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

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 53; no. 5; pp. 890 - 896
• Main Authors: Mauch, Lysann, Lun, Andreas, O'Gorman, Maurice R.G, Harris, John S, Schulze, Ilka, Zychlinsky, Arturo, Fuchs, Tobias, Oelschlagel, Uta, Brenner, Sebastian, Kutter, Dolphe, Rosen-Wolff, Angela, Roesler, Joachim
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01.05.2007; American Association for Clinical Chemistry; Oxford University Press
• Subjects: Acridines; Analytical, structural and metabolic biochemistry; Apoptosis; Biological and medical sciences; Chemiluminescence; Coloring Agents; Diagnosis, Differential; Diagnostic tests; Eosinophils - enzymology; False Positive Reactions; Female; Flow Cytometry; Fluorescence; Fundamental and applied biological sciences. Psychology; Genetic disorders; Granulomatous Disease, Chronic - diagnosis; Granulomatous Disease, Chronic - enzymology; Humans; Immune system; Investigative techniques, diagnostic techniques (general aspects); K562 Cells; Laboratory methods; Luminescent Agents; Luminescent Measurements; Male; Medical sciences; Methods; Monoclonal antibodies; NADPH Oxidases - analysis; NADPH Oxidases - genetics; Neutrophils - enzymology; Patients; Peroxidase - analysis; Peroxidase - deficiency; Recombinant Proteins - analysis; Rhodamines; Scatter diagrams; Transfection; Immunopathology; Chronic granulomatous disease; Enzyme; Complete; Deficiency; Routine analysis; Exploration; Hemopathy; Biochemistry; Medical screening; Immune deficiency; Genetic disease; Signal transduction; Peroxidases; Clinical biology; Peroxidase; Oxidoreductases; Molecular biology; Leukocyte disease
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2006.083444

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). 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. 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. 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.