation of reduced nicotinamide adenine dinucleotide phosphate oxidase function in patients with chronic granulomatous disease☆,☆☆,★
Section snippets
METHODS
The materials and methods used in this study were identical to those described by Vowells et al.7 After erythrocyte lysis, the leukocyte pellet was resuspended in 400 μl of Hanks balanced salt solution (without calcium, magnesium, and phenol red) with albumin and ethylenediamine tetraacetic acid. Cells were then loaded with 1.8 μl of 29 mmol/L DHR for 5 minutes at 37° C in the presence of 1000 U/ml catalase. After DHR loading, cells were stimulated with phorbol myristate acetate at a
RESULTS
We used the DHR assay to evaluate 26 healthy subjects, 18 patients with CGD with the X-linked gp91-phox–deficient genotype, and 17 patients with CGD with the autosomal recessive p47-phox–deficient genotype. Increase in stimulated granulocyte fluorescence, as reflected by the geometric mean SI of the 26 normal subjects, was 127.9 (range, 85.2 to 264.6; standard error of geometric mean, 1.06). Those for the gp91-phox–deficient and p47-phox–deficient patients were 1.32 (range, 0.9 to 2.2; SEM,
DISCUSSION
When DHR was used in a flow cytometric assay of granulocyte NADPH oxidase activity, the combination of histogram SI and CV established a clear distinction between the two CGD genotypes studied, as well as between each patient group and the normal group. Nonfluorescent assays of superoxide production fail to provide such a distinction.
The increase in fluorescence observed in the stimulated granulocytes of the p47-phox–deficient patients could be related to an increased sensitivity of DHR in the
Acknowledgements
We thank Drs. John Gallin and Steven Holland, who provided blood samples for patients with CGD throughout the study.
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Cited by (0)
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From the Laboratory of Host Defenses, National Institute of Allergy and Infectious Disease, and the Immunology Service, Clinical Center, National Institutes of Health, Bethesda, Maryland
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Reprint requests: Sarah J. Vowells, MD, Department of Health and Human Services, National Institutes of Health, Bldg. 10, Room 2C-140, Bethesda, MD 20892.
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