Discussion
Peanut allergy is one of the most frequent causes of severe food-associated anaphylaxis. To prevent or minimize such systemic, potentially life-threatening allergic reactions, accurate and reliable diagnostic tools that are easy to implement and safe are urgently required. Moreover, diagnostic accuracy and the reliability of negative values in sensitized individuals is needed to avoid unnecessary diagnosis with all the consequences that come with the diagnosis of peanut allergy. In this study, we sought to investigate the clinical utility of the recently developed in vitro Hoxb8 MAT for the diagnosis of peanut allergies and to compare it to established and validated clinical diagnostic tests including SPT, sIgE measurements and BAT. For this purpose, we analyzed serum samples that have been prospectively collected in the context of MONAS and were subsequently stored long term in a frozen state (-80°C) in a blinded manner. Importantly, the BAT has previously shown high accuracy in the diagnosis of peanut allergy in multiple studies . Our results show here strongly suggest that the Hoxb8 MAT performs as well as the BAT in terms of diagnostic accuracy in a mixed cohort of severe and clear peanut allergic patients and those with an equivocal history. The respective test is at the same time more accurate as commonly performed tests like the SPT and sIgE measurement.
While the idea to perform MAT for diagnostic purposes is not new , its broader implementation has been hampered by scalability limitations. In one study, Bahri et al. generated mature mast cells from isolated human primary blood stem cells. Their results concerning diagnostic accuracy were similar to those we present here using Hoxb8 MCs. However, their assay relied on a sophisticated mast cell differentiation protocol, which required 8-10 weeks to generate a limited number of cells available for the diagnostic assay. Another challenge of this approach is the variability between different blood stem cell donors, which might affect assay robustness and diagnostic reproducibility, unless cells of various donors are pooled to minimize variability each time. These issues have been resolved with the Hoxb8 MC system, as mature mast cells can be repetitively differentiated from the same immortalized mast cell progenitor stock at unlimited numbers within only 5 days. In another study, Santos et al. have used the human LAD2 mast cell line for the diagnosis of peanut allergies . The result also looked encouraging. However, LAD2 cells generally feature slow growth characteristics with a doubling time of 2 weeks . Hoxb8 MC progenitors double roughly once every 24 hours .
Hoxb8 MCs showed optimal cut-off values ≤5.2%, which is comparable to the cut offs reported for BAT , as well as maximal activation of >89% in our diagnostic performance study. These findings suggest that the Hoxb8 MAT overcomes prior limitations of other MAT approaches while maintaining excellent diagnostic accuracy.
From a methodological standpoint the BAT and the MAT both represent similar cell-based functional in vitro tests, which rely on flow cytometric identification of activated allergic effector cells upon allergen stimulation. However, there are major differences between these two assays. The BAT is performed with patient’s whole blood, which must be processed and measured within maximally 24 hours to ensure valid results . The MAT, on the other hand, relies on serum samples that can be stored in the freezer over months (even years) and analyzed in large batches. This is particularly important for large studies or multi-center clinical trials as the analysis of serum samples can be done retrospectively in a centralized manner. The serum samples included in this study were analyzed in just 2 days. Thus, the Hoxb8 MAT clearly has the potential to overcome logistical challenges that have hampered a broader implementation of the BAT in clinics so far.
Direct comparison of activation results between the BAT and the Hoxb8 MAT revealed that the BAT requires roughly 100-times lower allergen concentrations to induce comparable percentage of cell activation. One potential explanation for this difference is linked to the number of allergic effector cells present per sample. There are an estimated 0.5 x 106 leukocytes in 50 µl human whole blood , which is the amount used for the BAT. Basophils make up roughly 0.1-0.5% of leukocytes , which means that there are 500-2500 basophils present in one BAT sample. In comparison, we were using 50 x 104mast cells per condition for the Hoxb8 MAT. Thus, there were 20 to 100-times more cells present in the Hoxb8 MAT than in the BAT, which could explain the observed difference in allergen concentration needed to reach an equivalent percentage of activated cells. However, this hypothesis has to be further investigated.
Despite losing intrinsic cellular information (i.e. patient’s own versus lab grown allergic effector cells), the Hoxb8 MAT showed comparable diagnostic accuracy as the BAT in this study, further supporting the notion that humoral components contained in the serum are sufficiently representative of a patient’s allergy status. Furthermore, in approximately 10% of cases the BAT results in false negative or false positive outcomes with basophils that do not show any activation upon FcεRI-mediated stimulation (non-responders) or with unspecific activation of basophils in the negative control . It has been demonstrated that basophils of so-called non-responders feature an impaired signaling cascade with low tyrosine kinase Syk levels . These individuals pose an important diagnostic challenge in the daily clinical allergy workup. It is also important to note, that many diagnostic performance studies using BAT exclude these non-responder samples and thereby do not entirely represent the real-life situation . As the initial dataset we used in this study did not contain non-responder samples, the diagnostic accuracy of the BAT in our ROC curve analysis might be overestimated as in many other studies. Importantly, we provided strong evidence in a separate experiment showing that the Hoxb8 MAT is able of accurately capturing these non-responder samples and might thereby overcomes this current diagnostic issue .
Overall, this study has demonstrated that the Hoxb8 MAT features excellent diagnostic accuracy and outperformed both SPT and sIgE measurements in the diagnosis of peanut allergy. While it represents a valuable alternative to established allergy diagnosis tests further studies including other allergens and allergen components will be conducted in near future.
References
Acknowledgements: We thank all the people from the Kaufmann, Eggel and Eiwegger laboratories. We also acknowledge members of ATANIS Biotech AG, Prof. Dr. med. Jean-Pierre Kinet, Charlène Niogret, Marianne Zwicker and especially Antonia Ferreira and Sophie Buchser for valuable scientific discussions and support with experimental procedures and protocols.
Competing interests : NBZ is a co-founder, shareholder, and employee of ATANIS Biotech AG. AC has nothing to declare. RvB is an employee of ATANIS Biotech AG. JAH has nothing to disclose. XY has nothing to disclose. CMD has nothing to disclose. LD has nothing to disclose. JEMU reports grants and personal fees from ALK-Abelló A/S, personal fees from Bausch Health, personal fees from Kaleo, grants from DBV Technologies, grants from Regeneron, and grants from Food Allergy Anaphylaxis Programme (SickKids), outside the submitted work; and Section Chair of Food Allergy and Anaphylaxis, Canadian Society of Allergy and Clinical Immunology; Healthcare Advisory Board, Food Allergy Canada. TK is a co-founder, shareholder and board member of ATANIS Biotech AG. AE is a co-founder, consultant and shareholder of ATANIS Biotech AG. AE received grants from ATANIS Biotech AG. TE reports grants from CIHR, FWF, Food Allergy and Anaphylaxis Program Sickkids, ALK. He is site PI of company sponsored trials by DBV, Novartis and Stallergenes Greer, EFSA and FARE. Personal fees from Danone/Nutricia/Milupa, ThermoFisher, Aimmune, Stallergenes Greer, ALK, MADX and Non-financial support from Novartis and MADX, all outside the submitted work. He is an associate editor of Allergy.
Author contribution: NZ and RvB performed Hoxb8 MAT and did data entry. AC analyzed data and critically reviewed the manuscript. JAH, XY and CMD performed BAT and did data entry. LD cross checked clinical accuracy and contributed to clinical data entry. JEMU cross checked clinical accuracy and contributed to clinical data entry, contributed to recruitment and contributed to manuscript revision. TK contributed to Hoxb8 MC generation, provided advise on study design and critically reviewed the manuscript. AE and TE co-designed the study, co-contributed to generation of the manuscript, and co-supervised the project and collaboration. All authors reviewed the results and commented on the manuscript.