Optimization of Basophil Activation Test in the Diagnosis and Qualification for Allergen-Specific Immunotherapy in Children with Respiratory Allergy to the House Dust Mite Dermatophagoides pteronyssinus.

The aim of this study was to optimize a basophil activation test in the detection of allergy to the house dust mite Dermatophagoides pteronyssinus in children with allergic respiratory diseases. This study involved 32 cases, 13 girls and 19 boys aged 4-17 years, with perennial asthma or allergic rhinitis caused by D. pteronyssinus. The control group consisted of 13 girls and 19 boys aged 4-17 years with seasonal allergic asthma or rhinitis provoked by Timothy or birch pollen. House dust mite (HDM) allergy was excluded in the controls based on their medical history, skin prick test (SPT) results and sIgE determination. In all patients, a basophil activation test (BAT) was performed with five dilutions of D. pteronyssinus allergen (the dilution series ranged from 22.5 to 0.00225 ng/mL). The results were analyzed by using the receiver operating characteristics (ROC) to determine the optimal allergen concentrations, outcome measures and cut-off points that would differentiate most accurately between HDM-allergic and non-allergic patients. As a "gold standard", criteria for allergen-specific immunotherapy with D. pteronyssinus or respective pollens were applied by an experienced pediatric allergist following the guidelines of the European Academy of Allergy and Clinical Immunology. The highest diagnostic efficiency was yielded by the protocol assuming a cut-off value of 9.76% activated basophils after activation with a single allergen concentration of 2.25 ng/mL (sensitivity 90.6%, specificity 100%). This protocol yielded 3 (4.7%) misclassifications, all false negative, when compared with the "gold standard". There was a strong correlation with the BAT results at 22.5, 2.25 and 0.225 ng/mL (respectively r = 0.90 and r = 0.78, p < 0.001), as well as between the BAT at 2.25 ng/mL and SPT (r = 0.82, p < 0.001) and between the SPT and sIgE levels (r = 0.78, p < 0.001). High cross-reactivity between D. pteronyssinus and D. farinae was confirmed based on the BAT at 22.5 ng/mL (r = 0.82, p < 0.001). In conclusion, the BAT showed very good concordance with the result of a meticulous process of decision-making that combined validated allergy tests (SPT, sIgE) with expert guidelines, specialist knowledge and experience. Facing the risk of the incorrect qualification of patients for costly, long-lasting and potentially risky allergen-specific immunotherapy, the inclusion of a basophil activation test into diagnostic process seems fully justified.

Blood stored in EDTA tubes provides accurate peanut basophil activation test results for 48 hours.

BACKGROUND: The peanut basophil activation test (BAT) has demonstrated excellent diagnostic accuracy with heparinized blood, but its clinical utility is limited by the short stability of samples stored in this anticoagulant. OBJECTIVE: Using EDTA anticoagulated blood, these investigations determined if Peanut BAT sample stability can be extended to 2 days, the minimum stability requirement for diagnostic tests currently offered through American reference laboratories. METHODS: Peanut non-allergic control (NAC), peanut IgE sensitized (PS), and peanut allergic (PA) children aged 6 months through 17 years were recruited from members of Kaiser Permanente Southern California. EDTA anti-coagulated blood samples were collected from participants, shipped to a centralized laboratory, and stored at 4oC for peanut BAT testing 1 and 2 days later. RESULTS: Peanut BAT results for 23 unblinded participants were used to establish sample rejection and interpretation criteria that were subsequently validated in a prospective double-blind study involving 112 additional children (39-NAC, 36-PS, 37-PA). Of 105 blinded blood samples tested on each study day, 88 (84%) day-1 and 90 (86%) day-2 peanut BAT results were considered interpretable, with diagnostic accuracies of 95.5% and 94.4%, respectively. Moreover, all interpretable PA results were considered positive (100% sensitivity). CONCLUSION: Using EDTA anti-coagulated blood samples collected remotely, 1 and 2 days before testing, study results highlight the favorable diagnostic performance characteristics of the peanut BAT and provide further evidence that the test could be readily operationalized for clinical use by interested commercial reference laboratories.

Evaluation of the passive mast cell activation test for identifying allergens in perioperative anaphylaxis: a study protocol for a prospective diagnostic accuracy study.

INTRODUCTION: Perioperative anaphylaxis (POA) can lead to significant complications. Therefore, accurate identification of allergens for POA patients is critical to ensure the safety of future surgical and anaesthetic procedures. Existing perioperative allergen detection methods face challenges in sensitivity and specificity. The passive mast cell activation test (pMAT) has recently emerged as a potential diagnostic tool. Our study aims to evaluate the diagnostic efficacy of pMAT for identifying perioperative allergens, with a focus on non-depolarising neuromuscular blocking agents, the most common culprits of POA. METHODS AND ANALYSIS: This prospective diagnostic accuracy study will measure the diagnostic accuracy of pMAT in POA patients. Participants will undergo skin testing (ST), basophil activation testing (BAT) and pMAT. The diagnostic validity of pMAT will be assessed based on the results of ST and BAT. The assessment of diagnostic accuracy will include sensitivity, specificity, likelihood ratios, and false-positive and false-negative rates while measurement of the consistency rate will assess reliability. ETHICS AND DISSEMINATION: This study has been approved by the Institutional Review Board of China-Japan Friendship Hospital (2023-KY-247). Results will be disseminated through academic presentations and peer-reviewed journal publications and will provide valuable scientific data and some new insights into the diagnostic accuracy of pMAT.

BAT and MAT for diagnosis of peanut allergy: A systematic review and meta-analysis.

Basophil activation test (BAT) or the mast cell activation test (MAT) are two in vitro tests that are currently being studied in food allergy as diagnostic tools as an alternative to oral food challenges (OFCs). We conducted a meta-analysis on BAT and MAT, assessing their specificity and sensitivity in diagnosing peanut allergy. Six databases were searched for studies on patients suspected of having peanut allergy. Studies using BAT or MAT to peanut extract and/or component as diagnostic tools with results given in percentage of CD63 activation were included in this meta-analysis. Study quality was evaluated with the QUADAS-2 tool. On the 11 studies identified, eight focused exclusively on children, while three included a mixed population of adults and children. Only one study provided data on MAT, precluding us from conducting a statistical analysis. The diagnostic accuracy of BAT was higher when stimulated with peanut extract rather than Ara h 2 with a pooled specificity of 96% (95% CI: 0.89-0.98) and sensitivity of 0.86 (95% CI: 0.74-0.93). The sensitivity and specificity of BATs in discriminating between allergic and sensitized patients were studied as well, with pooled analysis revealing a sensitivity of 0.86 (95% CI: 0.74; 0.93) and a specificity of 0.97 (95% CI: 0.94, 0.98). BATs, when stimulated with peanut extracts, exhibit a satisfactory sensitivity and specificity for the diagnosis of peanut allergy and can help to discriminate between allergic individuals and those only sensitized to peanuts. More investigations on the potential for MATs diagnostic methods are warranted.

Basophil Activation Test Is Inferior to Provocation Test in Diagnosing Aspirin Hypersensitivity.

INTRODUCTION: Diagnostic of aspirin (ASA) hypersensitivity is largely based on provocation tests. However, they have significant limitations including influence of medications, necessity of hospitalization, and safety issues. Basophil activation test (BAT) seems to be a promising in vitro alternative. It has already proven to be a useful tool for diagnosing IgE-mediated allergy to certain food and airborne allergens as well as insects venoms. The aim of the study was to assess performance of BAT in diagnosing aspirin hypersensitivity in comparison with current golden standard (oral provocation test, OPT). METHODS: The study group comprised 148 adult patients with suspicion of aspirin hypersensitivity, including 51 (36%) with chronic urticaria, 73 (51%) with asthma, and 55 (39%) with chronic sinusitis. The control group was 10 healthy adult patients who used NSAIDs during preceding year with good tolerance. BAT with ASA was conducted in all the participants. Additionally, in the study group, OPT was performed with cumulative dose of 1,000 mg of ASA. RESULTS: Out of 148 study group participants, 114 underwent BAT and ASA provocation with conclusive results acquired in both tests. In this group, the threshold for positive BAT was 4.9%. Sensitivity and specificity of BAT were found to be 55.9% and 75%, respectively, with a positive predictive value of 77% and a negative predictive value of 54%. The highest sensitivity (78%) was found in subgroup patients with chronic urticaria, while specificity was highest in the subgroup with chronic respiratory diseases (87%). CONCLUSION: Despite significant advantages of BAT such as safety, no influence of drugs, and objectivity, its performance makes it inferior to current standard in ASA hypersensitivity.

Basophil activation test in the diagnostic workup of perioperative anaphylaxis due to neuromuscular blocking agents: A case series and implications for practice.

In Australia, neuromuscular blocking agents are the leading cause of perioperative anaphylaxis. Current investigation of suspected anaphylaxis includes tryptase levels, serum immunoglobulin E (IgE) levels, and skin testing, including intradermal testing and skin prick testing. The gold standard for the diagnosis of a hypersensitivity reaction is a challenge test, but this poses a risk to patient safety. An alternative test, known as the basophil activation test (BAT) is a form of cellular in vitro testing using flow cytometry to measure the degree of basophil degranulation within a sample of blood following exposure to an allergen. This acts as a surrogate marker for mast cell and basophil activation, thereby identifying IgE-mediated allergy. It is most commonly used to supplement equivocal findings from initial in vitro testing to assist in confirming the diagnosis of a hypersensitivity reaction and identify the causative agent. We present a case series in which five patients with suspected anaphylaxis underwent a BAT, demonstrating its role and limitations in allergy testing within Australia.

Basophil activation in insect venom allergy: comparison of an established test using liquid reagents with a test using 5-color tubes with dried antibody reagents.

BACKGROUND: Flow cytometry-based basophil activation tests (BAT) have been performed with various modifications, differing in the use of distinct identification and activation markers. Established tests use liquid reagents while a new development involves the use of tubes with dried antibody reagents. The aim of this pilot study was to compare these two techniques in patients with insect venom allergy. METHODS: Seventeen patients with an insect venom allergy were included in the study. The established "BAT 1" utilizes conventional antibody solutions of anti-CCR3 for basophil identification and anti-CD63 to assess basophil activation, whereas "BAT 2" uses dried anti-CD45, anti-CD3, anti-CRTH2, anti-203c and anti-CD63 for identification and activation measurement of basophils. Negative and positive controls as well as incubations with honey bee venom and yellow jacket venom at three concentrations were performed. RESULTS: Seven patients had to be excluded due to low basophil counts, high values in negative controls or negative positive controls. For the remaining 10 patients the overall mean (± SD) difference in activated basophils between the two tests was 0.2 (± 12.2) %P. In a Bland-Altman plot, the limit of agreement (LoA) ranged from 24.0 to -23.7. In the qualitative evaluation (value below/above cut-off) Cohen's kappa was 0.77 indicating substantial agreement. BAT 2 took longer to perform than BAT 1 and was more expensive. CONCLUSION: The BAT 2 technique represents an interesting innovation, however, it was found to be less suitable compared to an established BAT for the routine diagnosis of insect venom allergies.

PD-BAT: A novel approach of pooling basophil donors for expansion of commercial laboratory testing of Chronic Spontaneous Urticaria.

The type II autoimmune subtype of Chronic Spontaneous Urticaria (CSU) is characterized by the presence of IgG autoantibodies targeting IgE or the IgE high-affinity receptor (FcεRI) on mast cells and basophils. In evaluation of CSU patients, indirect basophil activation testing (BAT), has been utilized, involving the mixing of patient serum with heterologous peripheral blood donors, followed by flow cytometric assessment of basophil markers. However, the reliability of the indirect BAT results hinges on the quality of the donor basophils utilized. In this study, we introduce an innovative approach where multiple potential basophil donors undergo rigorous BAT characterization alongside control samples. By selecting and pooling donors with optimal performance, we significantly enhance the inter-assay reproducibility of the indirect BAT test.

Mast cell versus basophil activation test in allergy: Current status.

In the past two decades, we witnessed the evolution of the basophil activation test (BAT) from mainly research applications to a potential complementary diagnostic tool to document IgE-dependent allergies. However, BAT presents some technical weaknesses. Around 10%-15% of tested patients are non-responders, BAT can be negative immediately post-reaction and the use of fresh basophils, ideally analysed within 4 h of collection, restricts the number of tests that can be performed per sample. The need for fresh basophils is especially limiting when conducting batch analyses and interlaboratory comparisons to harmonize BAT methodology. These limitations significantly hinder the wider application of BAT and urge the development of alternative testing, such as the mast cell activation test (MAT). The essential difference between BAT and MAT is the heterogeneity of the starting material used to perform the assays. Mast cells are tissue-resident, so cannot be easily accessed. Current alternative sources for functional studies are generating primary human mast cells, differentiated from donor progenitor cells, or using immortalized mast cell lines. Hence, the methodological approaches for MAT are not only vastly different from BAT, but also different among MAT protocols. This review summarizes the advantages and disadvantages of BAT and MAT assays, dedicating special attention to elucidating the key differences between the cellular sources used and provides an overview of studies hitherto performed comparing BAT and MAT in the diagnosis of IgE-mediated food and drug allergies.

Possible alternative strategies to implement basophil activation testing in multicentric studies.

The Basophil Activation Test (BAT) enables flow cytometry characterization of basophil reactivity against specific allergenic molecules. The focus now revolves around democratizing this tool, but, as blood sample stability could be challenging, after having developed a simplified approach, herein, we aimed to characterize two strategies for implementing BAT in multicentric studies: store and ship blood before or after sample processing. Fresh heparin- and EDTA-anticoagulated whole blood samples followed both BAT workflows: "collect, store, process & analyze" or "collect, process, store & analyze". Storage temperatures of 18-25 °C or 2-8 °C and preservation times from 0 to 7 days were considered. Interleukin-3 was also evaluated. With the "collect, store, process & analyze" workflow, heparin-anticoagulated blood and 18-25 °C storage were better than other conditions. While remaining possible, basophil activation exhibited a possible reactivity decay after 24 h. Under the conditions tested, interleukin-3 had no role in enhancing basophil reactivity after storage. Conversely, the "collect, process, store & analyze" workflow demonstrated that either heparin- or EDTA-anticoagulated blood can be processed and kept up to 7 days at 18-25 °C or 2-8 °C before being analyzed. Various strategies can be implemented to integrate BAT in multicentric studies. The "collect, store, process & analyze" workflow remains a simplified logistical approach, but depending on time required to ship from the clinical centers to the reference laboratories, it might not be applicable, or should be used with caution. The "collect, process, store & analyze" workflow may constitute a workflow improvement to provide significant flexibility without impact on basophil reactivity.

Basophil activation test in the food allergy clinic: its current use and future applications.

INTRODUCTION: The basophil activation test (BAT) has shown evidence of high sensitivity and high specificity to support the diagnosis of IgE-mediated allergy. It is a functional test that uses live cells analyzed by flow cytometry and thus needs to be performed within 24h of blood collection. BAT has shown to be reproducible and reliable when tested in a clinical diagnostic laboratory with standardized protocols and flow cytometry settings. AREAS COVERED: In this review, we summarize the evidence to support clinical use of BAT and the next steps required for clinical implementation for an improve clinical care for patients with suspected IgE-mediated food allergy. EXPERT OPINION: BAT has recently been included in Clinical Guidelines of Food Allergy Diagnosis and its implementation in clinical practice depends largely on availability. Proposed clinical applications of the BAT include: distinction between food allergy and asymptomatic IgE sensitization; determination of food allergic status to peanut, tree nuts and seeds in polysensitized children; evaluation of tolerance to baked egg and baked milk in egg and milk allergic children; identification of patients at high-risk of severe allergic reactions; monitoring for spontaneous resolution of food allergy; confirmation of eligibility for specific treatments of food allergy; prediction and monitoring of response to immunomodulatory treatments.

Targeting inhibitory Siglec-3 to suppress IgE-mediated human basophil degranulation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs. OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for 1 hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to N-formylmethionyl-leucyl-phenylalanine, providing evidence that this inhibition is IgE pathway-specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. CONCLUSIONS: Pretreating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions.

Drug hypersensitivity reactions (DHRs) to platinum-based compounds (PCs) are on the rise, and their personalized and safe management is essential to enable first-line treatment for these cancer patients. This study aimed to evaluate the usefulness of the basophil activation test by flow cytometry (BAT-FC) and the newly developed sIgE-microarray and BAT-microarray in diagnosing IgE-mediated hypersensitivity reactions to PCs. A total of 24 patients with DHRs to PCs (20 oxaliplatin and four carboplatin) were evaluated: thirteen patients were diagnosed as allergic with positive skin tests (STs) or drug provocation tests (DPTs), six patients were diagnosed as non-allergic with negative STs and DPTs, and five patients were classified as suspected allergic because DPTs could not be performed. In addition, four carboplatin-tolerant patients were included as controls. The BAT-FC was positive in 2 of 13 allergic patients, with a sensitivity of 15.4% and specificity of 100%. However, the sIgE- and BAT-microarray were positive in 11 of 13 DHR patients, giving a sensitivity of over 84.6% and a specificity of 90%. Except for one patient, all samples from the non-allergic and control groups were negative for sIgE- and BAT-microarray. Our experience indicated that the sIgE- and BAT-microarray could be helpful in the endophenotyping of IgE-mediated hypersensitivity reactions to PCs and may provide an advance in decision making for drug provocation testing.

Validation of the NUT CRACKER Diagnostic Algorithm and Prediction for Cashew and Pistachio Co-Allergy.

BACKGROUND: Because of the high cross-sensitization among tree nuts, the NUT CRACKER (Nut Co-reactivity-Acquiring Knowledge for Elimination Recommendations) study proposed a diagnostic algorithm to minimize the number of required oral food challenges (OFCs). OBJECTIVE: To validate the algorithm for cashew and pistachio allergy and determine markers for allergic severity. METHODS: Patients (n = 125) with a median age of 7.8 (interquartile range, 5.9-11.2) years with suspected tree nut allergy were evaluated prospectively with decision tree points on the basis of skin prick test (SPT), basophil activation test (BAT), and knowledge of the coincidence of allergies. Validation of allergic status was determined by OFC. Markers of clinical severity were evaluated using the combined original and prospective cohort (n = 187) in relationship to SPT, BAT, and Ana o 3-sIgE. RESULTS: Reactivity to cashew in SPT, BAT, and Ana o 3-sIgE and the incidence of abdominal pain on challenge were significantly higher in dual-allergic cashew/pistachio patients (n = 82) versus single cashew allergic patients (n = 18) (P = .001). All 3 diagnostic tests showed significant inverse correlation with log10 reaction doses for positive cashew OFC. The algorithm reduced overall the total number of OFCs by 72.0%, with a positive predictive value and negative predictive value of 93.0% and 99.0%, respectively. Cashew false-positives were observed primarily in hazelnut-allergic patients (P = .026). In this population, Ana o 3-specific IgE could diagnose cashew allergy with a sensitivity of more than 90% and a specificity of more than 95%. CONCLUSIONS: The NUT CRACKER diagnostic algorithm was validated and reduced the number of diagnostic OFCs required. Markers for severity phenotypes may guide oral immunotherapy protocols, improving the risk/benefit ratio for patients.