Diagnosis and carrier detection of chronic granulomatous disease in five families by flow cytometry.
BACKGROUND
The application of flow cytometric assays, for determination of phagocyte respiratory burst (ROB) activity, to the investigation of chronic granulomatous disease (CGD) may lead to improved laboratory detection of patients and carriers and indicate the nature of the molecular defect. To evaluate the diagnostic capability of flow cytometry an investigation of 5 CGD families was undertaken.


METHODS
Phorbol myristate acetate (PMA)-induced neutrophil ROB was determined using dihydrorhodamine 123 (DHR) and flow cytometric analysis in 26 members of 5 CGD families (2: X-CGD; 3: autosomal recessive CGD).


RESULTS
Neutrophils from X-CGD patients displayed absence of reactivity. Female carriers demonstrated dual fluorescence peaks of high and low intensity indicative of normal and abnormal populations, respectively. Normal ROB activity was observed in a boy whose X-CGD was successfully treated by bone marrow transplantion. Reduced ROB activity was observed in 3 patients with autosomal-recessive CGD compared with their parents and siblings. The patterns of flow cytometric reactivity correlated with the different molecular defects identified. Absence of the p22phox membrane component of the NADPH oxidase complex resulted in a significantly reduced level of respiratory burst activity which was comparable to that observed in X-CGD, whereas reduced but detectable levels of respiratory burst activity were observed in a patient with diminished levels of p22phox and in a patient with deficiency of the cytosolic p47phox component.


CONCLUSIONS
The DHR flow cytometric assay offers a sensitive diagnostic screening test for CGD and furthermore may provide an indication of the likely underlying molecular defect.
Crockard
A D
AD
Regional Immunology Laboratory, Royal Victoria Hospital, UK. a.crockard@qub.ac.uk
Thompson
J M
JM
Boyd
N A
NA
Haughton
D J
DJ
McCluskey
D R
DR
Turner
C P
CP
D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D002648:Child; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006105:Granulomatous Disease, Chronic / Q000175:diagnosis* / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D008297:Male; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009252:NADPH Dehydrogenase / Q000302:isolation & purification; D019255:NADPH Oxidases; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010375:Pedigree; D010750:Phosphoproteins / Q000302:isolation & purification; D016897:Respiratory Burst; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D026901:Membrane Transport Proteins; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D009580:Nitroblue Tetrazolium; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D013755:Tetradecanoylphorbol Acetate