Page 812 - Hematology_ Basic Principles and Practice ( PDFDrive )

698 Part VI Non-Malignant Leukocytes

TABLE Chronic Conditions Associated With Chronic Diagnosis of Chronic Granulomatous Disease
50.4 Granulomatous Disease a
The diagnosis of CGD is easily established by doing an NBT slide
Condition Relative Frequency (%)
test or flow cytometry of dihydroxyrhodamine (DHR) 123 fluorescence
Lymphadenopathy 98 to detect neutrophil NADPH oxidase activity. The NBT slide test is
very easy to set up, as is DHR flow cytometry. However, because
Hypergammaglobulinemia 60−90
the probability of getting an abnormal result is very low, there may
Hepatomegaly 50−90 be confusion in interpretation because of a lack of experience. In
the authors’ experience, incorrect positive and negative results have
Splenomegaly 60−80
been reported for both assays. Thus, if the index of suspicion is
Anemia of chronic disease Common † high, consultation should be obtained from a center with extensive
Underweight 70 experience with the test and with the disorder.
Neutrophil respiratory burst activity is preserved in anticoagulated
Chronic diarrhea 20−60 blood maintained at room temperature for several days; thus, DHR
Short stature 50 testing can be done 1 to 2 days later after shipping to a commercial
laboratory. A normal blood sample should always be shipped with
Gingivitis 50 the patient specimen as a control for problems in specimen handling
Dermatitis 35 during transport.
Hydronephrosis 10−25 NBT Slide Test
Granulomatous ileocolitis 10−15 • No NBT reduction (absence of cells with dark blue formazan
deposits) in both X-linked and AR forms of CGD (see Fig. 50.5B).
Gastric antral narrowing 10−15 • Usually no reduction in 50% of cells and normal in 50% for
Ulcerative stomatitis 5−15 X-linked carrier. The percent positive cells can vary if there is
unequal X inactivation and may appear normal or like CGD with
Granulomatous cystitis 5−10 b extreme lyonization (see Fig. 50.5C).
Pulmonary fibrosis <10 b • False-positive results can occur (i.e., apparent failure to reduce
NBT supporting the diagnosis of CGD) if the neutrophils do not
Esophagitis <10 b
adhere to the slide. This happens with greasy slides or with some
Granulomatous cystitis <10 cases of LAD. Using phorbol myristate acetate to stimulate the
cells will avoid this.
Chorioretinitis <10
Glomerulonephritis <10 DHR Flow Cytometry
• This approach has replaced the NBT slide test in many
Discoid lupus erythematosus <10
laboratories. It has the advantage of assessing large numbers
a The relative frequencies of chronic conditions associated with chronic of cells and can give quantitation of the amount of oxidant
granulomatous disease (CGD) were estimated from the series of reports listed in production.
Table 50.3. • The change in fluorescence channel number with stimulation is
b The incidence is estimated from the 50 cases of CGD followed at Scripps the critical number and not the percent positive cells.
Research Institute and Stanford University (unpublished data).
• X-linked CGD patients will not respond at all and show no
increase in fluorescence with stimulation (see Fig. 50.5F).
• X-linked carriers will show about 50% of the cells that respond
lupus nor does one find serologic evidence of even subclinical disease. with a normal increase in fluorescence, and the other half will
Those with severe discoid lupus can be treated with Plaquenil. Recur- have no response. Degrees of unequal X inactivation are much
rent stomatitis, significant gingivitis, or both have also been noted in more accurately quantified by this assay (see Fig. 50.5G).
phox
as many as half of X-CGD carriers. A few also have arthralgias, • AR patients, particularly those with absent p47 , have
polyarthritis, and Raynaud phenomenon. The second important some response to stimulation and show a small increase in
fluorescence (see Fig. 50.5H). This level of oxidant production is
complication of the X-linked CGD carrier state is serious infection usually not visible on the NBT test.
in women who have an unusually high degree of inactivation of the • AR carriers have a good response, but the histogram may be
normal X chromosome in their myeloid cells. If the circulating broader than normal and may even appear bimodal with a weakly
neutrophil population is skewed to the point that fewer than 10% to fluorescent peak and a strongly fluorescent peak. This is not
15% of the cells function, then the carrier has an increased risk of distinguishable on the NBT slide test.
bacterial infections that in some cases have been severe. 3 • Falsely negative results not supporting the diagnosis of CGD have
been reported in specimens that have been run a few days after
phlebotomy.
Diagnosis • Falsely abnormal results suggesting CGD can be seen in patients
with MPO deficiency because MPO is required to generate strong
DHR fluorescence.
The diagnosis of CGD is usually suggested by the unusual clinical
histories outlined earlier or by a family history of CGD. The NBT Genetic Analysis
slide test on fresh blood is the classic diagnostic test. A typical result • Genetic analysis for X-linked and AR CGD is clinically available
is shown in Fig. 50.5. Fig. 50.5A shows the normal positive staining and should be performed on at least the proband in each
of a group of seven neutrophils and one monocyte. Fig. 50.5B shows kindred.
the complete absence of NBT staining in a patient with X91° CGD, Those with fewer than 5% oxidase-positive cells have full-blown CGD.
the classic X-linked form of the disease. Fig. 50.5C shows the mixed
population of NBT-positive and NBT-negative cells observed in that
patient’s mother, reflecting random X chromosome inactivation. conversion of dihydroxyrhodamine (DHR) 123 to rhodamine 123,
Because nearly 100% of the normal cells in this test are positive, the can also provide both quantitative measurements of oxidant genera-
carrier state in X-linked CGD can be detected when as few as 5% of tion and the cell-by-cell distribution of activity (see Fig. 50.5D–G).
the cells are NBT negative. This test also permits detection of diffuse The DHR 123 assay for oxidase activity is now available in many
−
populations of weakly positive cells such as those seen in X91 CGD, referral centers and through reference laboratories. In addition to
−
which are characterized by a partial deficiency of flavocytochrome b. X91 CGD neutrophils, weak staining in the NBT test or a small
Because X-linked CGD can arise by new mutations in the maternal but measurable level of DHR fluorescence can be seen in A47° cells
germ line, one does not always see NBT-negative cells in the mother. (see Fig. 50.5H) because of a small amount of residual oxidant pro-
Flow cytometric assays of oxidase activity, such as those based on the duction. Regardless of diagnostic assay used, is important to have