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Allergy Diagnostic Testing

Updated: July 2014
Originally posted: November 2007

Dr. John Oppenheimer
Director of Clinical Research,
Pulmonary and Allergy Associates
Denville, NJ, USA

Prof. Stephen Durham
Department Allergy and Respiratory Medicine,
Imperial College, London, UK

Dr. Harold Nelson
National Jewish Medical and Research Center
Denver, CO, USA

Dr. Ole D. Wolthers
Clinical Institute, Health, Aarhus University
Asthma and Allergy Clinic, Children’s Clinic Randers 
Randers, Denmark

Credit for the first skin testing goes to Charles H. Blackley, who in 1865 abraded a quarter-inch area of his skin with a lancet, applied grass pollen on a piece of wet lint, and covered the scarified area with an occlusive bandage. This resulted in intense itching and a very large cutaneous response.

Percutaneous skin test ranks first in confirming the presence of IgE-mediated sensitization in the allergist's office. This should come as no surprise, as it has many advantages. Skin testing is minimally invasive, and when it is performed correctly it has good reproducibility, is easily quantified, and allows the evaluation of multiple allergens at one session. The results correlates with in vivo challenges. in vitro testing is an alternative, usually a back up tool for diagnosing allergic illness. Skin testing alone or in combination with in vitro testing is relied upon for the evaluation of allergic rhinitis, asthma, eczema, food allergy, insect sting allergy, drug allergy (especially beta-lactam and local anesthetic allergy), occupational disease and anaphylaxis. However, the reliability of these tests depends on a number of factors. In the case of skin testing, it is important that the technician performing the skin tests and the clinician ordering or interpreting these tests are aware of the advantages and pitfalls of the type of skin testing, the device used, the location of the tests on the body, the extracts used and the potential for suppression of the skin response by medications used to treat allergies or depression. These issues have been reviewed elsewhere in greater detail.1 For in vitro testing, it is imperative that quality standards be met. These include calibration of the assay, training and experience of the technician and the use of quality allergens in the solid phase.2 As in any diagnostic test, it is of paramount importance that the clinician consider the positive and negative predictive value of the tests performed. These tests should always be considered as adjuncts to the medical history and physical exam in formulating the diagnosis in each individual case, bearing in mind that both test types can yield false positive or, less commonly, false negative results.

Methods of Skin Testing

Skin testing may be performed using either the prick/puncture (percutaneous) or intradermal (intracutaneous) technique. Intradermal testing is far more sensitive than prick/puncture testing, which means that it requires about 1000-fold less concentrated extracts than those used for prick/puncture testing to achieve a similar response. Although direct comparisons indicate that intradermal testing is more reproducible than percutaneous testing, there are many factors that favor the routine use of percutaneous allergy tests. These include economy of time, patient comfort and patient safety. Percutaneous testing allows the use of extract in 50% glycerin, which provides greater extract stability. Intradermal testing cannot use this diluent, as it may incite a false-positive irritant response. However, the most important consideration is that results of percutaneous testing correlate better with clinical allergy. The higher sensitivity of intradermal skin tests does not usually offer added benefit, since the results of skin prick tests performed with potent extracts are of sufficient sensitivity for use in clinical practice.

Two studies reinforce this concept.3,4 Each study compared intradermal with skin prick tests by correlating their results with patients' responses to natural exposure to allergen as well as by allergen challenge testing. In the first study, three groups of patients with seasonal rhinitis were compared. These subjects were classified into 3 groups based on their degree of sensitization to Timothy grass pollen. They were either skin prick test positive, only intradermal test positive, or were negative by both skin prick and intradermal testing. Both nasal allergen provocation testing and symptom scores during the pollen season correlated best with a positive skin prick test (>60% of subjects with positive skin prick tests had symptoms on allergen exposure). The frequency of positive nasal provocation (11%) and symptom scores (21%) in subjects with positive intradermal testing alone were not different from subjects who were skin prick test and intradermal test negative. The authors conclude that under the conditions of this study, the presence of a positive intradermal skin test response to Timothy grass in the presence of a negative skin prick test did not indicate the presence of clinically significant sensitivity to this grass.

In the second study, patients were challenged with cat exposure for one hour.4 Both positive skin prick tests and in vitro tests to cat were highly predictive of the development of symptoms upon allergen exposure in the cat challenge room.4 Subjects with a negative skin prick test were just as likely to have a positive challenge result if they had a negative intradermal skin test (31%) as subjects with a positive intradermal skin test (24%). The authors conclude that, at least with regard to cat allergy, major therapeutic decisions, such as environmental control or immunotherapy, should never be based on a positive intradermal skin test alone.

Both of these studies were performed in adults and both relied upon skin testing with relatively potent allergens (Timothy grass and cat). The clinical applicability of these results to less potent allergens, such as dog, or to younger patients (especially infants) is a matter of clinical judgment, because no specific evidence is available for these groups.

Skin Testing Devices

Whereas intradermal skin tests are always performed using a hypodermic syringe and needle, percutaneous tests may be performed with a variety of devices. Comparisons of percutaneous devices have been reviewed elsewhere in greater detail.5 Some devices have a single stylus with one or several points, whereas others have multiple heads and allow up to 10 tests to be accomplished with one application. The degree of skin trauma created by these devices for percutaneous testing varies and so may result in differences in the size of positive reactions, and the likelihood of producing a reaction at the site of the negative control. Thus, they require different criteria for what constitutes a positive reaction (see Table 1).

Table 1.  Wheal size indicating a positive response to skin tests using various devices.a

a Positive response is defined as a wheal greater than 99% of wheals generated by the administration of saline to the subject's back by the same operator. Adapted from ref. 14.
b HS = Hollister Steir, Greer = Greer laboratories, Lincoln = Lincoln Diagnostics, ALK = ALK America, ALO = Labs of Ohio


Recording and Scoring of Skin Test Results

Skin test results are often reported by clinicians in semi-quantitative terms. They may record results only as positive or negative, or express them on a 0 to 4+ scale without any indication of the size of the reactions that these numbers represent. However, allergy patients may have to change their allergist for numerous reasons, and it is important that records of prior allergy testing be interpretable by the receiving physician. At the very least, a record of skin testing should contain sufficient information to allow another physician to interpret the results and avoid the need to repeat skin testing. Standardized forms have been developed and are available through the American Academy of Allergy Asthma and Immunology website (for an example AAAAI's Skin Test and Immunotherapy Forms).

Although the area of the wheal and erythema are the most accurate measurements, the longest diameter or two diameters at right angles to each other correlate with area (r > 0.9).8 The importance of performing such measurements is exemplified by the study of McCann and Ownby in which allergists were asked to interpret photographs of skin test reactions. The scoring and interpretation of the skin test results varied greatly.9 The authors of this study reinforce the idea that the most reliable method of reporting a skin test reaction is to measure and record the reaction size. At the very minimum, skin test results should be graded 0 to 4+, and the criteria for each grade of reaction clearly stated along with the skin test results.

Various investigators suggest different criteria for interpreting a skin test response as positive. To assess the reliability of different means of interpreting the results of skin prick testing, Vanto and colleagues studied a group of 202 children sensitive to dogs.10 A determination of sensitivity to dog was based on a composite score derived from the history, RAST, and bronchial or conjunctival allergen challenges. Although in this study the overall efficacy was greatest with the histamine reference method (in which the allergy skin test response is compared to a histamine control, with a positive response considered to be a response at least as great as that of the histamine control), maximal sensitivity was achieved when using a cutoff of a wheal 3 mm. If a clinician wishes to maximize sensitivity, the latter criterion would be most useful; however, adjustment must be made for the device used. Therefore, the criteria for a positive test should be: 1) the larger of a 3 mm mean wheal diameter or 2) equal to or greater than the 99th percentile reaction with that device at negative control sites (see Table 1).

Proficiency Testing

Like all other laboratory tests, it is imperative that quality assurance standards be met to ensure that the testing technique produces accurate results. To confirm such standards, it is recommended that all technicians performing skin testing undergo evaluation of their technique.11 Certainly, it would be comforting to know that skin test technicians achieve some degree of consistency in skin test performance. Although there are no formal standards available for skin test proficiency testing, several publications suggest some possible criteria. European publications suggest a coefficient of variation of less than 20% following repeated skin test control applications, and the Childhood Asthma Management Program study requires that a coefficient of variation of less than 30% be attained with repeated testing with histamine and consistently negative reactions to saline to confirm proficiency in skin testing.

The National Committee for Clinical Laboratory Standards recommends quality control procedures for daily performance of in vitro allergy testing, with a recommended coefficient of variation of less than or equal to 15%.2 Even with such calibration and the increased use of automation, in vitro assays still have flaws. Williams and colleagues examined the performance of 6 large commercial laboratories on tests of blinded samples of the same sera, both diluted and non-diluted.12 They found that only two of the laboratories demonstrated acceptable precision and accuracy.

Comparing in vivo to in vitro Testing:

The preponderance of comparative studies demonstrate skin tests to be more sensitive than in vitro tests. However, the majority of these studies were performed with earlier generation in vitro tests. The newer in vitro tests produce higher test sensitivity and specificity13 by using a matrix capsule containing antigen bound to a hydrophilic carrier to produce enhanced specific IgE binding with lower nonspecific IgE binding.2 Levels of specific IgE measured by different commercial assays are not equivalent, as each assay differs in the composition of allergen reagents, methods of measurement and standardization procedures.

The advantages of in vitro testing are largely related to use in patients with extensive dermatoses (e.g., atopic dermatitis), resulting in an inability to perform tests on unaffected skin, or in patients who are unable to discontinue medicines that block the histamine response, i.e., antihistamines or tricyclic antidepressants. The disadvantages of in vitro testing include a potential decrease in sensitivity, added cost, and lack of immediate and visible response. Performing both in vitro and in vivo tests may yield improved sensitivity.15

"Gold Standard" Confirmation of Allergy

Although there are challenge protocols available in the research setting to confirm allergic rhinitis and asthma, the standard tool available to the clinician is a careful history and physical exam. Skin testing correlates with results of nasal challenge and with bronchial challenges when allowance is made for nonspecific airway responsiveness.

When evaluating potential food allergy, the clinical history is the initial screening, with skin testing or in vitro tests used to corroborate the history. Oral food challenges represent the "gold standard" for the confirmation of food allergy. These can be performed as open challenges or in a single- or double-blind fashion. Food challenges are not without risk and thus require that appropriate supportive care be available. Several studies demonstrate that the magnitude of the in vitro test or the skin test reaction size may be useful in determining the utility of performing a food challenge.16,17 One additional advantage of skin testing for food allergies is the ability to perform skin testing with the fresh food, "prick-prick" test. Several reports demonstrate that fresh foods provide greater sensitivity for certain foods.18, 19 This is particularly important in assessing allergy to fruit; however, useful results have also been demonstrated for other foods, including seafood, peanut, tree nuts, vegetables, milk and eggs.

Molecular-based allergy diagnostic

It is hoped that the predictive value of allergy diagnostic testing can be improved with the use of molecular-based allergy diagnostics.  This methodology is used to map the allergen sensitization of a patient at the epitope level, using purified natural or recombinant allergenic molecules (components) 20,21Molecular-based allergy diagnostics is available either using singleplex platforms which utilize panels of single allergens together with the corresponding allergen extract or can also be performed using multiplex technology to measure serum IgE antibodies against multiple allergens in a single assay 20-22 The technique allows for the testing of a large number of allergens using a small amount of serum (as little as 20 µL; conventional specific IgE tests use 50 µL per allergen). Currently one multiplex platform is available on the market (the Immuno-Solid phase Allergen Chip (ISAC) platform) 23,24 Though a higher degree of variability in low IgE levels have been found, ISAC results have been similar to those obtained from singleplex platforms 25,26 At low serum IgE levels singleplex platforms may be more sensitive than ISAC and thus this should be considered when interpreting testing using the ISAC. Although more than 130 epitopes have been identified, the clinical relevance of many of these is not known. Evidence, however, has been provided for use of several epitopes in clinical practice, such as peanut.

In many cases of peanut sensitization detected solely by prick skin testing or by whole allergen specific IgE it is difficult to decide whether true allergy exists versus sensitization with no clinical symptoms as a manifestation of cross reactivity to pollen. In such cases there is good evidence for analyzing IgE to the epitopes Ara h 2 (genuine IgE mediated allergy) and Ara h 8 (Bet v 1 (birch pollen) homologue; a marker of cross-reactivity) 27,28 IgE sensitization to Ara h 2 often correlates with positive IgE against Ara h 1 and Ara h 3. If there were IgE antibodies in serum to Ara h 2 and/or Ara h 1/Ara h 3, more than 95% of patients have reported symptoms when ingesting peanuts 29 If there was IgE only to Ara h 2 and not to Ara h 1, 3 or 8, 87% reported symptoms. Whether there may be a threshold level of serum IgE to Ara h 2 above which peanut allergy may be diagnosed with a sufficient sensitivity and specificity which may abandon the need for oral provocation remains to be prospectively evaluated 30If there was only IgE to Ara h 8 and not to Ara h 1, 2 or 3 only around 18% of patients have reported symptoms, and these were usually very mild 29 More serious symptoms cannot be ruled out, however, in Ara h 8 sensitized patients. In the event of itching and swelling in the mouth and throat both Ara h 2 and Ara h 8 should be determined, and, at the same time, assessment of sensitization to birch pollen should be made by analyzing IgE to Bet v 1.

Bet v 1, PR-10 protein is the major allergen in birch pollen and approximately 95% of birch pollen sensitized patients have specific IgE antibodies to Bet v 1 31 Specific IgE to Bet v 1 may also be found in patients with primary sensitization to other tree pollens (e.g. elm: Aln g 1; hazel pollen: Cor a 1) as well as to foods (hazelnut, apple, soy, peanut (Ara h 8), kiwi, celery). IgE antibodies to Bet v 2 (profilin) and/or Bet v 4 (calcium-binding protein) are markers of cross-reactivity 31 and as opposed to Bet v 1 if increased are indicators that the patient is primarily sensitized to another pollen. IgE to Bet v 2 is a marker of cross-reactivity with many pollens and vegetable foods 32 while IgE to Bet v 4 is a marker of cross-reactivity only with other pollen allergens 33

      IgE antibodies to Phl p 1 and Phl p 5 are specific markers for sensitization to Phleum pratense (Timothy grass). Phl p 7 (calcium-binding protein) and Phl p 12 (profilin) are markers of cross-reactivity with fruits and vegetables. Increased IgE to these components and not to Phl p 1 and/or Phl p 5 indicates primary sensitization to a different species of grass pollen than Phleum pratense 34 It has been suggested that if relevant symptoms are present in addition to elevated IgE Phl p 1 and p 5 levels immunotherapy with phleum pratense extract would likely be clinically effective because phleum pratense extracts contain mainly Phl p5 and p6 24,35

      Molecular-based allergy diagnostics are also likely to be of utility when considering immunotherapy for dust mite allergy.  Der p 1 and Der p 2 are the most important component markers for sensitization to house dust mites 36as more than 80-90% of patients allergic to house dust mites have IgE antibodies to these epitopes. Approximately 10% of patients allergic to house dust mites, however, have increased IgE levels to Der p 10 37 These patients will not benefit from specific immunotherapy since house mite extracts contain mainly Der p 1 and Der p 2 and variable or low amounts of Der p 10.  Whether molecular-based allergy diagnostics may increase the effect ratio of immunotherapy of Phleum pratense and houst dust mite allergic patients has not, however, been tested in prospectively planned trials.

      Positive IgE to both bee and wasp venom is often due to cross-reactivity between cross-reactive carbohydrate-determining reagents (CCD) 38 shared in these two species. In the frequently occurring clinical situation of an uncertain history and positive IgE to both allergens, determination of specific molecular epitopes may be of aid. An increase in both Api m 1 and Ves v 5 would indicate a true double sensitization and immunotherapy with both bee and wasp extracts would be indicated 38,39

      Understanding the paucity of data, a recent consensus document concluded that molecular-based allergy diagnosis may be considered for investigation of 20

  • selected cases of suspected peanut allergy, birch pollen allergy and associated cross-reactivity (Ara h2, h8 (h1, h3) (Bet v1, v2, v4).  
  • patients with insect allergy (Api m1; Ves v5).
  • patients and triggering allergens for specific immunotherapy, specifically
    - grass, (Phl p1, p5, p12)
    - house dust mites, (Der p1, p2, p10)
    - hymenoptera venom (Api m1; Ves v5).

Rather than classic testing, alternative molecular-based allergy diagnostics should be seen as an adjunct to the traditional whole allergen specific IgE tests. It is important to remember that many patients can still be sufficiently assessed using conventional prick skin testing or specific IgE to whole allergens in the blood in addition to a thorough history and clinical examination 20 The clinical significance of sensitization detected via molecular-based allergy diagnostics should only be used in relation to the clinical history and physical signs. ISAC testing is likely to be most useful in poly-sensitized patients for evaluation of sensitization to cross-reacting food and airborne allergens. Prospectively planned studies should be undertaken to determine to what extent such extensive panel screening may be helpful in clinical practice. Robust evidence has not yet been provided to prove that molecular-based allergy diagnostics can be utilized in lieu of oral challenge testing in food allergy.

Conclusion Diagnostic testing remains an essential tool for the evaluation of the allergic patient. Several variables should be controlled to produce more reliable skin test results and improve the predictive values of allergy skin testing. It is also imperative that allergists ensure that the results of skin testing are reliable by conducting proficiency testing. In addition, the results must be properly documented to make them easily understandable by others. Similar standards must be applied to in vitro testing; as in the case of skin testing, it is imperative that the ordering physician be familiar with the operating characteristics that the in vitro lab employs. Lastly, it is likely that in the future, molecular based allergy diagnostics will play a bigger role in the evaluation of allergic patients.


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