Allergy Tests



Allergy Tests






Introduction

The techniques used for allergy diagnosis in vitro are those already discussed in Chapters 17 and 18, and include RIA and EIA. Flow cytometry, as discussed in Chapter 20, may also have a role. The technical principles are identical.

In vitro allergy tests do not substitute for clinical history-taking, examination, and direct patient testing (SPT, patch tests, challenges).


Allergen-specific IgE



  • Units: kAU/mL.


  • Range:



    • <0.35 = RAST score 0


    • 0.35-0.70 = RAST score 1


    • 0.70-3.50 = RAST score 2


    • 3.50-17.5 = RAST score 3


    • 17.5-50.0 = RAST score 4


    • 50.0-100 = RAST score 5


    • >100 = RAST score 6


Principles of testing



  • Detection of allergen-specific IgE in the blood is by sensitive RIA, EIA, or fluorescent assay. There are numerous acronyms for this process, depending on the method (RAST, MAST, FAST, etc.).


  • Radio-immunoassay is now little used.


  • Principles of all the tests are identical, with allergen bound to a solid phase that is then incubated with a labelled anti-IgE antibody.


  • The Pharmacia UniCAP/ImmunoCAP system is the most widely used system in the UK.


  • Tests are expensive due to costs of purification and standardization of allergen.


  • CE marking requirements have reduced the number of commercially available allergens.


  • EQA and international standards are available.


Indications for testing



  • Skin-prick testing (SPT) remains the gold standard.


  • In vitro EIA assays may be used for patients with a high risk of anaphylaxis (SPT contraindicated), drugs that interfere with SPT (antihistamines, calcium-channel blockers, antidepressants), patients with extensive skin disease, and small children.


  • Patients with a total IgE <20kU/L have a low (but not zero) probability of having positive specific IgE tests.


Interpretation



  • The assays are carried out quantitatively and reported in units.



  • There is a trend to reporting units rather than grades, although many laboratories will still report both.


  • RAST grades of 0 and 1 are usually considered to indicate a negative result.


  • High total IgE levels (>1000kU/L) may cause false-positive tests for allergen-specific IgE, because of non-specific binding of IgE to the solid phase. This is less common with newer systems, but applies particularly to cross-reactive food allergens.


  • Units are standardized against an international standard for birch pollen allergen, but this cannot validly be applied to reactions with other allergens.


  • Attempts are being made to standardize allergens in terms of defined proteins and protein nitrogen. With a potentially limitless list of allergens, this is a slow process.


  • The important clinical implication is that the detection of a grade 3 response to two different allergens does not indicate that the same amount of IgE is present against both and that equivalent clinical reactions might be expected.


  • There is no close relationship between the grade and the severity of reactions (either past or future).



    • The presence of allergen-specific IgE is a marker only of exposure.


    • Positives may be detected where there is no evidence of any clinical reaction.


  • Levels will fall with time if the offending allergen is avoided over a long period, so low or negative results may be obtained even with sensitized patients.


  • As with skin-prick tests, if the allergic reaction is highly localized, there may be insufficient spillover of specific IgE into the circulation to be detected, leading to a false negative.


  • Results must always be interpreted in the light of the clinical history (blood tests are not a substitute for proper history-taking!).


Limitations



  • Few allergens are available for robustly identifying IgE to drugs.


  • Reagents for penicillin contain major but not minor determinants. A negative result does not exclude significant allergy, and SPT and IDT are required with a minor determinant mixture.


  • Reagents for labile food allergens such as fruits are unreliable. Use SPT with the fresh fruit.



    • Recombinant allergens may assist in the diagnosis, where available (see Table 19.1 and Chapter 3).


  • If there is a good history for type I food allergy and an unexpected negative result, consider SPT with fresh food.


  • 15% of NRL-allergic patients will be negative by specific IgE testing.


Serial monitoring



  • Justified in cystic fibrosis: screen for IgE to Aspergillus (marker of colonization and associated with worse prognosis).



Component resolved diagnosis



  • Key recombinant allergens are now available for the Phadia systems (Table 19.1).


  • Other recombinant food and inhalant allergens are available, but the precise clinical roles of many still await further clinical studies.


  • There may be considerable differences between the results obtained using tests for whole foods compared with those obtained using recombinant allergens. For example, the standard soybean ImmunoCAP contains very little of the recombinant allergen rGly m4 (PR10), an important allergen which is a Bet v1 homologue.


Website



  • The following link gives details of all available Phadia Allergens with details of their composition and significance.


  • http://www.phadia.com/en-GB/Allergens/ImmunoCAP-Allergens/








Table 19.1 Key recombinant allergins for Phadia systems

































Recombinant allergens


Substance


Utility


rTri a19


Wheat omega-5-gliadin


Diagnosis of wheat-dependent exercise-induced anaphylaxis


rAra h1, h2, h3, h8, h9


Peanut allergens


Positives to h2 associated with severe clinical symptoms; responses to h8 and h9 associated with OAS symptoms


rBet v1 (PR-10)


Birch


Predictors for OAS


rBet v2 (profilin)


nGal d1(ovomucoid), d2 (ovalbumin), d3 (conalbulin)


Egg


Presence of antibodies to d1 and d2 are predictive of persistence of egg allergy


rHev b1, b3, b5, b6.01, b6.02, b8, b9, b11


Natural rubber latex


Useful back-up for skin-prick tests


rApi m1 (phospholipase A2)


Bee


Main allergen for bee, does not include cross-reactive carbohydrates




Allergen-specific IgG antibodies


Principles of testing



  • Measured using same techniques as used for specific IgE (CAP, UniCAP).


  • No EQA or standards are available.


Indications for testing



  • Uncertain.


  • Possibly for following the response to desensitization therapy.


Interpretation



  • It has been suggested that desensitization procedures work, in part, by producing blocking IgG antibodies which prevent the allergen from binding to cytophilic IgE:



    • these have been identified as IgG4 subclass.


  • image Success and duration of desensitization may be determined by measurement of such antibodies. This is controversial!


  • However, it is possible to measure allergen-specific IgG antibodies to allergens such as bee or wasp venom, grass pollen, and house dust mite.


  • Whether the results provide any clinically useful information is still unproven.


Basophil activation test


Principles of testing



  • Measured by flow cytometric expression of CD63, a membrane tetraspanin (deficiency in Hermansky-Pudlak syndrome), which is increased on degranulated basophils.


  • Blood is incubated with IL-3 and allergen (with appropriate controls) and the expression of CD63 is detected by FITC-labelled anti-CD63.



Indications for testing



  • Uncertain.


  • This is a very expensive way to test for allergy!


Interpretation



  • Depends on proper controls and knowing that there are no nonspecific degranulating effects of the allergens.



CD23, soluble (Fcε receptor)



  • Measurement of soluble CD23, the shed form of the Fcε receptor which has B-cell stimulatory activity, has been proposed as a useful marker of the activity of chronic allergic disease.


  • In asthma, elevated sCD23 may denote underlying chronic inflammatory activity even when the peak flow may be near that predicted.


  • Measurement of this marker cannot yet be recommended without reservation.


  • Assay is by EIA, but is not widely used.


C3a, C4a, and C5a (anaphylotoxins)




  • Measurement of the anaphylotoxins may be of value in the investigation of suspected acute allergic reactions, as a marker of complement activation.


  • This is particularly valuable in circumstances where IgE is not involved (anaphylactoid reactions).


  • Other conditions with raised anaphylotoxins include:



  • Samples must be collected into Futhan-EDTA, to prevent in vitro generation of anaphylotoxins.



    • These tubes are not readily available.


    • Samples need to reach the laboratory quickly as the breakdown products are labile.


  • Value of the tests in clinical practice is limited because of the lack of ready availability of appropriate tubes.


  • Other tests for complement activation may be more appropriate.


Challenge tests

Challenge tests form an important part of the diagnosis of allergic disease. Clearly, identification of the site of the reaction is important. Nasal, bronchial, and oral challenges may be performed. It is wise to avoid challenging someone who has had a severe systemic reaction or has pre-existing severe asthma with allergens. As for skin testing, it is essential that patients are not taking antihistamines and have been off treatment for a period of time appropriate to the half-life of their antihistamine (up to 4 weeks in some cases).




  • image Challenge tests are potentially dangerous and should be carried out by experienced staff prepared to deal with any adverse reactions that may arise.


  • Informed consent should always be sought from the patient prior to the test.


Nasal challenge tests



  • Usually performed by spraying a dilute solution (1:1000 of SPT solution) of the test allergen into one nostril, while spraying a similarly diluted solution of the buffer only into the other nostril.


  • Patient’s symptoms are recorded (running nose, sneezing, itching eyes, etc.) and the nasal mucosa inspected for signs of inflammation and oedema. Each nostril is inspected.


  • Process is limited to one allergen at a time, which restricts its use to confirming sensitivity to a single suspect allergen.


  • More complex challenge tests involve measurement of nasal airflow (rhinomanometry), but this is rarely available outside specialist research centres.


Bronchial challenge tests



  • Most common bronchial challenge is with methacholine or histamine.


  • Carried out by starting with very dilute solutions and gradually increasing the dose while measuring the forced expired volume (FEV1) sequentially.


  • Reduction of 20% from the control value is viewed as a positive test and is indicative of hyper-reactive airways. The dose causing this reduction is the PD20.


  • If a 25mg/mL dose of methacholine is tolerated without achieving a 20% reduction in FEV1, the test is unequivocally negative.


  • Allergen solutions may be substituted for methacholine, but the principles are the same.


  • Late reactions may occur 6-8 hours after the challenge as part of type I response. The patient must be kept under observation for this period.


  • Enthusiasts may carry out an endobronchial challenge through a bronchoscope, which allows them to observe the changes in the bronchial mucosa and also to carry out bronchoalveolar lavage to look at the release of mediators and the cellular response. This is important in research but not for routine diagnosis.


Food challenge



  • Food challenges are complex and must involve an experienced senior dietician.


  • The gold standard is double-blind placebo-controlled (DBPC) challenge. This is time-consuming.


  • The initial step is usually withdrawal of the suspect food(s) followed by open challenge.



  • For open incremental food challenge, incremental doses of food are given at 15-minute intervals, with monitoring of blood pressure, PEFR, and symptoms before each increment.



    • Steps are: food on lip (may be omitted), 1%, 4%, 10%, 20%, 20%, 20%, 25% of a normal portion (this may be variable!).


    • Over the course of the challenge, 100% of the normal portion size is administered.


  • Where multiple foods are suspected, an oligoallergenic (elimination) diet may be instituted for a period to see whether symptoms remit. If symptoms persist, food allergy/intolerance is not the cause.


  • If symptoms improve, foods may then be reintroduced one at a time as open challenges and the patient’s symptom response noted.


  • This should ideally be followed by a DBPC challenge where the patient, on an oligoallergenic diet, is challenged with the suspect food concealed in opaque gelatin capsules interspersed with identical capsules containing an innocuous substance in a random sequence determined by the dietician but unknown to the patient and doctor.


  • This is time-consuming, as some food allergic symptoms may require exposure for several days before they appear and, equally, may take several days to disappear when the food is withdrawn. There needs to be a washout period between the placebo and the active capsules.


  • A method of scoring symptoms needs to be decided in advance.


  • As for bronchial challenges, enthusiasts have directly instilled allergens into the small bowel and watched for inflammatory reactions.


Drug allergy testing


Principles of testing

Jul 22, 2016 | Posted by in GENERAL SURGERY | Comments Off on Allergy Tests

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