Chronic granulomatous disease (CGD) and complete myeloperoxidase deficiency both yield strongly reduced dihydrorhodamine 123 test signals but can be easily discerned in routine testing for CGD.
BACKGROUND
The flow cytometric dihydrorhodamine 123 (DHR) assay is used as a screening test for chronic granulomatous disease (CGD), but complete myeloperoxidase (MPO) deficiency can also lead to a strongly decreased DHR signal. Our aim was to devise simple laboratory methods to differentiate MPO deficiency (false positive for CGD) and NADPH oxidase abnormalities (true CGD).


METHODS
We measured NADPH-oxidase and MPO activity in neutrophils from MPO-deficient patients, CGD patients, NADPH-oxidase-transfected K562 cells and cells with inhibited and substituted MPO.


RESULTS
Eosinophils from MPO-deficient individuals retain eosinophilic peroxidase and therefore generate a normal DHR signal. The addition of recombinant human MPO enhances the DHR signal when simply added to a suspension of MPO-deficient cells but not when added to NADPH-oxidase-deficient (CGD) cells. Lucigenin-enhanced chemiluminescence (LCL) is increased in neutrophils from MPO-deficient patients, whereas neutrophils from patients with CGD show a decreased response.


CONCLUSIONS
A false-positive result caused by MPO deficiency can be easily ascertained because, unlike cells from a CGD patient, cells from MPO-deficient patients (a) contain functionally normal eosinophils, (b) show a significant enhancement of the DHR signal following addition of rhMPO, and (c) generate a strong LCL signal.
Mauch
Lysann
L
University Clinic Carl Gustav Carus, Department of Pediatrics, Dresden, Germany.
Lun
Andreas
A
O'Gorman
Maurice R G
MR
Harris
John S
JS
Schulze
Ilka
I
Zychlinsky
Arturo
A
Fuchs
Tobias
T
Oelschlägel
Uta
U
Brenner
Sebastian
S
Kutter
Dolphe
D
Rösen-Wolff
Angela
A
Roesler
Joachim
J
D000166:Acridines; D004396:Coloring Agents; D003937:Diagnosis, Differential; D004804:Eosinophils / Q000201:enzymology; D005189:False Positive Reactions; D005260:Female; D005434:Flow Cytometry; D006105:Granulomatous Disease, Chronic / Q000175:diagnosis* / Q000201:enzymology; D006801:Humans; D020014:K562 Cells; D049408:Luminescent Agents; D008163:Luminescent Measurements; D008297:Male; D019255:NADPH Oxidases / Q000032:analysis / Q000235:genetics; D009504:Neutrophils / Q000201:enzymology; D009195:Peroxidase / Q000032:analysis / Q000172:deficiency*; D011994:Recombinant Proteins / Q000032:analysis; D012235:Rhodamines; D014162:Transfection
D000166:Acridines; D004396:Coloring Agents; D049408:Luminescent Agents; D011994:Recombinant Proteins; D012235:Rhodamines; C058319:dihydrorhodamine 123; C033472:10,10'-dimethyl-9,9'-biacridinium; D009195:Peroxidase; D019255:NADPH Oxidases