Page 1317 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPtEr 94 Assessment of Neutrophil Function 1279

Neutrophils isolated from patients with CGD produce little, if
Principles and Interpretation of any, O 2 in response to PMA in 10 minutes (Fig. 94.6). However,
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Laboratory Assessments some patients with autosomal forms of CGD have low, but
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The nitroblue tetrazolium (NBT) test is a qualitative assay of detectable O 2 production in 60 minutes. Neutrophils isolated
ROS production. Either whole blood or isolated neutrophils are from X-linked heterozygous carriers of CGD can yield a full
•
mixed with NBT in a chamber slide and stimulated with PMA spectrum of O 2 production, whereas neutrophils from autosomal
for 15–30 minutes at 37°C. The neutrophils are allowed to recessive carriers of CGD generally yield a normal response (see
•
settle on the slide. The slide is air-dried, counterstained with Fig. 94.6). Although the detection of O 2 by reduction of cyto-
0.1% safranin, and examined under a microscope. The NBT chrome c is useful in the diagnosis of patients with CGD, it
test yields a visual record of the reduction of the NBT dye to cannot be used in the diagnosis of carriers because of the wide
the insoluble, blue-black deposits of formazan. Normal neutro- spectrum of responses that result from the degree of X chromo-
phils, but not neutrophils from patients with CGD, reduce the some lyonization.
yellow dye to black-brown-blue aggregates in the cells. Because
of the random inactivation of the X chromosome, X-linked
−
+
carriers of CGD exhibit both NBT and NBT neutrophils. The KEY COnCEPt
+
percentage of NBT neutrophils in X-linked carriers of CGD Reactive Oxygen Species
ranges from 5 to 95%. The drawback of the NBT test is the need
for manual counting to obtain an accurate reflection of the 2H +
percentage of positive cells. An alternative to the NBT test is a e − e − e − e −
flow cytometric assay using the dye dihydrorhodamine–123 ↓ ↓ ↓ ↓
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(DHR-123). Neutrophils are loaded with the nonfluorescent − • −
dye and then stimulated with PMA for 15 minutes at 37°C. The O 2 ↓ O 2 ↓ H 2 O 2 ↓ OH +OH ↓ H 2 O+ 1 / 2 O 2
H 2 O 2 produced oxidizes the dye and results in increased fluo- Oxygen Superoxide Hydrogen Hydroxyl Water
rescence, which is detectable with a flow cytometer. The assay anion peroxide radical
is dependent on endogenous myeloperoxidase in the primary
The stepwise reduction of oxygen (O 2 ) leads to reactive oxygen species
granules. Catalase is added to prevent cell-to-cell diffusion of and finally to water (H 2 O).
H 2 O 2 . Since dye is localized to the cytoplasm and catalase is
present in the extracellular fluid, the DHR-123 assay detects the
intracellular production of ROS. As shown in Fig. 94.5, stimula-
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tion of normal neutrophils (Fig. 94.5A) with PMA results Studies have shown that O 2 determinations sufficiently
in a two-log shift in the fluorescence intensity. Neutrophils from reliable to diagnose CGD can be obtained from neutrophils
an X-linked carrier of CGD (see Fig. 94.5B) exhibit mosaicism isolated from heparinized whole blood that has been stored
with a negatively stained (abnormal) population and a brightly overnight. Hence, analyses can be performed on blood samples
stained positive population. Neutrophils from a patient with shipped by overnight express. A normal control blood sample
X-linked CGD that lack gp91 phox (Fig. 94.5C) express little should accompany the sample to ensure adequate shipment
increase in fluorescence while neutrophils from a patient with handling. By 48 hours of storage, however, there are marked
a deficiency in p47 phox (see Fig. 94.5D) exhibit a slight increase reductions in the PMA response, and the data are no longer
in fluorescence. The major advantages of the DHR-123 assay valid.
are the sensitivity, the signal-to-noise ratio, and the ease of An alternative assay to measure ROS production is luminol-
counting a large number of cells. Moreover, it has been shown enhanced chemiluminescence. This versatile assay, in addition
that the DHR-123 assay yields reliable results on ethylenediami- to its quick and easy setup, offers the capability to test several
netetraacetic acid (EDTA) or heparin-treated blood samples that different subjects and stimuli on the same plate using reduced
have been stored overnight. In general, more than 90% of numbers of cells while providing high sensitivity. The lumines-
the neutrophils from the control blood samples will exhibit cence readings are taken every 1–5 min for up to 2 hours, and
increased DHR-123 fluorescence. For this same reason, however, data are expressed as relative light units (RLUs). Different stimuli
overnight samples should not be used to rule out X-linked exhibit different kinetics (e.g., fMLF induces a rapid respiratory
heterozygosity, since a highly lyonized CGD carrier (>90%) could burst that decays quickly, whereas PMA [20–100 ng/mL] induces
yield similar results. a peak of luminescence within 5–15 minutes that slowly decays
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The production of O 2 can be detected using the reduction by 2 hours). Results from normal subjects and patients can be
•
of cytochrome c. Because O 2 causes a one-to-one stoichiometric assessed simultaneously and monitored kinetically or using the
reduction of ferricytochrome c to ferrocytochrome c, the resul- area under the curve (AUC). Luminol-enhanced chemilumines-
tant increase in the absorption spectrum at 550 nM can be used cence is a measure of both intracellular and extracellular ROS
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to quantitate the production of O 2 . Superoxide dismutase is production, although it may not detect them with equivalent
added to an identical tube to control for the nonspecific reduc- efficiency. Addition of superoxide dismutase significantly reduces
tion of cytochrome c. However, since cytochrome is not perme- both peak height and AUC after stimulation with PMA, suggesting
•
able to the cells, the detection of O 2 is limited to that released that at least a portion of the response can be attributed to
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into the extracellular milieu. Neutrophils isolated from normal O 2 . Typically, patients with CGD who are deficient in gp91 phox
6
volunteers produce 0.42 ± 0.67 nmol/10 neutrophils/10 min have little to no detectable luminescence in this assay; however,
under resting conditions; treatment with PMA results in 35.92 as observed with the ferricytochrome c assay, patients with CGD
6
± 11.92 nmol/10 neutrophils/10 min (Fig. 94.6). An estimate who are deficient in p47 phox have detectable luminescence
•
of normal O 2 production over 60 minutes can be obtained by that becomes most evident at later readings (40–80 minutes)
5
reducing the number of neutrophils in the assay to 2 × 10 . (Fig. 94.7).