Towards a democratization of the use of flow cytometry for diagnosis and monitoring of allergy

L’allergie représente un problème majeur de santé public. Le test de provocation orale constitue l’étalon-or du diagnostic de l’allergie. Cependant, pouvant induire de graves réactions cliniques, il se réalise à l’hôpital. D’autre part, les tests cutanés et sérologiques permettent d’étudier le profil de sensibilisation d’un individu à des allergènes (présence d’IgE spécifiques). Ces approches nécessitent une interprétation rigoureuse et permettent d’orienter de diagnostic de l’allergie. Le test d’activation des basophiles (TAB) est un test cellulaire fonctionnel qui représente une approche unique en allergologie. Bien que sa pertinence et son utilité soient établies, du fait de limitations techniques, son utilisation reste limitée à des laboratoires spécialisés. Afin que toutes les capacités du TAB puissent être exploitées, une levée de ces limitations est indispensable. L’objectif de cette thèse a été de travailler à une amélioration technique du TAB. Nous avons simplifié la chaîne pré-analytique du TAB. L’approche développée utilise une innovation technologique (DURA Innovation, Beckman Coulter) permettant l’emploi de réactifs secs, stables à température ambiante et prêts à l’emploi. La méthode se fait sur du sang total, sans étape de lavage, ne nécessite que 4 étapes de pipetage et, malgré l’utilisation d’un cocktail d’anticorps de 5 couleurs, sans compensation lors de l’analyse au cytométre. La méthode développée a été mise en œuvre dans différentes études constituant le volet clinique de la thèse. Nous avons aussi démontré la possibilité de miniaturiser le TAB en le transposant au format plaque 96 puits avec une préparation des échantillons totalement automatisée.Allergy is a major public health issue. The oral provocation test is considered as the gold standard of allergy diagnoses. Nevertheless, it can induce severe clinical reaction and need to be performed inside an hospital facility. On the other hand, skin testing and serum quantification tests allow to study one’s sensibilisation profile to allergens (Specific IgE detected). These methods require meticulous interpretation and allow to guide the allergy diagnosis process.Basophil Activation Test (BAT) is a cellular functionnal test. By reproducing ex vivo allergic reaction, it represents a unique approach in allergology field. Even if the relevance and the utility of the BAT have already been established, the use of this test is currently limited to specialist laboratories. It is necessary to overcome some limitations of the BAT to fully leverage its capabilities.The main objective of this thesis was to work on technical improvements of BAT in flow cytometry. We simplified the whole pre-analytical workflow. The developped BAT procedure uses a technological innovation (DURA Innovation, Beckman Coulter) allowing to use dry reagents, stable at room temperature and ready to use. The process is done on whole blood, using 5 color compensation-free antibody panel, without washing step, and requires only four pipetting steps before cytometre analysis. This BAT method has been used for different studies during this thesis and constitues the clinical part of the study.In a second step, we wanted to go further into BAT process simplification. We demonstrated the possibility to miniaturize the BAT by transposing it to 96-well plate format with a fully automated sample preparation

Streamlining basophil activation testing to enable assay miniaturization and automation of sample preparation

Background: Numerous studies have demonstrated the capabilities of the basophil activation test (BAT) but various parameters such as a lack of standardization and a time consuming and labor intensive workflow continue to hinder the field to fully leverage the capabilities of this technique. When pediatric patients have to be considered, an additional limitation is related to blood volume consumption. Objectives: This work aimed at developing and characterizing a simplified and standardized whole-blood based BAT prototype procedure and at further assessing the feasibility of automating and miniaturizing the developed assay into a 96 well plate format. Methods: A dry and room temperature stable reagent technology was used to simplify and standardize BAT. Under optimized conditions, EDTA anticoagulated whole blood samples of non-allergic and allergic donors ( < 24 h old) together with calcium containing buffer were added to ready-to-use dry reagent tubes or 96 well plates (negative controls, positive controls and allergen tests) containing a 5 color compensation-free antibody panel (CD45-KrO/CD3-PC7/CRTH2-A647/CD203c-PE/CD63-PB). Upon mixing and incubation at 37 degrees C for 15 min, erythrocytes were lysed and samples were analyzed by flow cytometry without further washing steps. While it is important to precisely control the incubation time to minimize the assay variability, herein, a 15 min incubation time was chosen as it provides a suitable compromise for both the magnitude of basophil activation and the quality of the staining. A Biomek NXP robotic platform (Beckman Coulter) was used for automation and both CD203c and CD63 levels were monitored to characterize basophil reactivity. Results: This streamlined BAT protocol is no-wash, compensation free and only requires 4 pipetting steps to be completed. The assessment of assay performance characteristics showed wide applicability, satisfactory repeatability and a high degree of standardization as demonstrated by very low infra-assay and inter-operator variabilities (CVs < 10%). Leveraging these technical foundations, it was then proven that this new BAT procedure can easily be transposed into the 96 well plate format, thereby benefiting from a miniaturized format and full automation capabilities. When considering 8 dilution points to characterize the ex vivo basophil reactivity of a given whole blood sample, we found that as little as 51.1.L of blood per point could be used. Conclusions: A whole blood based and simplified procedure for BAT is proposed. It relies on a dry antibody formulation technology and requires only a few manual steps to be completed. This procedure can also be transposed in a 96 well plate format, fully automated and miniaturized, when sample volume reduction, throughput increase or unattended sample preparation is required

Basophil activation test with Pru p 7 is predictive of clinical severity in peach allergic patients: Meeting Abstract: PD0298

International audienc

Pru p 7 sensitization is a predominant cause of severe, cypress pollen‐associated peach allergy

International audienceBACKGROUND:Peach is a common elicitor of food allergic reactions. Peach-induced immediate reactions may occur as benign pollen-food syndromes, usually due to birch pollen-related PR-10 cross-reactivity in temperate climates, and as potentially severe primary food allergies, predominantly related to nsLTP Pru p 3 in Mediterranean regions. The newly described peach allergen Pru p 7 has gained recent attention as a potential peach allergy severity marker. Sensitization to Pru p 7 and its allergenic homologues of the gibberellin-regulated protein family occurs in areas with high Cupressaceae tree pollen exposure.OBJECTIVE:We sought to investigate the distribution, clinical characteristics and molecular associations of Pru p 7 sensitization among subjects with suspected peach allergy in different regions of France.METHODS:Subjects with suspected peach allergy (n = 316) were included. Diagnostic work-up was performed according to current guidelines, including open food challenge when required. IgE antibody measurements and competition experiments were performed using the ImmunoCAP assay platform.RESULTS:Sensitization to Pru p 7 was present in 171 (54%) of all subjects in the study and in 123 of 198 (62%) diagnosed as peach allergic, more than half of whom were sensitized to no other peach allergen. Frequency and magnitude of Pru p 7 sensitization were associated with the presence of peach allergy, the clinical severity of peach-induced allergic reactions and the level of cypress pollen exposure. Cypress pollen extract completely outcompeted IgE binding to Pru p 7. Pru p 7 was extremely potent in basophil activation tests.CONCLUSION AND CLINICAL RELEVANCE:A subtype of Cupressaceae pollinosis, characterized by Pru p 7 sensitization, can be an underlying cause of severe peach allergy