Clinical Chemistry 53:5
890 – 896 (2007)
Hematology
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
Lysann Mauch,1 Andreas Lun,2 Maurice R.G. O’Gorman,3 John S. Harris,4
Ilka Schulze,5 Arturo Zychlinsky,6 Tobias Fuchs,6 Uta Oelschlägel,7
Sebastian Brenner,1 Dolphe Kutter,8 Angela Rösen-Wolff,1 and Joachim Roesler1*
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.
© 2007 American Association for Clinical Chemistry
Chronic granulomatous disease (CGD)9 is caused by a
genetic defect in 1 of the components of the NADPHoxidase multienzyme complex that produces reactive
oxygen intermediates (ROIs). In CGD, the inability to
produce ROIs leads to recurrent life-threatening opportunistic infections and uncontrolled inflammation, often
accompanied by granuloma formation. In contrast, myeloperoxidase (MPO) deficiency is generally considered to
be innocuous and was recently removed as a primary
immune deficiency disease as classified by the Primary
Immunodeficiency Diseases Classification Committee of
the International Union of Immunological Societies (1 ).
MPO and the NADPH-oxidase multienzyme complex are
1
University Clinic Carl Gustav Carus, Department of Pediatrics, Dresden,
Germany.
2
Institut für Laboratoriumsmedizin und Pathobiochemie, Berlin, Germany.
3
The Children’s Memorial Hospital, Chicago, IL.
4
Valley Medical Center, Lewiston, ID.
5
Department of Pediatrics, Charite, Berlin, Germany.
6
Max Planck Institute for Infection Biology, Berlin, Germany.
7
Department of Haematology, Technische Universität Dresden, Dresden,
Germany.
8
Laboratoires Reunis Kutter-Lieners-Hastert, Luxembourg, Luxembourg.
* Address correspondence to this author at: University Clinic Carl Gustav
Carus, Department of Pediatrics, Fetscherstrasse 74, 01307 Dresden, Germany.
Fax: 49-351-458-6333; e-mail: roeslerj@rcs.urz.tu-dresden.de.
Received November 23, 2006; accepted February 15, 2007.
Previously published online at DOI: 10.1373/clinchem.2006.083444
9
Nonstandard abbreviations: CGD, chronic granulomatous disease; ROI,
reactive oxygen intermediate; MPO, myeloperoxidase; DHR, cytometric dihydrorhodamine 123; SHA, salicylhydroxamic acid; ABAH, 4-aminobenzoylhydrazide; PMA, phorbol-myristate-acetate; mAb, monoclonal antibody; LCL,
lucigenin-enhanced chemiluminescence.
890
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Clinical Chemistry 53, No. 5, 2007
involved in ROI metabolism according to the following
grossly simplified reaction chain:
O2 ⫹ e⫺ ⫹ NADPH-oxidase 3 O2⫺ 3, 3
H2O2 ⫹ Cl⫺ ⫹ MPO 3 HOCl 3, 3 other ROIs
H2O2 oxidizes dihydrorhodamine to rhodamine when a
peroxidase or an equivalent catalyst is present. Flow
cytometric measurement of ROIs by the in vitro stimulation of cells loaded with dihydrorhodamine 123 (DHR) (2 )
was introduced by our group (3, 4 ). The DHR assay is
easier to perform, more reliable, and more sensitive than
the nitroblue tetrazolium dye reduction assay (5, 6 ), and
DHR is also more sensitive than other ROI-indicating
fluorescent dyes, such as dichlorofluorescein (7 ). DHR
oxidation is not strictly dependent on NADPH-oxidase
activity, however, because the oxidation requires (myelo-)
peroxidase availability (8 ). In spite of this limitation, the
DHR assay has been successfully used to (a) measure
transfused non-CGD neutrophils in CGD patients (9 ), (b)
measure X-inactivation ratios (lyonization patterns) in
X-linked CGD carriers (10 –16 ) even when small percentages of the normal X-linked genes are present (17 ), (c)
measure low concentrations of residual NADPH-oxidase
activity in CGD patients (10, 18 ), and (d) measure improved granulocyte activity in preclinical (19 ) and clinical
gene therapy trials (20, 21 ). In addition, the DHR assay is
superior to other methods, such as chemiluminescence,
for analyzing shipped blood samples (22, 23 ).
CGD is a life-threatening disease, and the diagnosis has
significant implications regarding therapy (24 ). It is therefore critical that the limitations of the DHR screening
diagnosis assay (and of all other diagnostic tests for CGD)
be understood and that confirmatory testing be considered when appropriate. It is in this context that we
demonstrate for the 1st time that complete MPO deficiency can lead to a false-positive result for CGD diagnosis using the DHR flow cytometry assay and describe how
MPO deficiency can be easily discerned from CGD.
Patients, Materials, and Methods
patients
We evaluated 4 patients with complete MPO deficiency, 2
with partial MPO deficiency, and 3 with CGD (2 without
and 1 with residual NADPH-oxidase activity) and compared them in each of the assays described below (Table
1). All patients and/or their parents consented to blood
drawing and genetic analysis.
materials
We purchased salicylhydroxamic acid (SHA) and 4-aminobenzoylhydrazide (ABAH) from Sigma-Aldrich and
TCI America, respectively, and DHR, lucigenin, horseradish peroxidase type VI-A, rhMPO, lactoperoxidase, phorbol-myristate-acetate (PMA), and zymosan A from SigmaAldrich. Transfected K562 cells that permanently express
gp91-phox, p47-phox, and p67-phox and have endogenous p22-phox were a kind gift from Dr. T. Leto, Laboratory of Host Defenses, National Institute of Allergy and
Infectious Diseases, NIH, Bethesda, MD.
flow cytometric dhr assay
K562 cells were adjusted to 109/L in PBS (137 mmol/L
NaCl, 10 mmol/L phosphate, 2.7 mmol/L KCl, pH 7.4),
as were neutrophils that had been isolated from lithium-heparin– containing whole peripheral blood by
dextran sedimentation followed by hypotonic erythrolysis or from buffy coat cell layers. We incubated all
cells in 500-␮L aliquots with 20 ␮L of 1.6 ␮mol/L PMA
for 5 min at 37 °C with or without rhMPO (1 kU/L) or
MPO inhibitors as indicated. Thereafter, we added 7.5
␮L of 30 ␮mol/L DHR followed by another 15-min
incubation period at 37 °C, storage on ice, and flow
cytometric analysis as described (22 ). Experiments performed in whole blood with the commercially available
Phagoburst reagent set (Orpegen Pharma) yielded similar results in all cases (data not shown).
Table 1. Genes affected and effects on functions in reactive oxygen metabolism as measured by DHR and LCL.
Patient
no.
1–3
4
Sex
Status
Mutation
2 M, 1 F MPO deficiency Ex12–2A3C/Ex12–
2A3C
F
MPO deficiency Ex12–2A3C/R569W
5
6
7
F
F
M
8
M
9
M
MPO deficiency Ex12–2A3C/A332V
MPO deficiency R569W/A332V
CGD
IVS6–1157A3G
X chromosome
CGD
962T3G and 970delG
X chromosome
CGD
delGly533
X chromosome
Gene
affected
MPO
MPO
MPO
MPO
CYBB
CYBB
CYBB
MPO activity
DHR signal in
neutrophils
None, e.g., Fig. 1, 2(s),
B and E
variable
None, e.g., Fig. 1, s Fig. 2A
B and E
Trace, Fig. 1F
Normal
Trace
Normal
Normal
s, Fig. 2C,
4D
Normal
Absent, Fig.
4C
Normal
Absent
DHR signal
enhanced by
rhMPO
LCL signal
Ref., effect
of mutation
Yes, n ⫽ 2,
Fig. 4A
ND
a, Fig. 2B
(28 )
a, Fig. 2B
(28–30 )
ND
ND
No, Fig. 4D
a
a
s, Fig. 2D
(28 )
(28, 29 )
(18 )
No, Fig. 4C
Absent
No
Absent
(24, 27 )
Ex (exon) 12–2A3 C (c0.2031–2A⬎C), splice mutation with no in-frame splicing; IVS6 –1157A3 G, complex splice mutation with residual normal splicing; 962T3 G
and 970delG, complex null mutation; s, strongly decreased, but present; a, strongly increased; ND, not done.
892
Mauch et al.: CGD and MPO Deficiency
modified dhr assay
We added 20 ␮L of 1.6 mol/L PMA or 20 ␮L of a 10 g/L
nonopsonized vortex-mixed zymosan A suspension and
20 ␮L of a 60 ␮mol/L DHR solution to 200-␮L cell
suspensions (2 ⫻ 106/mL) in a 96-well plate together with
different concentrations of MPO inhibitors as indicated.
We detected rhodamine fluorescence resulting from oxidized DHR fluorescence by excitation at 485 nm and
measured emitted light at 535 nm by use of the Mithras
LB 940-luminometer (Berthold Technologies).
lucigenin-enhanced chemiluminescence
Neutrophils isolated as described above or transfected
K562 cells were adjusted to 2 ⫻ 106/mL in PBS, divided
into 200-␮L aliquots, and preincubated with 4 ␮L of a 10
mmol/L solution of lucigenin at 37 °C for 30 min. We
added 20 ␮L of 1.6 ␮mol/L PMA or 20 ␮L of a 10 g/L
nonopsonized vortex-mixed zymosan A suspension, followed by 30 min of counting at 37 °C by use of the
Mithras LB 940-luminometer with or without MPO
inhibitors.
mpo activity
We detected and verified MPO deficiency by (a) the
Advia-120 hematology analyzer (25 ), (b) flow cytometry
after staining of permeabilized cells with the MPO-7
fluorescein isothiocyanate monoclonal antibody (mAb;
DakoCytomation, Kopenhagen, Denmark) according to
conventional techniques (Fig. 1), (c) staining of cell smears
with o-dianisidine modified from Fujinami et al. (26 ), and
(d) photometry. We verified inhibition of MPO activity by
SHA and ABAH in assays c and d.
Using primers to amplify individual exons including
intron boundaries of MPO (myeloperoxidase)10 and CYBB
(cytochrome b-245, also called gp91-phox) (18, 27 ), we
performed nucleotide sequencing of genomic DNA from
MPO-deficient patients and CGD patients.
Results
The DHR signal yielded by flow cytometry in the routine
diagnostic screening assay for CGD depends on intact
NADPH-oxidase activity as well as the presence of a
peroxidase such as MPO, as shown in Fig. 2. The DHR
fluorescence (Fl1) generated by PMA-activated neutrophils of the completely MPO-deficient patient (Table 1,
patient 1) was significantly below normal (Fig. 2A) and
was comparable to that of an X-CGD patient (Table 1,
patient 7) with some residual NADPH-oxidase activity
(Fig. 2C). Thus, complete MPO deficiency could be confused with CGD (false positive).
In the presence of complete MPO deficiency, a small
subpopulation of cells yielded a high DHR signal upon
activation with PMA (Fig. 2A, arrow). This subpopulation
consisted of eosinophils, which contain the same
10
Human genes: MPO, myeloperoxidase; CYBB; cytochrome b-245.
Fig. 1. Staining of MPO-deficient (A, B) and normal (C, D) neutrophils
with the MPO-specific mAb MPO-7 and position of completely (E) and
partially (F) MPO-deficient neutrophils in the Advia-120 display.
(A, C), isotype control; (B, D), addition of mAb MPO-7. Completely MPO-deficient
neutrophils localize in the upper part of the upper left field (E), whereas traces of
MPO activity are indicated by their localization in the field next to it (F). The line
depicting an oval shape marks the position where neutrophils with normal
amounts of MPO would normally localize if present. FSC, forward scatter.
NADPH-oxidase as neutrophils, but they contain eosinophilic peroxidase instead of MPO.
Measurement of lucigenin-enhanced chemiluminescence (LCL) indicated that the signals were significantly
increased (consistent with normal NADPH-oxidase) in
the MPO-deficient patient (Fig. 2B) but were significantly
decreased from normal in patients with a confirmed
diagnosis of CGD (Fig. 2D).
We confirmed MPO deficiency in patient 1 by use of
mAb staining (Fig. 1, A and B), the Advia-120 automated
hematology analyzer (25 ) (Fig. 1E), and o-dianisidine
staining (data not shown). Three additional patients were
confirmed to be completely MPO deficient (Table 1,
patients 2, 3, and 4) by 2 or 3 of these methods.
We confirmed the mutations in the gene leading to
MPO deficiency by genomic sequencing (Table 1). We
detected the frequent exon12–2A3 C splice mutation
(c.2031–2A⬎C) in 8 alleles, and the A332V and R569W
missense mutations in 2 alleles each. None of the patients
were related. The c.2031–2A⬎C and the R569W mutation
have been published as null mutations, i.e., no residual
MPO activity was detected (28 –30 ). The neutrophils iso-
893
Clinical Chemistry 53, No. 5, 2007
Fig. 2. DHR assay (left panel) and LCL (right panel) on granulocytes
from a completely MPO-deficient patient (A, B), a CGD patient (C, D),
and a healthy blood donor (E).
(A, C, E), gray lines, no stimulation; black lines, maximal stimulation with PMA;
arrow, PMA-activated eosinophils. (B, D), black symbols, patients; open symbols,
normal, healthy blood donor; diamonds, maximal PMA activation; triangles,
activation with zymosan A; background on abscissa (crosses), no stimulation.
Note the different scale (right ordinate).
lated from all of the patients with complete MPO deficiency generated variable but significantly decreased
PMA-induced DHR fluorescence signals. Traces of detectable MPO resulted in the generation of normal DHR
fluorescence (Table 1).
When we added the MPO inhibitors SHA and ABAH
to suspensions of normal neutrophils and performed a
modified DHR flow assay (Fig. 3), both inhibitors markedly decreased the zymosan A–induced DHR fluorescence signal (Fig. 3A). The inhibition was not attributable
to cell damage or cell death, because the same concentrations of the inhibitors actually enhanced the LCL signal
(Fig. 3B). Fluorescence in these experiments was measured by luminometry, because zymosan particles interfered with the light scatter detection of cells by flow
cytometry. Experiments combining PMA, SHA, and
ABAH were inconclusive, presumably owing to their
combined toxicity.
K562 cells containing no endogenous peroxidase and
transfected to express gp91-phox, p47-phox, and p67phox [and having endogenous p22-phox (31 )] provided a
model to investigate the peroxidase dependency of PMAinduced DHR oxidation by H2O2. The transfected components of NADPH-oxidase allowed for the production of
high amounts of ROIs, as demonstrated by a strong LCL
signal (31 ), but yielded no PMA-induced DHR oxidation,
as demonstrated by the lack of increase in the fluorescence
signal (Fig. 4B). The addition of rhMPO, however, led to a
significant increase in the intensity of DHR fluorescence
following in vitro activation. A similar result was observed in patients with the false-positive CGD DHR result
who had no residual MPO activity (Fig. 4A; Table 1).
Addition of rhMPO enhanced the DHR signal in the
Fig. 3. Effect of MPO inhibitors on DHR oxidation (A) and LCL (B) in
normal neutrophils.
(A), fluorescence of rhodamine 123 (oxidized DHR). (B), LCL counts in percentage of uninhibited samples (ordinate). Abscissa, concentration of inhibitors;
open columns, ABAH; striped columns, SHA; error bars, SD; 15 independent
experiments.
MPO-deficient patients’ neutrophils, thus providing a
clear discrimination between MPO deficiency and CGD.
As shown in Fig. 4, addition of rhMPO did not change the
DHR signal in CGD patients with (Fig. 4D) or without
(Fig. 4C) detectable residual NADPH oxidase activity.
We detected considerable variability in the DHR fluorescence of the complete MPO-deficient patients (as well
as in different samples from 1 individual, compare Figs. 2
and 4). Therefore, the DHR assay may not be reliable in
detecting MPO deficiency.
In contrast to rhMPO, the addition of horseradish
peroxidase (2 different forms) or lactoperoxidase led to an
increase in DHR fluorescence even in the absence of a
functional NADPH-oxidase (data not shown). Therefore,
such peroxidases should not be used to investigate MPO
deficiency in the DHR assay.
Discussion
The incidence of MPO deficiency is estimated to be ⬃1 in
2000 individuals. Although the MPO system is highly
conserved in the evolution of mammals, and decreases in
the early stages of in vitro killing of certain bacteria, such
as Staphylococcus aureus, by MPO-deficient neutrophils
have been observed, the vast majority of MPO-deficient
individuals in industrialized countries do not exhibit
signs of overt immunodeficiency (32 ). One study de-
894
Mauch et al.: CGD and MPO Deficiency
Fig. 4. Addition of rhMPO can enhance the DHR signal in MPO-deficient
cells (A, B) but not in cells from CGD patients (C, D).
Gray lines, DHR and rhMPO alone (position never deviated from cells incubated
with DHR alone; omitted for clarity); dashed lines, activation with PMA, no MPO;
black lines, activation with PMA in the presence of rhMPO. Abscissa, DHR
fluorescence. (A), MPO-deficient patient 1; (B), K562 cells with functional
NADPH-oxidase, representative result of 4 experiments; (C), CGD neutrophils
without any NADPH-oxidase activity; (D), CGD neutrophils with residual NADPHoxidase activity.
signed to avoid disease detection bias reported a significantly enhanced frequency of severe, though heterogeneous, infections in MPO-deficient individuals (33 ), but
such events were rare. An association of MPO deficiency
with Candida infections in diabetic patients has been
described, and associations with decreased or increased
frequencies of noninfectious diseases have also been reported (32, 33 ). It remains possible that the MPO system is
a very important host defense system when the host is
challenged by high doses of infectious agents.
In contrast to defects in the MPO gene, mutations in the
genes encoding the components of NADPH-oxidase result in CGD, a life-threatening diagnosis that demands
appropriate clinical management and therapeutic decisions (24, 34 ). Life-threatening conditions include both
severe infections and uncontrolled inflammation associated with granuloma formation even in the absence of
overt infection. In comparison with classic CGD, CGD
with residual NADPH-oxidase activity is usually less
severe and can manifest at an older age, with less severe
infection and longer intervals between infections. This
does not apply to all such patients, however, and manifestations can be as severe as in classic CGD. Because of
the dire consequences of a CGD diagnosis, it is imperative
that MPO deficiency not be falsely diagnosed as CGD.
As indicated in this study, complete MPO deficiency
can lead to a significantly reduced fluorescence signal in
the DHR assay that is consistent with the results observed
in some CGD patients (Fig. 2). In contrast, cells obtained
from partial MPO deficiency containing only minimal
residual MPO activity generated fluorescence signals consistent with normal (non-CGD) individuals (8, 22 ). This
was confirmed in patients 5 and 6, whose neutrophils
contained only trace amounts of MPO but generated
PMA-induced DHR fluorescence signals consistent with
those of control individuals (Table 1).
Our investigation provides direct evidence that the
oxidation of DHR by H2O2 depends on a peroxidase in
cellular systems (Figs. 2A and 4, A and B). These results
are in accordance with those of inhibitor experiments of
MPO (Fig. 3) and the results of van Pelt et al. (8 ). As
expected, eosinophilic peroxidase can replace MPO in
mediating DHR oxidation (Fig. 2A, arrow). Most patients
will have detectable eosinophils, although at low frequency in peripheral blood. Therefore, strong DHR staining of eosinophils after activation with PMA is an extremely facile method to exclude CGD. It is important,
however, to discern eosinophils from cell aggregates in
the forward/side scatter diagram. It is also important not
to confuse a subset of normal ROI-producing granulocytes present in X-linked CGD carriers (that can be small;
see Introduction) (35, 36 ).
Identification of eosinophils by flow cytometry can be
challenging and may not always allow for differentiation
between CGD and MPO deficiency. In such cases, alternative methods can be adopted for the rapid identification
of false-positive CGD due to complete MPO deficiency.
The simple addition of rhMPO to the assay (Fig. 4) enables
a very rapid and clear discernment between MPO deficiency and CGD, as cells from patients with true CGD will
not be affected by rhMPO. MPO deficiency can be clearly
demonstrated using a monoclonal anti-MPO antibody
with conventional intracellular staining. MPO is strongly
expressed in granulocytes, less so in monocytes, and not
at significant levels in lymphocytes, thus providing
built-in staining controls.
Clinical Chemistry 53, No. 5, 2007
Finally, all measurements of ROIs in neutrophils that
use indicators independent of MPO yield considerably
enhanced readouts in the presence of MPO deficiency.
This is attributed to a regulatory effect of MPO, the
products of which are presumed to down-regulate
NADPH-oxidase (37 ). In the experiments shown here,
LCL was used to measure O2⫺ independent of MPO. As
demonstrated in Fig. 2B, the O2⫺ production plateaus at a
level more than 3 times higher in MPO-deficient neutrophils than in those cells obtained from non-CGD control
blood donors (Fig. 2, B and D). Another possible complementary assay is luminol-enhanced chemiluminescence
(38 ) comparing luminol oxidation by PMA-activated neutrophils in the absence and presence of horseradish peroxidase (1.2 kU/L). In the case of MPO deficiency, but not
in the case of CGD, the added peroxidase strongly enhances luminol oxidation (unpublished results), a reaction
that parallels the effect of rhMPO in the DHR assay
(Fig. 4).
We do not recommend the DHR assay as a screen for
MPO deficiency because of the variable results observed
and the lack of sensitivity in detecting partial MPO
deficiency. It is possible that there are redundant peroxidases that may substitute for MPO, and it has been
reported that DHR may be less dependent on MPO than
dichlorofluorescein diacetate (39 ).
In summary, it is important to consider complete MPO
deficiency when a result consistent with CGD is obtained
using the DHR assay, especially if patient history is not
consistent with a diagnosis of CGD. It is easy to discriminate between the 2 enzyme deficiencies by (a) consideration of eosinophils, (b) addition of rhMPO and observation of the effect on the DHR-123 assay, (c) intracellular
staining with anti-MPO antibody, and/or (d) the use of
MPO-independent ROI detection methods such as LCL.
In cases in which a diagnosis of CGD is suspected, we
highly recommend that confirmatory tests (sequencing or
others) be performed.
Grant/funding support: None declared.
Financial disclosures: None declared.
Acknowledgements: We thank Katrin Höhne for excellent
assistance; Dr. Thomas Leto, NIH, Bethesda, MD, for the
transfected K562 cells; and Andrea Gross for excellent
graphic design.
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