Food Allergy Diagnosis and Therapy

Where Are We Now?

Aleena Syed; Arunima Kohli1; Kari C Nadeau


Immunotherapy. 2013;5(9):931-944. 

In This Article

Diagnostic Tools for FA

The need for precise clinical tools is necessary to detect not only the presence of a possibly life-threatening allergy, but to also predict the severity and prognosis of disease. The mechanisms of FA are not well understood and it is yet to be determined whether FA represents pathological immune deviance in allergic children or the absence of protective mechanisms normally found in a healthy child.

In one of few studies to date, Turcanu et al. analyzed the immune profile of peanut-allergic subjects, allergic subjects who had outgrown allergies and nonallergic subjects. Analysis of peanut-specific lymphocytes revealed the cytokine profile was polarized towards Th2 cells in peanut-allergic children, while nonallergic children and those that had outgrown allergies exhibited a Th1 response to stimulation.[53] All subjects were Th1-biased in response to nonallergic foods, suggesting an association between Th1 response and allergy resolution. In a subsequent study, Thottingal et al. argued against the idea of a protective Th1 bias in healthy individuals after finding insignificant differences in Th1 response between healthy and allergic subjects.[54] Taken together, these studies suggest allergen sensitivity does not innately alter the immune system, but instead determines immune response. Among children with milk allergy, 80% outgrow allergy by the age of 5 years,[33] while only 20% of peanut-allergic patients outgrow their allergies.[7] Refined diagnostic tools are needed to assess predictive factors for spontaneous resolution versus allergy persistence, as well as for determining candidates for immunotherapy. There is still limited knowledge on the conditions that cause this shift from a healthy to allergic state, highlighting the need for increased studies profiling these differences.

Comprehensive guidelines have been developed to assist clinicians in differentiating between IgE-mediated FA and intolerance (adverse reactions that are not immune-mediated).[33,55] The primary concern in diagnosing FA is patient safety, emphasizing caution to prevent false-negative diagnoses. Current diagnostic techniques emphasize the importance of clinical history, family history, presence of other allergic conditions and the timing of allergic symptoms following ingestion.[55] This history serves as a pretest assessment; if allergy is probable, diagnostic tests can be used for further evaluation. At present, skin prick testing (SPT) and measures of food-specific IgE (sIgE) are widely used in clinical settings.[33,56,57] SPT and sIgE are considered safe and can be used to predict the probability of a positive reaction to an oral food challenge (OFC).

SPT involves the application of food extracts to the skin accompanied with a slight puncture. Allergic patients present with a wheal on the skin when stimulated with an allergen. Wheal size is used to determine the likelihood of a positive reaction to OFC, with a positive predictive value (PPV) of >90%.[55,57] SPT can also be used to predict the likelihood of milk allergy resolution.[58] While SPT is safe, rapid and highly sensitive, it does not provide specific information regarding severity.[55,59] Extracts are often crude and unstandardized. Accuracy can be affected by factors like antihistamine use (false-negative) and pre-existing atopic dermatitis (false-positive).[55,59] Diagnostic accuracy in SPT can be enhanced by focusing on single protein components, for example, casein in milk-allergic patients.[60] Multiple components, however, must be tested to ensure that allergy to any protein is not missed. Titrating allergen extracts in serial dilutions has also increased accuracy up to 99%.[61] Studies suggest a high PPV for SPT, however, it is important to note that this predictability varies from study to study given variations in age, allergen and the methods used in the food challenge. In addition, high PPV for SPT is generally associated with a large wheal size; many patients present with small-to-medium reactions, which often do not fall within the cut-off value described.[62]

Mechanistically, levels of sIgE have been positively correlated with the production of antigen-specific Th2 cytokines.[63] In IgE-mediated FA, measurements of sIgE have been shown to correlate with the likelihood of a clinical reaction.[7] In patients aged 4–11 months, measurements of sIgE are more sensitive than SPT.[64] However, levels of sIgE do not always correlate with reaction severity or clinical threshold of tolerance.[7,59,65] Concordance between positive SPT and high sIgE in milk and egg allergies was found to be very low, indicating that these two diagnostic tools are not interchangeable, but work best in conjunction.[66] sIgE is measured in the serum via solid-phase ELISA using commercial technology such as ImmunoCAP® (Phadia AB, Uppsala, Sweden). A limitation of this technique is that results must be interpreted on an individual basis based on clinical presentation, since specific cut-off values for sIgE are hard to identify. In addition, these cut-off values are based on small study groups and vary from individual to individual.[55,67] While some guidelines are predictive of a 95% chance of reaction,[57] it is important to note that in 10–25% of reactions, sIgE can be virtually undetectable.[7]

As with SPT, sIgE measurements are being refined to look at specific epitopes using component resolved diagnostics (CRD).[7] In CRD, a pure allergen is generated either from a natural source or through recombinant expression of allergen-encoding DNA and used for subsequent testing. Measuring the sIgE to specific proteins, such as Ara h 2 for peanut, is much more precise (97% accurate; sIgE >0.35 kUA/l) with a narrower cut-off than the use of the entire food (82% accurate; sIgE >15 kUA/l).[56] Specific epitopes are also useful in predicting the persistence and severity of a FA.[56,68,69] In milk allergy, the binding diversity of IgE has been linked to increased allergy severity.[68] sIgE to Ara h 1, Ara h 2 and Ara h 3 are indicative of severe and persistent peanut allergies, while Ara h 8 binding was associated with allergy in only 17% of patients.[56] In an additional study, monosensitization to Ara h 8 was found to indicate tolerance, suggesting that CRD could be useful in discriminating between allergic phenotypes.[69] Sensitization to Ara h 9 is linked to peanut allergy in the Mediterranean, suggesting that CRD can also be used to investigate regional differences.[70] For egg and milk allergies, ovomucoid-sIgE and casein-sIgE, respectively, are markers for persistent allergy.[56] CRD could also help clinicians identify patients who will have persistent allergies and advise them to permanently avoid the causative allergen, possibly preventing life-threatening anaphylaxis.

Protein microarrays can simultaneously measure IgE binding to a number of different components. The ImmunoCAP ISAC® system (Phadia AB) can measure sIgE for up to 112 allergens, requires 30 µl of plasma and takes less than 4 h. In this system, serum is added to a chip coated with immobilized allergen components. sIgE is measured based on luminescence.[56,71] Research is underway to develop automated microarray systems using photoimmobilized allergens.[72] While micorarrays may not enhance diagnostic capacity,[73] this technique requires little sera, making it ideal for detecting sIgE in young children. Microarray technology allows for rapid measurements of many components to allow diagnoses to be made precisely, accounting for geographic location, individual sensitization and cross-reactivity.[71] This technology could be useful for identifying candidates for therapy and as a monitoring tool during treatment.

IgE is measured routinely in clinical laboratories; however, measurements of other antibodies such as IgG are generally limited to research settings. Similar to IgE, IgG is measured with ELISA and compared with a standard curve generated using purified human IgG. High levels of serum IgG have been reported in tolerant individuals, but were also found in allergic patients.[74] In addition, IgG4 levels may reflect past allergen exposure and, thus, are not indicative of tolerance level in patients undergoing oral immunotherapy (OIT).[65] Binding of IgG4 epitopes to milk-specific proteins had no correlation with disease severity[68] and baseline levels of serum IgG4 were not predictive of allergy resolution.[58] In one study, casein-specific IgE/IgG4 ratio was used to accurately discriminate between tolerant patients and those reactive to baked milk, but was not effective in discerning desensitization to heat-inactivated protein (baked milk) from those who had fully outgrown allergy.[65] Measurements of IgG have not shown optimal predictive value, but routinely measuring IgG alongside IgE in allergy testing could provide further insight into the immune profile of allergic, desensitized and tolerant patients. Measurements of IgE/IgG4 ratios appear more promising than measurements of IgG4 alone. In a study of patients undergoing sublingual immunotherapy (SLIT) for peanut allergy, Kulis et al. found salivary peanut-specific IgA correlated with food challenge outcomes, although serum sIgA did not.[75] This study is the first to measure salivary IgA in patients undergoing SLIT with peanut protein, and suggests that salivary sIgA and serum IgA levels are correlated. Expanding studies on salivary levels of other antibody subclasses may present a minimally invasive technique in the study of FA.

One suggestion for improving diagnostic resolution of antibody quantifications would be to test functionality through in vitro tests such as the basophil activation test.[65,76,77] IgE binding to FcεRI receptors on the basophil results in activation, marked by increased CD63 and CD203c expression. Basophil studies are especially promising, given findings that basophil suppression is associated with desensitization in immunotherapy.[78,79] However, basophil activation tests are time- and resource-intensive, and the use of flow cytometry limits them to a laboratory setting.[56] This assay does hold promise for monitoring allergic patients on therapy,[77] but must be standardized to allow for comparison between patients and different studies.

Despite new developments, OFC is still considered the 'gold standard' for confirming positive or negative diagnoses. Types of OFC include open, single-blind and double-blind. Blinding of patients and observers minimizes bias. The most meticulous strategy is the double-blind placebo-controlled food challenge (DBPCFC). In a DBPCFC, patients and observers are blinded and all materials are prepared by a third party. DBPCFC is especially useful for patients with multiple allergens in order to differentiate between true allergies and food aversions. If a patient passes a DBPCFC (i.e., has no reaction), an open food challenge should be performed before reintroducing the food. An OFC is particularly useful when, based on SPT and sIgE testing, a positive reaction is highly unlikely and the challenge can be used to effectively convince patients and family that the food can be safely reintroduced. OFC is also useful to confirm if a patient has outgrown their allergies as is relatively common in allergies such as milk and egg.[57,59]

While an OFC can accurately confirm a diagnosis, procedural variability exists and efforts have been made to standardize stopping criteria to maximize both safety and consistency.[57,59] Despite monitoring precautions based on clinical preassessment and extensive guidelines, there is always risk of adverse reaction and constant medical supervision is needed. Reactions can be latent, so patients must be monitored for several hours after the challenge. OFC can also cause anxiety in patients who have experienced life-threatening anaphylaxis following accidental ingestion. In addition, for patients with sensitivity to multiple allergens (~30% of the allergic population),[26] OFC for each food must be spaced out and can take days when testing cross-reactive foods.[57,59]

Refined diagnostic techniques may minimize the need for OFC and maximize safety by helping predict reaction severity. Using sIgE and SPT measures coupled with CRD, the likelihood of reaction during OFC can be predicted with a PPV of up to 99%. We predict that the refinement of CRD and high-resolution epitope microarrays will allow for accurate, individualized results, allowing clinicians to confidently suggest exclusion of the antigenic food without the time, cost and anxiety associated with OFC.