Is the Quantification of Antigen-Specific Basophil Activation a Useful Tool for Monitoring Oral Tolerance Induction in Children With Egg Allergy?

OBJECTIVES: To assess modifications in baseline specific IgE- and anti-IgE- and antigen-specific-mediated basophil activation in egg-allergic children. The values were compared before and after the children completed specific oral tolerance induction (SOTI) with egg. PATIENTS AND METHODS: We studied 28 egg-allergic children who completed SOTI with egg. The basophil activation test and specific IgE determinations with egg white, ovalbumin, and ovomucoid were performed in all 28 children. RESULTS: A decrease in antigen-specific activation with egg white, ovalbumin, and ovomucoid was observed only at the 2 lowest concentrations used (5 and 0.05 ng/mL). Baseline activation was higher in patients with multiple food allergies and in those who developed anaphylaxis during SOTI; this activation decreased in both groups after completion of SOTI. A significant decrease was also observed in specific IgE values for egg white, ovalbumin, and ovomucoid after tolerance induction. CONCLUSIONS: Food tolerance induction is a specific process for each food that can be mediated by immunologic changes such as a decrease in specific IgE values and in specific and spontaneous basophil activation.

In vivo and in vitro testing with rAni s 1 can facilitate diagnosis of anisakis simplex allergy

Background: Traditional diagnostic tests such as skin prick tests (SPT) and specific IgE (sIgE) against whole Anisakis simplex extract have low specificity. Consequently, allergy to A simplex is overdiagnosed. Objective: Our aim was to compare tests used in component-resolved diagnosis. Methods: We evaluated 34 patients with allergy to A simplex, 15 patients with acute urticaria who were sensitized to A simplex but had no clinical history of allergy to A simplex, and 10 patients allergic to seafood. SPT, sIgE (ELISA and ISAC-112), and the basophil activation test (BAT) were performed with A simplex whole extract and the molecular components rAni s 1, rAni s 3, and nPen m 1. Sensitivity and specificity were calculated and compared with different cutoffs. Results: With the A simplex whole extract, SPT, sIgE, and BAT yielded specificity values of 72%, 68%, and 70%, respectively, with a cutoff (wheal size) of 11.2 mm, an sIgE value of 7.9 kUA/L, and a stimulation index of 1.9. Specificity increased to 100% using the molecular component rAni s 1 with SPT, sIgE by ELISA, and ISAC-112. Neither rAni s 3 sensitization nor cross-reactivity with Pen m 1 was observed in patients sensitized to A simplex. Conclusion: rAni s 1 is recognized by 100% of our patients and is able to distinguish between patients allergic to A simplex and patients with acute urticaria who are sensitized to A simplex but have no clinical history of allergy to this parasite (AU)

Introducción: Las pruebas diagnósticas tradicionales como pruebas cutáneas (PC) e IgE específica (sIgE) con el extracto completo de Anisakis simplex tienen una baja especificidad. Esto conlleva a un sobrediagnóstico de alergia a A simplex. Objetivo: Nuestro objetivo fue comparar diferentes pruebas de diagnóstico basado en componentes moleculares. Métodos: Se estudiaron 34 pacientes con alergia a A simplex, 15 con urticaria aguda sensibilizados a A simplex pero sin historia clínica compatible con alergia a A simplex y 10 alérgicos a mariscos. A todos ellos se les realizaron PC, sIgE mediante ELISA e ISAC-112 y TAB con el extracto completo de A simplex y los componentes moleculares rAni s 1, rAni s 3 y nPen m 1. Se calculó y se comparó la sensibilidad y especificidad de cada prueba con diferentes puntos de corte. Resultados: Las PC, la sIgE y el TAB con el extracto completo de A simplex mostraron una especificidad del 72%, 68% y 70% con un punto de corte de 11,2 mm de tamaño de pápula, 7,9 kUA/L de sIgE y un índice de estimulación de 1,9, respectivamente. La especificidad incrementó al 100% utilizando el componente rAni s 1en PC e sIgE mediante ELISA e ISAC-112. No se observó sensibilización a rAni s 3 ni reactividad cruzada con nPen m 1 en los pacientes sensibilizados a A simplex. Conclusión: el alérgeno rAni s 1 es reconocido por el 100% de nuestros pacientes y nos permite distinguir entre pacientes alérgicos a A simplex y pacientes con urticaria aguda sensibilizados a A simplex sin historia clínica de alergia a éste parásito (AU)

In vivo and in vitro testing with rAni s 1 can facilitate diagnosis of Anisakis simplex allergy.

BACKGROUND: Traditional diagnostic tests such as skin prick tests (SPT) and specific IgE (slgE) against whole Anisakis simplex extract have low specificity. Consequently, allergy to A simplex is overdiagnosed. OBJECTIVE: Our aim was to compare tests used in component-resolved diagnosis. METHODS: We evaluated 34 patients with allergy to A simplex, 15 patients with acute urticaria who were sensitized to A simplex but had no clinical history of allergy to A simplex, and 10 patients allergic to seafood. SPT, slgE (ELISA and ISAC-I 12), and the basophil activation test (BAT) were performed with A simplex whole extract and the molecular components rAni s 1, rAni s 3, and nPen m 1. Sensitivity and specificity were calculated and compared with different cutoffs. RESULTS: With the A simplex whole extract, SPT, slgE, and BAT yielded specificity values of 72%, 68%, and 70%, respectively, with a cutoff (wheal size) of 11.2 mm, an slgE value of 7.9 kUAIL, and a stimulation index of 1.9. Specificity increased to 100% using the molecular component rAni s 1 with SPT, slgE by ELISA, and ISAC-112. Neither rAni s 3 sensitization nor cross-reactivity with Pen m 1 was observed in patients sensitized to A simplex. CONCLUSION: rAni s 1 is recognized by 100% of our patients and is able to distinguish between patients allergic to A simplex and patients with acute urticaria who are sensitized to A simplex but have no clinical history of allergy to this parasite.

Recommendations for the Use of In Vitro Methods to Detect Specific Immunoglobulin E: Are They Comparable?

Total and specific immunoglobulin (Ig) E can be detected in vitro using several commercially available methods. The largest share of the global market for these methods is held by the ImmunoCAP technique (Thermo Fisher, previously Phadia), Immulite (Siemens), and Hytec-288 (Hycor). Most comparative studies examine Immulite and ImmunoCAP, which differ methodologically but use similar units of measurement relative to the same standard of total IgE (WHO IgE Standard 75/502). Despite their similarity, these kits differ in their quantification of specific IgE, which varies depending on the allergen studied. Thus, specific IgE results obtained with ImmunoCAP and Immulite are not interchangeable. It is important to bear this in mind, especially when determining cutoff points as predictors of a response to oral challenge with specific food allergens. The method used in practice must be the same as the one in the publication guiding clinical decision making. We analyze differences between ImmunoCAP and ISAC microarray, 2 methods from the same manufacturer used to detect IgE to specific proteins (purified or recombinant). The results show that the IgE values obtained with ImmunoCAP are not equivalent to the corresponding values obtained with the ISAC microarray system (AU)

Existen disponibles en el mercado distintos métodos para la detección de la IgE total y específica. Los métodos con mayor cuota de mercado son método ImmunoCAP deThermofi sher (anteriormente Phadia), Immulite de Siemens y Hytec-288 de Hycor. La mayoría de los estudios comparativos se han realizado con Immulite e ImmunoCAP, que si bien difieren metodológicamente, emplean similares unidades de medida relativas al mismo estándar de IgE total (IgE Estándard OMS 75/502). Aunque estas técnicas estiman la cantidad de IgE total de forma similar, difieren en la cuantificación de la IgE específica. Se ha observado que estas diferencias varían en función del alérgeno al que se une la IgE específica. De esta forma, los resultados de la IgE específica para un alérgeno concreto obtenido por ImmunoCAP y por Immulite no son equiparables. Es importante tener en cuenta esta realidad, especialmente en el caso de puntos de corte determinados como predictores de la respuesta a una provocación oral con un alimento. El método empleado en la práctica debe ser idéntico al publicado como predictor. También analizamos las diferencias en la determinación de IgE frente a proteínas específicas (purificadas o recombinantes) por la misma casa comercial pero empleando distintas tecnologías, ImmunoCAP y micromatriz ISAC. Los datos demuestran que los resultados obtenidos por ImmunoCAP para la IgE específica no son equivalentes a los obtenidos mediante la micromatriz ISAC (AU)

Recommendations for the use of in vitro methods to detect specific immunoglobulin E: are they comparable?

Total and specific immunoglobulin (Ig) E can be detected in vitro using several commercially available methods. The largest share of the global market for these methods is held by the ImmunoCAP technique (Thermo Fisher, previously Phadia), Immulite (Siemens), and Hytec-288 (Hycor). Most comparative studies examine Immulite and ImmunoCAP, which differ methodologically but use similar units of measurement relative to the same standard of total IgE (WHO IgE Standard 75/502). Despite their similarity, these kits differ in their quantification of specific IgE, which varies depending on the allergen studied.Thus, specific IgE results obtained with ImmunoCAP and Immulite are not interchangeable. It is important to bear this in mind, especially when determining cutoff points as predictors of a response to oral challenge with specific food allergens. The method used in practice must be the same as the one in the publication guiding clinical decision making. We analyze differences between ImmunoCAP and ISAC microarray, 2 methods from the same manufacturer used to detect IgE to specific proteins (purified or recombinant).The results show that the IgE values obtained with ImmunoCAP are not equivalent to the corresponding values obtained with the ISAC microarray system.

Diagnostic utility of components in allergy to Anisakis simplex.

BACKGROUND: In our region, Anisakis allergy is responsible for 8% of acute urticarial reactions, 25% of which progress to anaphylactic shock. The poor specificity of skin tests and in vitro specific immunoglobulin (Ig) E means that Anisakis allergy is frequently overdiagnosed. OBJECTIVE: We studied the diagnostic value of 2 Anisakis allergens: rAni s 1 and rAni s 3. METHODS: Skin tests, the basophil activation test (BAT), and specific IgE determination were performed with rAni s 1 and 3 in 25 patients allergic to Anisakis, 17 atopic controls, and 10 controls with acute urticaria and positive skin test and sIgE results for Anisakis, but no allergy to Anisakis. RESULTS: For rAni s1, skin tests had a sensitivity and specificity of 100% and specific IgE had a sensitivity and specificity of 100% in the atopic control group and 90% in the urticaria control group. BAT had a sensitivity of 96.8% and a specificity of 100% in the atopic control group and 66.7% in the urticaria control group. For rAni s 3, only 1 patient had positive specific IgE results to rAni s 3. All other techniques gave negative results in patients and controls CONCLUSIONS: rAni s 1 is the major allergen of Anisakis and the target allergen when diagnosing allergy to Anisakis, rAni s 3 is not relevant when attempting to explain false-positive results.

Lupin allergy: a hidden killer in the home.

This review addresses the problem of lupin sensitization in the home environment. We summarize the data currently available on allergy to lupin, which has become, in recent years, a hidden killer in our homes. Since 2006, when lupin was included in European regulations as a food whose presence must be declared, the situation may have changed. Nevertheless, we must take into account the possibility of undeclared allergenic ingredients or the presence of 'hidden' allergens, given that contamination during food production processes may be a great risk for sensitized individuals. Furthermore, the United States, Japan, Australia and New Zealand still do not include lupin among the ingredients that must be listed on foodstuff labelling. Our responsibility is to educate the public so that they are aware of the danger and look for lupin in the labels of products that run the risk of containing it. Lupin allergy can manifest itself in isolation or in parallel to peanut allergy. Identification of the proteins causing possible cross-reactivity is complicated, and new structural studies are needed. To date, it has not been possible to clearly identify the allergens responsible for isolated lupin sensitization in relation to parallel and/or cross-sensitization between lupin and peanut. Most of the allergenic proteins of lupin are α- and ß-conglutins, with a lesser presence of γ- and δ-conglutins. A ß-conglutin corresponding to Lup an 1, with a sequence similar to Ara h 1, has been identified as a major allergen of lupin in patients with allergy following lupin ingestion.

In vitro diagnosis of anaphylaxis.

The application and development of new in vitro techniques aims to enable a diagnosis to be reached while incurring no risk for the patient, a situation which is particularly desirable in the case of severe reactions like anaphylaxis. The in vitro diagnosis of anaphylaxis includes, among other aspects, the serial measurement of mediators which are released in the course of an anaphylactic reaction such as tryptase, histamine, chymase, carboxypeptidase A3, platelet-activating factor and other products from mastocytes. The detection of agents which trigger the anaphylactic reaction can be made with the use of serologic methods: serum-specific IgE or with cellular tests which measure the release of basophil mediators (leukotrienes, histamine) or with the analysis of the expression of basophil markers, a technique known as the basophil activation test. These techniques offer interesting alternatives in the diagnosis of anaphylaxis. The basophil activation test provides important advantages in patients with anaphylaxis to beta-lactams, non-steroidal anti-inflammatory drugs, neuromuscular blocking agents and drugs where there is no technique to measure specific IgE.

In vitro diagnosis of immediate allergic reactions to drugs: an update.

Evaluation of allergic reactions to drugs is difficult because of the poor sensitivity of in vivo tests, which makes controlled administration of the drug necessary to confirm the diagnosis. In vitro tests are important in order to avoid the risks of in vivo testing. In the present review, we describe the different methods for detecting immunoglobulin (Ig) E antibodies that are specific to drugs involved in the development of type I (immediate) reactions. The 2 main in vitro methods are immunoassays and the basophil activation test, both of which have sufficient sensitivity and specificity for the detection of specific IgE antibodies, although with a limited number of drugs, and they have proven complementary to in vivo methods. We show the importance of the allergological workup of the patient within less than 1 year from the occurrence of the allergic reaction in order to obtain positive results in both in vivo and in vitro tests.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome: a multicenter study. II. Basophil activation by nonsteroidal anti-inflammatory drugs and its impact on pathogenesis.

BACKGROUND: Patients who are clinically hypersensitive to nonsteroidal anti-inflammatory drugs (NSAIDs) sometimes present basophil activation in vitro, and in 50% of cases a parallel response to release of sulfidoleukotrienes (cellular allergen stimulation test) is observed. These phenomena occur not only in clinically hypersensitive patients, but also in some healthy controls who tolerate NSAIDs. MATERIAL AND METHODS: We studied 16 clinically hypersensitive patients, 22 controls tolerating NSAIDs, and 29 healthy blood donors (clinical NSAID status unknown) using 2 different basophil isolation techniques (buffy coat or plasma leukocytes). RESULTS: In a population of 13 aspirin-tolerant healthy controls and 29 healthy blood donors, basophil activation with aspirin, diclofenac, and naproxen was analyzed at 4 different concentrations. The results in the 2 groups were quite similar in qualitative terms. Choosing a cutoff of 5% and a stimulation index >2, the proportion of positive results increased with the concentration. There were more positive results at all concentrations using the plasma leukocyte technique. CONCLUSIONS: The most important finding of this study is that basophil activation by NSAIDs occurs not only in clinically hypersensitive patients but also, to a very variable extent and on an individual basis, in apparently normal healthy individuals who tolerate NSAIDs. The phenomenon is clearly dose-related, and hypersensitive patients seem to react to lower NSAID concentrations.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome. A multicenter study. I. Clinical findings and in vitro diagnosis.

BACKGROUND: We present the results obtained from the largest series of in vitro diagnostic tests ever reported in patients with clinically validated hypersensitivity to acetylsalicylic acid (ASA)/nonsteroidal anti-inflammatory drugs (NSAID) compared with various categories of controls tolerating ASA/NSAIDs. This multicenter study, which was performed within the framework of the European Network for Drug Allergy (ENDA) group, showed that the basophil activation test (BAT), particularly when used with the 3 NSAIDs aspirin (ASA), diclofenac (DIC), and naproxen (NAP), allows us to confirm the diagnosis of NSAID hypersensitivity syndrome. The results of the cellular allergen stimulation test (CAST) frequently correlate with those of the BAT, although not always. An unexpected finding was that basophil activation by NSAIDs is not an all-or-nothing phenomenon restricted to clinically hypersensitive patients, but that it also occurs in a dose-related manner in some NSAID-tolerant control individuals.Therefore, NSAID hypersensitivity appears as a shift in the normal pharmacological response to NSAIDs. These findings allow us to formulate a new rational hypothesis about the mechanism of NSAID hypersensitivity syndrome, a mechanism that most authors continue to describe as "unknown." METHODS: We enrolled 152 patients with a history of hypersensitivity to NSAIDs and 136 control participants in 11 different centers between spring 2003 and spring 2006. Flowcytometric BAT was performed. RESULTS: The most noteworthy results of our study were that 57% of 140 patients presented very clear-cut positive BAT results to multiple NSAIDs, and 16% were entirely negative. In about 27% of cases, positive results were obtained with 1 or 2 concentrations of a single NSAID. There is clearly a correlation between the results of BAT and CAST. CONCLUSIONS: BAT seems particularly indicated in patients with a clinical history of NSAID intolerance, and in whom a provocation test is not advisable for ethical, clinical, or other reasons. Clear-cut positive results can be considered as confirming a history of NSAID hypersensitivity, although negative results may not exclude it.

Allergenicity and cross-reactivity of pine pollen.

BACKGROUND: Pine pollen has long been considered a non-allergenic pollen. The large size of the grain and its low levels of proteins are the main reasons invoked to explain this low allergenicity. The aim of this study was to describe the main allergenic bands of Pinus radiata (PR) and its cross-reactivity with other pine species, other conifers and grass pollen. METHODS: Sixty-five pine-pollen-allergic patients (51% also sensitized to grass pollen) were studied. Skin prick tests (SPT) to a battery of allergens including PR, Pinus pinea, Pinus sylvestris, Pinus nigra and Cupressus sempervirens pollens and specific IgE determination to PR and Pinus strobus were performed. IgE-immunoblotting to a PR extract and other pine pollens was also carried out. UniCAP inhibition and immunoblotting inhibition studies were performed to assess the cross-reactivity between different pollens. RESULTS: The SPTs were positive with all the pine pollen extracts tested in 69% of the patients. Specific IgE was positive to PR or P. strobus in 77% of the patients, and to Lolium perenne in 51%. Nine different allergenic bands were detected. The two main allergens were a 42 kDa band recognized by 85% of the patients and a band of approximately 6-8 kDa recognized by 40%. A high degree of cross-reactivity was observed between different pine pollen species, but not between pines and C. sempervirens pollen. A partial cross-reactivity could be seen between pine and grass pollens only in patients also sensitized to L. perenne. CONCLUSIONS: Pine pollen should be considered as a potential allergenic pollen especially where this pollen is abundant. The detection of a high number of patients that were monosensitized to pine pollen suggests the possibility of treating these patients with specific immunotherapy.

The epidemiology of drug allergy-related consultations in Spanish Allergology services: Alergológica-2005.

BACKGROUND: Allergic reactions to drugs constitute one of the most frequent reasons for consultations in Allergology services with an increasing prevalence in recent years. OBJECTIVES: To describe the results of the Alergológica-2005 study on the clinical characteristics of patients consulting for suspected drug allergies. METHODS: Alergológica-2005 was a descriptive, cross-sectional, prospective, observational study undertaken with 4991 patients treated for the first time in Allergology services in Spain. RESULTS: Seven hundred thirty-two patients (mean age 41.4 +/- 19.4 years) presented for drug allergies (62% females, 38% males). Diagnosis was confirmed in 26.6% of cases and rejected in 37.2%. Seventy five percent reported only cutaneous symptoms and 10% anaphylaxis. Forty-seven percent of reactions were caused by beta-lactams (63% of which were due to amoxicillin), 29% by nonsteroidal anti-inflammatory drugs (NSAIDs) and 10% by pyrazolones. Sixty nine children were treated for this reason, 8 of whom were diagnosed as drug allergic (5 to beta-lactams, 2 to NSAIDs and 1 to pyrazolones). CONCLUSIONS: Drug allergies are the third most important reason behind consultations in Allergology services, after bronchial asthma and rhinitis. Females are predominantly affected and the beta-lactams, NSAIDs and pyrazolones are the 3 drug families most responsible.

Diagnosis of immediate-type beta-lactam allergy in vitro by flow-cytometric basophil activation test and sulfidoleukotriene production: a multicenter study.

INTRODUCTION: This multicenter study aimed to evaluate the diagnostic value of 2 cellular tests based on basophil reactivity--the basophil activation test (BAT, Flow-CAST) and the sulfidoleukotriene release assay (CAST-ELISA)--in immediate-type beta-lactam allergy, particularly in patients with a clinical history of allergy and a negative skin test result. MATERIAL AND METHODS: In a multicenter study encompassing 10 European centers, 181 patients with a history of immediate-type beta-lactam allergy, and 81 controls, we evaluated the diagnostic efficiency of specific IgE determinations and of 2 cellular tests based on basophil reactivity, the BAT and the sulfidoleukotriene release assay. RESULTS: With Flow-CAST, sensitivity varied for individual beta-lactam allergens from 16% for penicilloyl-polylysine to 33% for amoxicillin, reaching 50% when all 5 allergens were considered. In beta-lactam-allergic patients with negative skin test results (22.8%), Flow-CAST showed positive results for at least 1 of the 5 allergens in 37%. Specificity varied from 89% to 97%, depending on the allergens used. In CAST-ELISA, the overall sensitivity in skin test-positive patients was 41.7%; in patients with negative skin test results it was 27.9%. Both tests were not absolutely correlated, so that when all the results were considered together, sensitivity increased to 64.3% and specificity varied for both tests combined from 73% to 92%. In contrast, specific IgE determinations in the same population yielded a lower sensitivity (28.3%). CONCLUSIONS: A diagnostic algorithm including skin tests and specific IgE, followed by cellular tests in negative patients and controlled challenge enabled us to confirm beta-lactam allergy in 92% of cases. This procedure would also allow us to avoid two-thirds of the required controlled challenges.

Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen-specific immunotherapy.

BACKGROUND: Specifically designed recombinant allergens with reduced IgE reactivity are promising candidates for a more defined, effective, and safer specific immunotherapy (SIT). OBJECTIVE: We sought to obtain hypoallergenic hybrid molecules which could potentially be applied to house dust mite (HDM) allergy treatment. METHODS: Two hybrid molecules (QM1 and QM2) derived from the two major Dermatophagoides pteronyssinus allergens, Der p 1 and Der p 2, were engineered by PCR, produced in Escherichia coli, and purified. The overall IgE-binding capacity of the hybrids was compared with their single components by Western blot, specific IgE, skin prick test (SPT), and IgE-inhibition assays. T cell proliferation assay were performed to confirm their retention of T cell reactivity. Immune responses to the hybrid molecules were studied in BALB/c mice. RESULTS: The IgE reactivity of both hybrid proteins was strongly reduced as evaluated by in vitro methods. Furthermore, in vivo SPTs performed on 106 HDM-allergic patients showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the individual components. Hybrid molecules induced higher T cell proliferation responses than those produced by an equimolecular mixture of Der p 1 and Der p 2. Immunization of mice with the hybrid proteins induced Der p 1- and Der p 2-specific IgG, which inhibited the binding of allergic patients' IgE to these natural allergens. CONCLUSION: QM1 and QM2 hybrids exhibited less IgE-binding activity but preserved immunogenicity and fulfilled the basic requirements for hypoallergenic molecules suitable for a future SIT of HDM allergy.