First Aid, Chapter 4 Laboratory Tests, Mediator Detection Flashcards
What is the source of leukotrienes, and which leukotrienes, during IgE and non-IgE mediated reactions?
Basophils are the main source of LTC4, LTD4, and LTE4 during IgEmediated reactions, as well as during some non-IgE-mediated reactions (e.g., nonsteroidal anti-inflammatory drug [NSAID] hypersensitivity).
How is the basophil activation test done?
Basophil activation test uses IL-3-primed basophils that are stimulated by allergens in vitro. The release of LTC4, LTD4, and LTE4 is measured by ELISA.
What does the cellular allergen stimulation test (CAST) measure? Is it sensitive/specific?
This test became available commercially in 1993 and provides a quantitative measurement of leukotriene release. CAST demonstrates high sensitivity but low specificity for diagnosis of IgEmediated allergy to inhalant allergens.
What other test can the cellular allergen stimulation test CAST test be used in correlation with?
May be used in conjunction with the flow cytometric basophil activation test (FAST), which analyzes surface expression of CD63 on basophils following allergen stimulation.
What percentage of people with stinging insect allergies have negative skin tests? What testing can be used in this scenerio?
Up to 33% of patients with stinging insect allergy proven by provocation have negative skin tests. The combined CAST and FAST test appears to have high diagnostic efficiency in this scenario.
What method used in the future may be used to overcome the problem of mixed results of various studies of the CAST assay?
The mixed results found in various studies may be due to the lack of standardized allergens used and the varying dose of allergen used to perform the CAST assay. The future use of recombinant allergens may overcome these problems.
What are drawbacks of the histamine release test?
Although the histamine release test (HRT) has been used as a research tool for 40 years, it has not been applied routinely in laboratory diagnosis, mainly because it is relatively cumbersome and not very sensitive. In principle, it is very similar to CAST and shows good correlation with it; however, it has been found to be less sensitive than CAST. An additional drawback is that histamine release may be nonspecifically high (often observed in food allergy) and lead to false positives in cases of allergen cytotoxicity and/or following recent in vivo allergen exposure.
How are interleukins and TNF measured?
Measured using commercially available sandwich ELISA.
How are prostaglandins measured?
Measured using radioimmunoassay (RIA).
How is TLR expression measured? How is TLR functionally measured?
- Real-time reverse transcription polymerase chain reaction (RT-PCR) can be performed on ribonucleic acid (RNA) samples using commercially available TaqMan assays for TLR1 through TLR10.
- Flow cytometry can also be performed to analyze cell surface and intracellular expression of TLR1 through TLR10.
- Functional Study: The responsiveness of cells to TLR stimulation can be assessed using incubation with a panel of ligands for TLR1 through TLR9, followed by measurement of inflammatory molecule production by real time RT-PCR, ELISA, and flow cytometry.
How is Fas (CD95 or Apo 1) measured?
- Fas and FasL expression on the cell surface can be measured using flow cytometry.
- sFas and sFasL can be measured via sandwich ELISA using monoclonal human antibodies.
How is apoptosis quantified?
Several assays quantify apoptosis. Labeling of DNA strand breaks by the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) reaction is the most specific of all the methods tested.
How is bradykinin measured?
RIA may be used for the measurement of plasma bradykinin levels.
