The Diagnosis of Allergy to Lipid Transfer Proteins.

PURPOSE OF REVIEW: To provide an update on the diagnosis of non-specific Lipid Transfer Protein (nsLTP) allergy. RECENT FINDINGS: More publications report the presence of nsLTP allergy in Northern European countries and nsLTP sensitisation in children. Individuals are more likely to have severe reactions if there is recognition of increasing numbers of LTP components. Diagnosis is problematic; not all those with nsLTP allergy will have a positive test to a peach extract containing Pru p 3, the peach nsLTP. Sensitisation to nsLTP is being reported in more countries, including to the nsLTP in Cannabis Sativa in North America. Meals containing multiple nsLTP foods are more likely to be involved in co-factor reactions. Component-resolved diagnostics are superior to skin prick tests, to determine sensitisation to the individual nsLTP allergens causing symptoms and, in the future, the Basophil Activation test may best discriminate between sensitization and clinical allergy.

Procedure for a standardized preparation of skin prick test solutions for the diagnosis of occupational type I allergies in the absence of commercial extracts.

In order to ensure valid diagnostics for occupational test allergen solutions despite the ongoing reduction in the availability of commercial test extracts, a plan B was initiated for the possible production of skin prick test (SPT) solutions in public pharmacies. For important occupational allergen sources (wheat and rye, storage mites, animal epithelia, mold material) laboratory extraction methods were analyzed in comparison to pharmacy compatible extraction methods regarding protein quantity and quality in SDS-PAGE combined with silver staining. Subsequently, using the example of bovine epithelia, adapted extraction procedures as well as in-process and final product controls were transferred to a public pharmacy. Allergen sources with a high protein content, such as wheat and rye grains as well as storage mites, showed good comparability of the extractable protein quantity and protein pattern, regardless of the applied extraction method. In contrast, allergen source materials with a low total protein content, such as animal epithelia and molds, can benefit from laboratory extraction conditions such as mechanical disruption and specific buffer additives. In the qualitative protein silver staining, characteristic protein patterns were identified for each allergen source. Depending on the extraction method, only minor differences in total protein patterns were observed in animal epithelia and molds. Using source materials from two suppliers, the resulting allergen extracts displayed clear differences in protein content in storage mites and quantitative and qualitative differences in molds. A practical preparation attempt of SPT solutions in a public pharmacy was successful. SPT solutions prepared with adapted pharmacy extraction methods showed a comparable protein and Bos d 2 allergen content and equivalent qualities in the protein pattern compared to a previously available commercial SPT solution. Accordingly, it can be assumed that standardized SPT solutions with sufficient allergen quality for occupational allergen sources can be prepared in public pharmacies if certified allergen sources with appropriate protein content are available.

Diagnostics of IgE-mediated occupational allergies: Between reality, requirements, and opportunities.

Occupational skin and respiratory allergies are among the most common occupational diseases in Germany. The identification of the allergy trigger is essential for the recognition of an occupational allergy as well as for effective individual prevention. However, occupational type I allergens are among the "rare" allergens and the possibilities of guideline-compliant diagnosis using quality-tested skin test solutions is becoming increasingly difficult due to the reduction in commercially available test allergens. In order to guarantee meaningful diagnostic workup for all affected insured persons with suspected occupational type I allergies and to ensure this in the future, a durable optimization, standardization, and availability of allergy tests for occupational allergic diseases is urgently required. The need for action has been recognized by the German Social Accident Insurance (DGUV), and steps to eliminate the diagnostic gaps have been initiated by a joint research project at the Institute for Prevention and Occupational Medicine of the DGUV (IPA) and the Paul Ehrlich Institut (PEI). The evaluation of alternative methods for the production of standardized test allergen solutions can also be used for newly emerging allergens in the workplace. New allergen sources at workplaces and thus also sensitization and allergies among employees can be expected as a result of changes in work processes and the introduction of new technologies and/or working materials, which are also introduced in connection with climate change and the concept of sustainability.

History of food allergy and where we are today.

The food allergy (FA) entity went through a long difficult road which led to much delay in its recognition. After long periods of denial and misdiagnosis, it attained its current designation as food hypersensitivity or allergy. This review will briefly address the evolution of the FA entity from the early BC era until our 21st century and highlight the milestones in the main aspects of diagnosis, treatment, prevention, and research. A great recognition of the allergy specialty was gained by the discovery of its main mediator -immunoglobulin E in 1967 - which also helped in classifying FA into IgE-mediated (immediate-type) and non-IgE-mediated. The cause of the increasing prevalence during the past few decades may be attributed to an increased food consumption and the consequences of modern lifestyle (the hygiene hypothesis). In addition to a skillful medical history-taking, helpful tests have been developed involving the skin or blood. The scratch test was modified to the prick test and in certain instances prick-by-prick. The use of intradermal test has been markedly reduced. Blood testing began by measuring specific-IgE antibodies (sIgE) in the serum using the radioallergosorbent test which went through multiple modifications to avoid radioisotope material and increase the test's sensitivity. The test was advanced to measure sIgE to individual allergen components. Recently, cellular tests were developed in the form of basophil activation or mast cell activation. In most cases, FA needs verification by appropriately-designed challenge testing. Regarding treatment, strict avoidance remains the basic approach. Certain food-labeling regulations led to some improvement in the problem of hidden food allergens but more is desired. Recently some protocols for oral immunotherapy (OIT) showed reasonable safety and efficacy in preventing reactions to accidental exposures. The protocol for peanut has been approved in the United States and other foods are expected to follow. Epicutaneous immunotherapy showed higher safety and promising efficacy. Sublingual immunotherapy might follow as well. Studies on the use of certain biologicals, alone or in combination of OIT, showed promising findings. Very recently, omalizumab was approved in the United States for patients with multiple FA. A major change in the strategy of prevention is the benefit of introducing allergenic foods at an early age (4-6 months). Research on FA markedly flourished in recent decades with increasing numbers of investigators, funding, publications, and education. Despite the major strides, still more awaits exploration with expected better understanding and practice of FA.

Novel IgE crosslinking-induced luciferase expression method using human-rat chimeric IgE receptor-carrying mast cells.

BACKGROUND: The measurement of antigen-specific serum IgE is common in clinical assessments of type I allergies. However, the interaction between antigens and IgE won't invariably trigger mast cell activation. We previously developed the IgE crosslinking-induced luciferase expression (EXiLE) method using the RS-ATL8 mast cell line; however, the method may not be sensitive enough in some cases. METHODS: In this study, we introduced an NF-AT-regulated luciferase reporter gene into the RBL-2H3 rat mast cell line and expressed a chimeric high-affinity IgE receptor (FcεRI) α chain gene, comprising an extracellular domain from humans and transmembrane/intracellular domains from rats. RESULTS: We generated multiple clones expressing the chimeric receptor. Based on their responsiveness and proliferation, we selected the HuRa-40 clone. This cell line exhibited significantly elevated human α chain expression compared to RS-ATL8 cells, demonstrating a 10-fold enhancement of antigen-specific reactivity. Reproducibility across different batches and operators was excellent. Moreover, we observed a detectable response inhibition by an anti-allergy drugs (omalizumab and cyclosporin A). CONCLUSIONS: HuRa-40 cells-which carry the human-rat chimeric IgE receptor-comprise a valuable reporter cell line for the EXiLE method. Their versatility extends to various applications and facilitates high-throughput screening of anti-allergy drugs.

PEGylated liposomes for diagnosis of polyethylene glycol allergy.

BACKGROUND: Polyethylene glycol (PEG) is a nonprotein polymer that is present in its native (unbound) form as an excipient in a range of products. It is increasingly being utilized clinically in the form of PEGylated liposomal medications and vaccines. PEG is the cause of anaphylaxis in a small percentage of drug reactions; however, diagnosis of PEG allergy is complicated by the variable and poor diagnostic performance of current skin testing protocols. OBJECTIVE: We assessed the diagnostic performance of PEGylated lipid medications as an alternative to currently described tests that use medications containing PEG excipients. METHODS: Nine patients with a strong history of PEG allergy were evaluated by skin testing with a panel of PEG-containing medications and with a PEGylated lipid nanoparticle vaccine (BNT162b2). Reactivity of basophils to unbound and liposomal PEG was assessed ex vivo, and specificity of basophil responses to PEGylated liposomes was investigated with a competitive inhibition assay. More detailed information is provided in this article's Methods section in the Online Repository available at www.jacionline.org. RESULTS: Despite compelling histories of anaphylaxis to PEG-containing medications, only 2 (22%) of 9 patients were skin test positive for purified PEG or their index reaction-indicated PEG-containing compound. Conversely, all 9 patients were skin test positive or basophil activation test positive to PEGylated liposomal BNT162b2 vaccine. Concordantly, PEGylated liposomal drugs (BNT162b2 vaccine and PEGylated liposomal doxorubicin), but not purified PEG2000, consistently induced basophil activation ex vivo in patients with PEG allergy but not in nonallergic controls. Basophil reactivity to PEGylated nanoparticles competitively inhibited by preincubation of basophils with native PEG2000. CONCLUSION: Presentation of PEG on the surface of a lipid nanoparticle increases its in vivo and ex vivo allergenicity, and improves diagnosis of PEG allergy.

Molecular allergology: a clinical laboratory tool for precision diagnosis, stratification and follow-up of allergic patients.

Identification of the molecular culprits of allergic reactions leveraged molecular allergology applications in clinical laboratory medicine. Molecular allergology shifted the focus from complex, heterogeneous allergenic extracts, e.g. pollen, food, or insect venom, towards genetically and immunologically defined proteins available for in vitro diagnosis. Molecular allergology is a precision medicine approach for the diagnosis, stratification, therapeutic management, follow-up and prognostic evaluation of patients within a large range of allergic diseases. Exclusively available for in vitro diagnosis, molecular allergology is nonredundant with any of the current clinical tools for allergy investigation. As an example of a major application, discrimination of genuine sensitization from allergen cross-reactivity at the molecular level allows the proper targeting of the culprit allergen and thus dramatically improves patient management. This review aims at introducing clinical laboratory specialists to molecular allergology, from the biochemical and genetic bases, through immunological concepts, to daily use in the diagnosis and management of allergic diseases.

Sensitization to Food and Aero-Allergens in Children with Coeliac Disease Assessed with the Use of a Multiplex Molecular Diagnostic Technique.

(1) Background. Coeliac disease (CD) often co-occurs with autoimmune conditions or genetic syndromes, but there are few studies on the co-existence of CD and immunoglobulin E (IgE)-mediated allergies. The purpose of this study was to assess sensitization to food and aero-allergens in pediatric patients with CD. (2) Methods. A multiplex ALEX®2 test was used to determine specific IgEs (sIgEs). (3) Results. The study included 108 children newly diagnosed with CD. Allergen extract- and/or allergen molecule-sIgEs were detected in 49.1% of children. Most children (41.5%) were sensitized to both inhalant and food allergens. The three most common aero-allergens (timothy pollen, ryegrass, silver birch) were molecules Phl p 1, Lol p 1, and Bet v 1. The most common food allergens (hazelnut, apple, and peanut) were Cor a 1, Mal d 1, and Ara h 8 molecules of the PR-10 subfamily. Patients were not sensitized to cereal allergens containing gluten. Spearman's rank correlation analysis of sensitized patients showed a significant positive relationship (r = 0.31) between the patients' age and the occurrence of positive sIgEs (≥0.3 kUA/L) for inhalant allergen molecules (p = 0.045). In sensitized patients, mainly symptoms of inhalant allergy were observed, such as hay fever, conjunctivitis, and bronchial asthma. (4) Conclusions. The current study indicates the co-occurrence of IgE sensitization to food and inhalant allergens in children with CD. The study highlights the need to take a closer look at the diagnosis of IgE-mediated allergy in patients with CD, which may help in their care and lead to a better understanding of the relationship between CD and IgE-mediated allergy.

Global assessment of the knowledge and confidence in managing allergic disorders among primary care pediatricians across Europe: An EAACI task force report.

BACKGROUND: Pediatricians are often the first point of contact for children in Primary Care (PC), but still perceive gaps in their allergy knowledge. We investigated self-perceived knowledge gaps and educational needs in pediatricians across healthcare systems in Europe so that future educational initiatives may better support the delivery of allergy services in PC. METHOD: A multinational survey was circulated to pediatricians who care for children and adolescents with allergy problems in PC by the EAACI Allergy Educational Needs in Primary Care Pediatricians Task Force from February to March 2023. A 5-point Likert scale was used to assess the level of agreement with questionnaire statements. Thirty surveys per country were the cut-off for inclusion and statistical analysis. RESULTS: In this study, 1991 respondents were obtained from 56 countries across Europe and 210 responses were from countries with a cut-off below 30 participants per country. Primary care pediatricians (PCPs) comprised 74.4% of the respondents. The majority (65.3%) were contracted to state or district health services. 61.7% had awareness of guidelines for onward allergy referral in their countries but only 22.3% were aware of the EAACI competencies document for allied health professionals for allergy. Total sample respondents versus PCPs showed 52% and 47% of them have access to allergy investigations in their PC facility (mainly specific IgE and skin prick tests); 67.6% and 58.9% have access to immunotherapy, respectively. The main barrier to referral to a specialist was a consideration that the patient's condition could be diagnosed and treated in this PC facility, (57.8% and 63.6% respectively). The main reasons for referral were the need for hospital assessment, and partial response to first-line treatment (55.4% and 59.2%, 47% and 50.7%, respectively). Learning and assessment methods preference was fairly equally divided between Traditional methods (45.7% and 50.1% respectively) and e-learning 45.5% and 44.9%, respectively. Generalist physicians (GPs) have the poorest access to allergy investigations (32.7%, p = .000). The majority of the total sample (91.9%) assess patients with allergic pathology. 868 (43.6%) and 1117 (46.1%), received allergy training as undergraduates and postgraduates respectively [these proportions in PCPs were higher (45% and 59%), respectively]. PCPs with a special interest in allergology experienced greater exposure to allergy teaching as postgraduates. GPs received the largest amount of allergy teaching as undergraduates. Identifying allergic disease based on clinical presentation, respondents felt most confident in the management of eczema/atopic dermatitis (87.4%) and rhinitis/asthma (86.2%), and least confident in allergen immunotherapy (36.9%) and latex allergy (30.8%). CONCLUSION: This study exploring the confidence of PCPs to diagnose, manage, and refer patients with allergies, demonstrated knowledge gaps and educational needs for allergy clinical practice. It detects areas in need of urgent improvement especially in latex and allergen immunotherapy. It is important to ensure the dissemination of allergy guidelines and supporting EAACI documents since the majority of PCPs lack awareness of them. This survey has enabled us to identify what the educational priorities of PCPs are and how they would like to have them met.

Evaluation of skin prick location on the forearm using a novel skin prick automated test device.

Background: The skin prick test (SPT) is the gold standard for identifying allergic sensitization in individuals suspected of having an inhalant allergy. Recently, it was demonstrated that SPT using a novel skin prick automated test (SPAT) device showed increased reproducibility and tolerability compared to the conventional SPT, among other benefits. Objective: This study aimed to evaluate prick location bias using the novel SPAT device. Methods: A total of 118 volunteers were enrolled in this study and underwent SPATs with histamine (nine pricks) and glycerol control (one prick) solutions on the volar side of their forearms. Imaging of the skin reactions was performed using the SPAT device, and the physician determined the longest wheal diameter by visually inspecting the images using a web interface. Prick location bias was assessed along the medial vs. lateral and proximal vs. distal axes of the forearm. Results: In total, 944 histamine pricks were analyzed. Four medial and four lateral histamine pricks were grouped, and wheal sizes were compared. The longest wheal diameters were not significantly different between the medial and lateral prick locations (p = 0.41). Furthermore, the pricks were grouped by two based on their position on the proximal-distal axis of the forearm. No significant difference was observed among the four groups of analyzed prick locations (p = 0.73). Conclusion: The prick location on the volar side of the forearm did not influence wheal size in SPAT-pricked individuals.

Corrigendum: Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays.

[This corrects the article DOI: 10.3389/fimmu.2023.1155613.].

Problems Encountered Using Fungal Extracts as Test Solutions for Fungal Allergy Diagnosis.

Fungal allergy is a worldwide public health burden, and problems associated with a reliable allergy diagnosis are far from being solved. Especially, the lack of high-quality standardized fungal extracts contributes to the underdiagnosis of fungal allergy. Compared to the manufacturing processes of extracts from other allergen sources, the processes used to manufacture extracts from fungi show the highest variability. The reasons for the high variability are manifold as the starting material, the growth conditions, the protein extraction methods, and the storage conditions all have an influence on the presence and quantity of individual allergens. Despite the vast variety of studies that have analyzed the impact of the different production steps on the allergenicity of fungal allergen extracts, much remains unknown. This review points to the need for further research in the field of fungal allergology, for standardization and for generally accepted guidelines on the preparation of fungal allergen extracts. In particular, the standardization of fungal extracts has been and will continue to be difficult, but it will be crucial for improving allergy diagnosis and therapy.

Why the role of mHealth in allergy diagnosis and treatment adherence cannot be overlooked.

BACKGROUND: Allergic diseases-rhinitis and asthma-are the most common chronic conditions affecting adults. Traditional approaches to allergy diagnosis and treatment do not meet the health needs of all patients. Treatment adherence remains a challenge for physicians. The ubiquity of Internet access paired with limited in-person contact with medical personnel in the course of the COVID-19 pandemic demonstrated the potential of mHealth in communicating health information. BODY: The abundance of new applications dedicated to various medical specialties encourages reflection on the informed use of such tools. The paper takes a closer look at the potential of mHealth and presents conclusions of selected studies focusing on the use of good apps. The strength weakness opportunities threats analysis was used to illustrate the strengths of the mHealth strategy, as well as its advantages, limitations and areas in need of further development. CONCLUSION: The strength of mHealth depends on the quality and quantity of the collected patient data, its reliable processing, as well as publication of outcomes and conclusions from analyses. Therefore, it is necessary to promote the use of validated applications among patients, physicians and medical staff.

Allergy patient-specific IgE antibody shows significantly stability during 3 months of storage at multiple temperatures from -80 to 25°C.

The detection of allergen-specific IgE antibodies is an important biomarker for the diagnosis and treatment monitoring of allergic diseases. And the pre-analytical phase is an important part of the overall quality of the laboratory. In this study, 44 patients with allergic diseases (including 23 patients with allergic rhinitis, 12 patients with allergic rhinitis and asthma, and 9 patients with allergic dermatitis) were included in the outpatient center of the Department of Allergy, the First Affiliated Hospital of Guangzhou Medical University. We mixed the serums of the above 44 patients (approximately 0.8 ml of serum volume per patient) into a large volume of serum pool (about 35 ml in total) and divided into 26 parts. And 26 serum samples were stored at 4 different temperatures for 90 days to observe the stability of sIgE antibodies to 16 allergens in serum. The results show that serum sIgE antibody titers in patients with allergic diseases show significant stability during 90 days of storage, even at room temperature. Good stability even after up to 10 freeze-thaw cycles under low temperature storage conditions.

Evolution Toward Chip-Based Arrays in the Laboratory Diagnosis of Human Allergic Disease.

Multiplex-based specific IgE antibody assays have emerged into the clinical immunology laboratory through the combined use of pure, recombinant allergenic molecules and new methods to simultaneously and accurately analyze specific IgE antibodies to hundreds of allergens. This review traces the historical development and examines outstanding questions related to the strengths and limitations of these new molecular allergen multipex technologies for the assessment of human allergic sensitization. Multiplexed technologies are poised to provide the most cost-effective and comprehensive evaluation of patients with suspected allergy as compared with the commonly used singleplex autoanalyzers. How analytically sensitive and quantitative are the multiplex technologies, down to 0.1 kUA/L? Because each allergen is viewed as a unique assay, how will analytical and clinical performance be documented at the manufacturing and clinical laboratory levels to guarantee reproducibility and obtain government regulatory clearance? Will interference by naturally occurring allergen-specific IgG compromise analytical performance? Successful resolution of these and other questions covered in this review will position multiplex technologies to become the single most-effective means of screening patients for allergic sensitization, assessing IgE antibody cross-reactivity, and planning therapy directed at the patient with allergy.