Editorial

A Lesson from Component-resolved Testing: We Need Better Extracts Joost A. Aalberse, MDa, and Rob C. Aalberse, PhDb,c Utrecht and Amsterdam, The Netherlands

How should we deal with the rapid increase in the availability of allergenic components for diagnostic testing? The title of the article by Kattan et al1 seems to suggest to us to consider a full switch to components and stop using hazelnut extract. However, the investigators state in their conclusion that “additional studies with larger cohorts in different geographic regions will be needed to establish specific diagnostic protocols.”1 This conclusion invites 2 questions: how should we do these additional studies, and what should we do in the meantime?

HOW SHOULD WE DO THESE ADDITIONAL STUDIES One question to be addressed in such studies is how much the application of a new diagnostic modality (component testing) increases or decreases the statistical likelihood that a patient will have symptoms on exposure. The major impact of the pretest statistical likelihood of clinical food allergy on the optimal diagnostic protocol has been stressed by other investigators.2-4 In the study by Kattan et al,1 42 patients were tested for specific IgE to hazelnut components. Of the 13 patients who were classified as “clinically reactive” in the posttest analysis, 9 had a clinical history that was so convincing that food challenge was considered unnecessary, that is, the pretest likelihood of a positive reaction was already 100%. The remaining 4 patients were part of a group of 116 subjects with a pretest likelihood of a positive reaction of 7.8%. Their likelihood increased to 75% when the results of component testing were used, which indicates a substantial increase in likelihood, but the numbers are too small for a reliable estimate. The point is that patients with a very high or a very low likelihood of sensitization are not informative for deciding how useful it is to add another test to a diagnostic protocol and that it is not statistically helpful to increase numbers by mixing groups of subjects with very different pretest likelihoods. a

Laboratory for Translational Immunology, Department of Pediatric Immunology, University Medical Center, Utrecht, The Netherlands Sanquin Research, Department Immunopathology, Amsterdam, The Netherlands c Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands No funding was received for this work. Conflicts of interest: R. C. Aalberse has received lecture fees from the American Academy of Allergy, Asthma & Immunology, European Academy of Allergy and Clinical Immunology, and ALK-Abello. J. A. Aalberse declares that he has no relevant conflicts of interest. Received for publication April 28, 2014; revised July 4, 2014; accepted for publication July 8, 2014. Corresponding author: Rob C. Aalberse, PhD, Department of Immunopathology, Sanquin Blood Supply Foundation, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail: [email protected]. J Allergy Clin Immunol Pract 2014;2:635-6. 2213-2198 Ó 2014 American Academy of Allergy, Asthma & Immunology http://dx.doi.org/10.1016/j.jaip.2014.07.006 b

Although subjects with a moderate pretest likelihood are most informative, subjects with a very low or a very high likelihood can help us to decide which components might be useful in diagnostic protocols. Tolerant subjects are informative in 2 ways. First, to establish which allergens are least likely to be clinically relevant. Second, to establish which characteristics make subjects likely to be food sensitized without symptoms on oral exposure. Sera from patients who are evidently symptomatic are important to identify the spectrum of allergenic proteins and to check whether all these components are adequately present in diagnostic extracts. If not, we need to decide whether these deficient components (such as Cor a 1, a prominent allergen in the majority of the patients who are provocation positive) are useful to add to our diagnostic protocol. The proposed “additional studies” require oral provocation. To recruit patients for provocation studies, most protocols rely on tests with extracts for inclusion. We have discussed this potential bias elsewhere in relation to peanut components.5 Our conclusion was that it is impossible to judge the clinical relevance of an allergic component if the extract used for inclusion of patients is inefficient in detecting sensitization to this component (such a Cor a 1 in hazelnut extracts). Another issue is how to deal with potential cross-reactivity, for example, between the birch pollen allergen Bet v 1 and Cor a 1. Should patients with pollen sensitization be excluded from these studies, and, if so, how? IgE induced by exposure to birch pollen often reacts with Cor a 1.01 from hazel pollen and Cor a 1.04 from hazelnut.6 Because many patients who are allergic to birch tolerate hazelnuts despite such cross-reactive antibodies, it is tempting to conclude that IgE to hazelnut with a patient who is sensitized to birch is clinically irrelevant. However, 85% of the clinically reactive patients in the article by Kattan et al1 are Cor a 1 positive. The effect of sensitization to cross-reactive pollen components will be an important aspect of the future studies. Similarly, the effect of sensitization to fruit-derived lipid transfer protein (eg, peach) should be taken into account.7,8 The quantitative aspect of specific IgE to allergenic components is another topic that needs to be addressed. Would it suffice to use nonquantitative component tests (dot blots, microarrays)? Analysis of the preliminary data from the article by Kattan et al1 suggests that well-defined cutoff values (ie, quantitative data) might be needed for an optimal diagnostic evaluation. Moreover, a quantitative comparison of specific IgE levels to components and extract enables an evaluation of the completeness of the component panel and the adequacy of allergen extracts.5

WHAT SHOULD WE DO IN THE MEANTIME? Based on their results from more than 100 hazelnut food challenges, Kattan et al1 stress the limitations of skin prick testing and food specific IgE levels.9,10 One limitation is the poor quality 635

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of diagnostic extracts.11 The results obtained by component testing indicate that the standard protocols for the preparation of extracts are suboptimal not only for the Bet v 1-related components but also for some of the other low-abundance food allergens, notably lipid transfer proteins and oleosins.7,8,12 In addition to these well-defined components, it is probable that other components are underrepresented.5 Because we will most likely have to live with extracts for many years to come, it makes sense to see how we can improve extractbased testing. We have all the tools and protocols to make moreefficient extracts, but the obvious objection is that a substantially more efficient extract will give even more false-positive responses. Hazelnut is a good example because the extract used has been engineered to improve diagnostic sensitivity.13-15 This was found to increase the apparent level of IgE to Cor a 1 on average more than 10-fold,14 which clearly indicates a substantial deficiency in the content of this allergen in the extract used. In this article, sensitivity was not an issue, because the goal of the testing of the majority of the patients was to identify problematic nuts for patients with an established nut allergy.14 In the initial diagnostic phase, however, sensitivity is a problem because, even with the conventional extract, more than 20% of the patients have a falsenegative test.10 Before moving to the sensitivity issue, we first want to discuss the specificity problem. The lack of specificity is often a very visible problem, particularly with epidemiologic studies: the test is positive, but food-related symptoms are absent. To reduce the number of false-positive results, we need ways to recognize signs that point to asymptomatic sensitization. Tests with allergen components will undoubtedly be helpful, but a careful (quantitative) evaluation of sensitization to cross-reactive allergens (eg, pollen allergens in the case of a positive hazelnut test) should also be informative. In this scenario, food challenges, either controlled or accidental, will be needed to distinguish cases with symptomatic and asymptomatic sensitization. The results of these food challenges in combination with the results of component tests and cross-reactivity tests will be invaluable to improve our ability to distinguish these cases and thus to decrease the subsequent need for food challenges. Back to the less-visible problem of sensitivity. This is particularly a problem during the initial testing of subjects with an inconclusive history and a relatively low risk to be sensitized. Because, with a negative test, such subjects would presumably not be tested further, a protocol with a high sensitivity is needed to avoid false-negative results. A target might be the recognition of >95% of the patients with positive food challenge not only in a high-risk population but also in a low-to-intermediate risk population. The availability of more-efficient extracts for testing (in vitro and possibly also in vivo) is likely to be needed, even if this results in the increased recognition of asymptomatic sensitization. Improving deficient extracts by spiking with recombinant allergens

J ALLERGY CLIN IMMUNOL PRACT SEPTEMBER/OCTOBER 2014

is a surrogate solution, because this does not help to identify unknown allergens needed to improve test sensitivity. Although we seem to have too many allergens already, our current component panel is still deficient. We need a diagnostic test protocol with an adequate sensitivity for subjects with a nonconvincing exposure history. This is hard to do without better extracts. REFERENCES 1. Kattan JD, Sicherer SH, Sampson HA. Clinical reactivity to hazelnut may be better identified by component testing than traditional testing methods. J Allergy Clin Immunol Pract 2014. Epub ahead of print. 2. DunnGalvin A, Daly D, Cullinane C, Stenke E, Keeton D, ErlewynLajeunesse M, et al. Highly accurate prediction of food challenge outcome using routinely available clinical data. J Allergy Clin Immunol 2011;127:633-9. 3. Sicherer SH, Wood RA. Advances in diagnosing peanut allergy. J Allergy Clin Immunol Pract 2013;1:1-13. 4. Sicherer SH, Sampson HA. Food allergy: epidemiology, pathogenesis, diagnosis, and treatment. J Allergy Clin Immunol 2014;133:291-307. 5. Aalberse JA, Meijer Y, Derksen N, van der Palen-Merkus T, Knol E, Aalberse RC. Moving from peanut extract to peanut components: towards validation of component-resolved IgE tests. Allergy 2013;68:748-56. 6. Lüttkopf D, Müller U, Skov PS, Ballmer-Weber BK, Wüthrich B, Skamstrup Hansen K, et al. Comparison of four variants of a major allergen in hazelnut (Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01. Mol Immunol 2002;38:515-25. 7. Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, et al. Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results. J Allergy Clin Immunol 2002;109:563-70. 8. Schocker F, Lüttkopf D, Scheurer S, Petersen A, Cisteró-Bahima A, Enrique E, et al. Recombinant lipid transfer protein Cor a 8 from hazelnut: a new tool for in vitro diagnosis of potentially severe hazelnut allergy. J Allergy Clin Immunol 2004;113:141-7. 9. Ortolani C, Ballmer-Weber BK, Hansen KS, Ispano M, Wüthrich B, BindslevJensen, et al. Hazelnut allergy: a double-blind, placebo-controlled food challenge multicenter study. J Allergy Clin Immunol 2000;105:577-81. 10. Maloney JM, Rudengren M, Ahlstedt S, Bock SA, Sampson HA. The use of serum-specific IgE measurements for the diagnosis of peanut, tree nut, and seed allergy. J Allergy Clin Immunol 2008;122:145-51. 11. Akkerdaas JH, Wensing M, Knulst AC, Krebitz M, Breiteneder H, De Vries S, et al. How accurate and safe is the diagnosis of hazelnut allergy by means of commercial skin prick test reagents? Int Arch Allergy Immunol 2003;132: 132-40. 12. Zuidmeer-Jongejan L, Fernández-Rivas M, Winter MG, Akkerdaas JH, Summers C, Lebens A, et al. Oil body-associated hazelnut allergens including oleosins are underrepresented in diagnostic extracts but associated with severe symptoms. Clin Transl Allergy 2014;4:4. 13. Andersson K, Ballmer-Weber BK, Cistero-Bahima A, Ostling J, Lauer I, Vieths S, et al. Enhancement of hazelnut extract for IgE testing by recombinant allergen spiking. Allergy 2007;62:897-904. 14. Sicherer SH, Dhillon G, Laughery KA, Hamilton RG, Wood RA. Caution: the Phadia hazelnut ImmunoCAP (f17) has been supplemented with recombinant Cor a 1 and now detects Bet v 1-specific IgE, which leads to elevated values for persons with birch pollen allergy. J Allergy Clin Immunol 2008;122: 413-4. 15. Masthoff LJ, Pasmans SG, van Hoffen E, Knol MJ, Bruijnzeel-Koomen CA, Flinterman AE, et al. Diagnostic value of hazelnut allergy tests including rCor a 1 spiking in double-blind challenged children. Allergy 2012;67: 521-7.

A lesson from component-resolved testing: we need better extracts.

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