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Chronic Granulomatous Disease (CGD)

Clinical Features and Laboratory Testing



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Dihydrorhodamine (DHR) Flow Cytometric Assay for Diagnosis of CGD

Slide 12

January 2010

The dihydrorhodamine flow cytometry based assay is the more commonly used diagnostic screening test for CGD in reference laboratories and larger medical centers.

This test is based on the principle that nonfluorescent DHR (dihydrorhodamine) 123 when phagocytosed by normal activated neutrophils (after stimulation with PMA – phorbol myristate acetate) can be oxidized by hydrogen peroxide, produced during the activated neutrophil respiratory oxidative burst, to rhodamine 123, a green fluorescent compound, which can be detected by flow cytometry.

Therefore, the detected fluorescence is an indirect measure of neutrophil function and oxidative burst. The top left-hand panel demonstrates normal neutrophil activation resulting in a robust oxidative burst and a complete shift of the peak (containing the cells) to the right indicating DHR oxidation to rhodamine. This shift can potentially be reported as a stimulation index (SI), which is the ratio of the mean fluorescent intensity of stimulated cells over unstimulated cells. In other words, the SI is the ratio of cells with rhodamine to the cells with DHR.

The top right panel depicts an X-linked CGD female carrier demonstrating two populations – one with no fluorescent shift, ie no oxidative burst and conversion of DHR to rhodamine, and the second normal peak, showing a typical oxidative burst. This would be expected in carrier females of X-linked diseases, since one allele is mutant and the other allele is normal. CGD is one of the PIDs where there is significantly skewed lyonization (random X-chromosome inactivation) resulting in female carriers with a clinical phenotype of disease. While it has been reported that even the presence of ~10% neutrophils with normal oxidative burst is sufficient to prevent a clinical phenotype, sometimes that is not always the case and therefore, clinical correlations should be performed with the DHR assay results. Further, it has been reported that there can be age-related changes in lyonization, with skewing more apparent with increasing age, resulting in clinically symptomatic CGD in older carrier females.

The lower left panel demonstrates the typical pattern of oxidative burst seen in patients with the autosomal recessive forms of CGD. This tends to be more challenging to interpret since there is not a complete absence of oxidative burst as is seen in the classic X-linked CGD patient (lower right panel). Rather, the autosomal recessive CGD patient shows at least a population of neutrophils with significantly reduced oxidative burst. The X-linked CGD patient does not usually have any neutrophils capable of mounting oxidative burst resulting in a SI that is almost close to one.

DHR

 


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