This text is reprinted by permission of the publisher.
Zimmerman, B. Methods of Evaluation of Atopy. Seminars in Asthma Management 1998;7-10.
Editor Dr. P. O'Byrne, Publisher BC Decker, Hamilton, Ontario, Canada.
From the Gage Occupational & Environmental Health Unit, Health Sciences Research Centre, St. Michael's Hospital and the Asthma Centre, Toronto Hospital (Western Division), The University of Toronto.
Atopy has been defined as an inherited tendency to produce excess IgE antibody to common environmental allergens such as mites, dander, pollens. However this definition is too restrictive in our current understanding of allergy. The atopic immune response is now known to be a separately inherited compartment of the immune response regulated by a subset of T cells termed Th2 cells. Thus the atopic immune system is a component of the immune response that creates inflammation, especially eosinophilic inflammation in diseases such as asthma, eczema and allergic rhinitis. The tests for evaluating atopy must be used in the context of the clinical history. As in any branch of medicine a thorough history and a physical examination are the starting point for the evaluation of atopy. When the history suggests allergy then the further investigation can involve several distinct tests including: 1. Serum level of total IgE; 2. Tests for determining the presence of allergen-specific IgE antibody, e.g. in vivo skin tests and in vitro measurement of serum antibodies; 3. Tests which induce the disease in the shock organ (conjunctival, nasal and bronchial provocation); and 4. Tests which measure eosinophils and eosinophil activation. In the next few years we should see a marked increase in the number of tests available for evaluating atopy.
Total IgE determination is a crude method for screening of atopic disease. Normal levels of serum IgE do not rule out the presence of atopic disease while elevated levels may in 20- 30% of instances not be associated with atopic disease. Thus it is difficult to set an exact level that always indicates atopy and below which atopy is always ruled out. It is fair to say that the higher the serum total IgE, the more likely the presence of atopy. Initially it was suggested that a serum IgE level above 100 KU/L was strong evidence for the presence of atopic disease while a value below 20 KU/L could rule out atopy (1). In children it has been shown that geometric mean IgE levels rose in association with different allergic disease states from atopic dermatitis without other allergic diseases to pure allergic rhinitis, to allergic asthma and then the combination of asthma with rhinitis. The highest levels occurred in patients with atopic dermatitis associated with respiratory allergy (2). However most studies have concluded that the spread of IgE values is very wide and reliable upper limits distinguishing groups i.e. non-atopic and atopic could not be described. In an epidemiologic study of asthma there was an association between the prevalence of the disease and the total IgE level standardized for age and sex, with no asthma present in the subjects with the lowest IgE levels for their age and sex (greater than 1.46 SD below the mean) (3). This suggests that the total serum IgE is useful in large epidemiologic studies while less applicable in the clinical evaluation of individual patients. Moreover the elevation of serum IgE is also confounded by association with other disease states such as parasitic diseases; neo-plastic diseases, e.g. Hodgkin's disease; infectious diseases e.g. allergic broncopulmonary aspergillosis, HIV infection; and immune deficiency disorders e.g. IgE myeloma, hyper IgE syndrome, Wiscott Aldrich syndrome.
The simple skin tests remains the workhorse method for determining the presence of IgE antibody. The modified prick test is performed by placing a drop of antigen extract on the skin and then using a hypodermic needle, or lancet or other device to gently tent the skin and puncture it in order to introduce the allergen. The allergen binds to IgE antibody on the surface of skin mast cells leading to release of histamine from the mast cells which in turn leads to the development of a wheal and flare at the site within twenty minutes. Subsequently this reaction fades over the next half hour but in some instances is followed by a "late phase" reaction occuring four to six hours later and characterized by a diffuse swelling of the test area due to edema and an influx of cells (4).
The prick skin test is simple, rapid to perform, highly sensitive and cheap. It is only semi- quantitative and unless controlled carefully can yield false positive or false negative results. It must be kept in mind that positive skin test indicates the presence of IgE antibody but this by itself does not mean that the individual has an allergic disease. The prick test has largely replaced the scratch test as it is less invasive and gives fewer false positives. The intradermal test is more sensitive but yields more false positives. It tends to be reserved for situations where the allergens to be tested are poor in quality such as drug and anesthetic testing. As yet there is no one standardized method for the prick test that has been universally adopted, although the modified prick test is certainly adequate. There have been numerous studies examining different techniques and different devices for performing prick tests. When standardized allergen extracts of appropriate concentrations are used for testing by trained personnel, skin testing can produce reproducible results with good specificity and sensitivity. Appropriate positive and negative controls must be applied each time skin testing is performed. Skin test reactivity is less pronounced on the forearm than on the back for both histamine and allergen. Prick tests can be performed and interpreted without difficulty in infants, although the wheal size induced by both positive control solutions and allergen-induced prick tests tend to be smaller in infants than adults.
Studies have been performed to assess the value of skin prick testing in epidemiological surveys and the reproducibility of such testing. In a large scale study to assess the prevalence and distribution of allergic skin-test reactions in a general population sample in the Tucson region of the United States (5), allergy prick tests were applied to 3101 subjects older than 2 years of age. No difference in the prevalence of measurable reactions was found between males and females but a definite age relation was found, with the peak prevalence occurring during the third decade, and falling after age 50. These authors concluded that when present, positive skin tests tended to be multiple, highly reproducible, and more frequent among those in higher socioeconomic strata. The prick test was judged to be a useful tool for the assessment of atopy.
The size of the skin test reaction increases linearly with the log of the antigen dose injected into the skin resulting in the dose response curve. There is also some correlation between the size of the skin test, the degree of IgE sensitization and the clinical symptoms. Several studies have related allergen provocation results to skin test results. The studies demonstrate that the larger the skin test result the more likely there will be a clinical reaction on provocation. In one study (6) it was found that more than 90% of patients with a wheal greater than or equal to 5 mm in diameter on skin testing by puncture technique using 1:20 w/v allergen extract experienced positive reactions on provocation testing for most antigens. Pastorello et al (7) attempted to determine the sensitivity and specificity of skin testing to a clinical history of symptoms. They determined the optimal size for the wheal area to seasonal as well as perennial allergens. Their data suggested that the larger the wheal size the more likely the allergen was involved in clinically relevant disease. Nevertheless one of the limitations of the skin test is that a final consensus on the threshold concentration of allergen for positive skin test or for that matter a method for standardization of allergen has not been reached. Studies are ongoing using standardized allergens and attempting to provide uniform standards.
In 1967 Wide (8) described the vitro test known as the radioallergosorbant technique (RAST) in which an allergen was coated on a disc which would then be incubated with an unknown serum. IgE along with other classes of antibody would bind to the allergen on the disc and then could be recognized by the subsequent binding of a labeled anti-IgE antibody. This technique has proven very useful despite its limitations. It does not allow exact quantitation of the IgE and is measured in units relative to a control antigen, usually birch pollen antigen. Generally the RAST test has been found to be less sensitive than prick skin testing.
A number of modifications have been made to develop in vitro assays with better sensitivity such as the modified RAST test or more convenient methodology i.e. by measuring the binding by chemiluminescence or enzyme activity rather than isotope. Some in vitro tests were developed to allow simple assessment of a panel of allergens from the same sample of blood i.e. the Mast test. In most instances the tests performed well when compared with a gold standard skin prick test with good specificity but somewhat less sensitivity.
The RAST technology has also been modified by coating a panel of aero-allergens to a single disc and using the test to screen for the presence of any specific IgE antibody thus diagnosing atopy (Phadiatop, Pharmacia Diagnostics, Pharmacia-Upjohn Ltd.). This test has performed well in studies except in children under age three where antibody to aero-allergens has not yet developed (9). As a result the Phadiatop disc was modified to include several common food allergens (Phadiatop-pediatric) making it more suitable for screening younger children.
More recently Pharmacia Diagnostics, Pharmacia-Upjohn Ltd. introduced a fully automated system (CAP system) that replaces the RAST disc with a high capacity hydrophilic polymer backbone material that by virtue of its high binding capacity can be made fully quantitative. The actual amount of IgE antibody can be quantitated in Kunits/L. Several studies have now been performed to compare the high capacity CAP system with the older RAST and skin test or other in vitro tests. All have found the CAP system to yield better performance characteristics ( ImmunoCAP, Pharmacia Diagnostics ).
When Sampson and Ho (10) applied the newer CAP system to the assay of IgE antibody to food allergens, they found it was possible to define a concentration of specific antibody to different food allergens that would predict clinical reactivity with greater than 95% certainty. This suggested that quantifying the amount of antibody present might eliminate the need to perform a blind oral challenge in some cases of food sensitivity.
Different allergen provocation test have been developed for research purposes including bronchial, nasal and conjunctival provocation tests. None of these tests are used in routine clinical practice but have been used to examine skin prick testing in order to define the characteristics associated with positive provocation challenges. While it has been suggested that a simple nasal provocation test in which allergen was instilled into the nasal passages and a positive response was measured as increased nasal resistance measured by anterior passive rhinomanometry could prove to be a simple test for clinical assessment of patients with multiple sensitivities, the test has never been widely utilized (11). Allergen inhalation provocation test has been instrumental in the description and characterization of the late phase reaction to allergen. The technique of allergen inhalation has also improved our understanding of the pathogenesis of asthma and its relation to inflammation particularly eosinophilic inflammation in the airways.
While positives skin tests mark the presence of an atopic immune system and the degree of sensitization, eosinophils and their activation products reflect the activity of this atopic immune response. Asthma is defined as a clinical syndrome associated with inflammatory cells, especially eosinophils in the bronchial mucosa or submucosa. The presence of eosinophils has been established by bronchoalveolar lavage and biopsy. Elevated eosinophil numbers have also been documented in sputum and peripheral blood. This suggests the possibility of monitoring the inflammation by sampling sputum or blood.
Induced sputum: the analysis of cells and mediators in sputum induced by inhalation of hypertonic saline in asthmatics is relatively non-invasive method for examining the inflammation in the airways of patients (12 ). This technique has been shown to be reproducible and yields quantitative data on cells in the sediment and cytokines in the supernatant. This technique is currently being perfected and in the next few years could become more routinely available for characterizing the sputum of patients with respiratory complaints. The sputum is induced by patients inhaling hypertonic saline nebulized over 20 minutes. The patients are encouraged to cough up sputum which is collected and processed by first separating saliva from the sputum plugs. Cells are released from the plugs by treatment with dithiothreitol and vortexing. This results in a supernatant fluid and cellular phase; the former can be used for measurement of chemistry and the latter for microscopic analysis of the stained cells.
Peripheral blood eosinophil and serum ECP: several studies have related peripheral blood eosinophilia (13 ) or eosinophil granular proteins, in particular serum eosinophil cationic protein, ECP (14 ) to the severity of asthma. Patients with symptomatic asthma had higher mean serum ECP and total eosinophils than asthmatics who are asymptomatic or normal controls. Treatment with steroid decreased the levels of serum ECP and the initial ECP values correlated with the change in the FEV1as the patients responded to therapy (15). As a result of the studies it has been suggested that measurement of peripheral eosinophil counts and more recently serum ECP or other eosinophil granular proteins might serve as a measure of the activity of the atopic immune response. The studies are still in their early stages, and reliability and predictive value of these measurements in asthma or atopic dermatitis of differing severity is not known. It must be remembered that these measurements are an assessment of eosinophilic activity and do not distinguish between different atopic diseases although it has been suggested that the serum ECP measurements are related to the activated component of the total eosinophils and as a result correlates more closely to asthma than rhinitis.
Future possibilities: The atopic immune response is governed by activated T helper cells of the Th2 subtype and a series of cytokines and chemokines that regulate the various cells involved in this immune response. It is possible that measurement of interleukins such as IL-4 and IL-5 in serum or secretions might provide assays of this immune activity in future.