Page 1331 - Clinical Immunology_ Principles and Practice ( PDFDrive )

1292 Part ElEvEn Diagnostic Immunology

Recommended serum collection times for tryptase quantifica- assay is not sufficiently sensitive for effective clinical use in the
tion range from 30 minutes to 4 hours after the onset of an diagnosis of IgE-mediated sensitivities to β-lactam or nonsteroidal
acute event. Because serological tests for mature tryptase are not antiinflammatory drugs (NSAIDs). Its utility in the diagnosis
widely available, it is important to compare an acute event total of sensitization to other allergen specificities appears more
tryptase level (within 4 hours) with a baseline total tryptase 24 promising, but further documentation involving clinical studies
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hours after all signs and symptoms of the event have subsided. is required.
Postmortem specimens are often difficult to analyze for tryptase
because of high viscosity related to gross hemolysis. However, Utility of Mediator Release Assays as Diagnostic Tests
reported mature tryptase levels in postmortem cases of fatal Despite their unquestioned value as research methods, the basophil
anaphylaxis have ranged from 12 to 150 µg/L in all nine fatalities histamine and LTC4 release assays are rarely used clinically in
caused by Hymenoptera venom and in six of eight food-induced the routine diagnosis of human allergic disease, and it is unlikely
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fatalities. The frequency of a severe reaction to venom from that this trend will change in the foreseeable future.
a Hymenoptera sting increases significantly with higher serum
baseline total tryptase concentrations. 68,69 The clinical indications Flow Cytometry Basophil Activation Assays
for and diagnostic value of tryptase as a marker of anaphylaxis In the late 1990s, basophils were shown to upregulate the expres-
and mastocytosis are reviewed elsewhere. 68 sion of a number of surface proteins (e.g., CD45, CD63, CD69,
and CD203c) when activated by allergen. 76-78 CD63 is a member of
Basophil Mediator Release Assays the transmembrane-4 superfamily that is expressed on basophils,
There are a number of diagnostic assays for sensitization that mast cells, macrophages, and platelets. 76,77 In resting basophils
use the basophil as the indicator modality. Some assays monitor from individuals without atopia and allergies, CD63 is attached
the magnitude of mediator release from basophils, and others to intracytoplasmic granules. Activated IgE-sensitized basophils
assess the degree of cell surface marker expression following express a high density of CD63 on their surface. By quantifying
exposure to allergen. ~500 basophils with gated flow cytometry, the percentage of
activated basophils can be identified, after correcting for spontane-
Histamine Release Assay ous CD63 expression. A variety of criteria are used to identify an
The potent vasoactive mediator histamine is stored in cytoplasmic allergen-induced positive response, such as a stimulation index
granules of basophilic leukocytes and mast cells. It is released (allergen-induced/basal ratio) >2. One confounding variable in
along with other mediators of inflammation in response to both this analysis is the adherence of CD63-expressing platelets in
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immunological and nonimmunological stimuli. The basophil basophil preparations that can sometimes generate false-positive
histamine release (BHR) assay has been particularly useful as a results.
quantitative assay of allergen potency and as an in vitro model The flow assay stimulation test (FAST), which is also referred
for the study of triggering mechanisms of mediator release from to as the basophil activation test (BAT) or BasoTest, uses CD63
basophils. In its most basic form, peripheral blood leukocytes as a marker for activation. Its performance was evaluated by
are isolated from a donor and incubated with varying concentra- using minor determinant mixture, benzyl penicilloyl polylysine,
tions (e.g., 3–10-fold dilutions) of allergen extract or antihuman penicillin, ampicillin, amoxicillin, and cephalosporin as stimulat-
IgE as a positive control. Histamine release is complete within ing antigens. 78,83 Subjects (n = 58) with a history of immediate-type
30 minutes, and then histamine in the supernatant is measured reactions to β-lactam antibiotics or nonallergic controls (n =
by enzymatic, radiometric, or spectrophotofluorometric tech- 30) were evaluated in the CD63 FAST/BAT and Phadia Immu-
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niques. Details of the BHR assay are given elsewhere. 70,72 noCAP system for drug-specific IgE levels. Relative to the clinical
Patient sensitivity for a given allergen can be determined with history, the CD63 FAST/BAT displayed a diagnostic sensitivity
a positive BHR test. The results are highly correlated with those and specificity of 50% and 93%, respectively. Diagnostic sensitivity
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determined by skin testing and bronchoprovocation. Although was marginally increased to 66% by simultaneously using the
the BHR test has been almost exclusively used in research labo- drug-specific IgE antibody serology result. The study concluded
ratories because of its expense, time-consuming nature, and the that the CD63 basophil activation test could be helpful in sup-
need for fresh blood (<24 hours old), it can be successfully applied porting the diagnosis of IgE-mediated allergy to β-lactam drugs
to the clinical diagnosis of allergic disease in selected cases. Its when used in conjunction with an additional diagnostic test,
results parallel those of other IgE antibody tests. BHR has also such as IgE antibody serology. 79
been a useful tool for clarifying discrepancies between skin test CD203c (also known as neural cell surface differentiation
and serological IgE antibody test results. antigen, E-NIPP3 PD-1β, 97A6, B10, and gp130rb13–6) is a
member of the ectonucleotide pyrophosphate phosphodiesterase
Leukotriene C4 (LTC4) Release Assay (E-NPP) family. It is expressed only on IgE-bearing basophils,
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An assay method for measuring LTC4 released from allergen- mast cells, and their progenitors and is upregulated after activation
activated basophils has been reported as the cellular antigen of IgE-sensitized basophils with allergen or anti-IgE in a manner
stimulation test (CAST)–enzyme-linked immunosorbent assay similar to CD63. The CD203c-based flow assay is performed in
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(ELISA). The LTC4 assay is designed for use with either a manner analogous to the CD63-based assay. Its advantage over
whole blood preparations or washed leukocytes. Using dust-mite, CD63 is its restricted expression on basophils in peripheral blood,
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food, Hymenoptera venoms and drugs as challenge allergens, which minimizes concern about the adherence of platelets that
the observed diagnostic sensitivity of the CAST compared with may produce false-positive CD63 results. Additionally, there is no
the combination of a clinical history and skin test ranged from need for the use of an additional fluorescent anti-IgE reagent to
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18% with aspirin and 50–85% for selected food allergens. The gate the basophils. Basophil activation as determined by CD203c
reported diagnostic specificity of the CAST in the same studies expression was studied in patients with allergy to Hymenoptera
ranged from 67% to 100%. These data indicate that the CAST venom and healthy controls in which their basophils were