Diagnostična uporabnost določanja alergogene aktivnosti preko aktivacije senzibiliziranih mastocitov pri alergiji za strup čebele ali ose

Dejanović, Luka (author), Lunder, Mojca (mentor), Koren, Ana (co-mentor)

Alergija za strupe kožekrilcev je poleg hrane in zdravil najpogostejši vzrok za nastanek močnih alergijskih reakcij. Pomembno vlogo pri diagnosticiranju alergij ima tudi laboratorijska diagnostika, ki temelji na potrjevanju IgE-mehanizma alergijske reakcije. Novost na področju testov in vitro za diagnostiko alergij je test aktivacije mastocitov (angl. mast cell activation test – MAT). S testom MAT preverjamo alergogeno aktivnost protiteles IgE preko aktivacije senzibiliziranih mastocitov. V literaturi MAT v povezavi z alergijo za strupe kožekrilcev še ni dobro opredeljen. Glavni namen tega dela je bil diagnostična opredelitev MAT v povezavi z alergijo za strup čebele in ose ter preveriti njegovo uporabnost pri bolnikih z neodzivnimi bazofilci pri testu aktivacije bazofilcev (BAT). V prvi del raziskave smo vključili skupino bolnikov s klinično in laboratorijsko potrjeno alergijo za strup čebele ali ose. Vsi bolniki so imeli jasno anamnezo in pozitivne rezultate kožnih testov, specifičnih protiteles razreda IgE (sIgE) in BAT za strup čebele ali ose. Za primerjavo smo vključili skupino zdravih posameznikov, ki so imeli nezaznavna sIgE za strup čebele in ose. MAT je bil izveden z uporabo celične linije mastocitov LAD2 in vzorcev plazme bolnikov oz. kontrol. Aktivacijo celic LAD2 smo analizirali preko označevalca CD63 s pretočno citometrijo. Pri obeh skupinah smo izvedli MAT za strup čebele in ose ter opredelili njegovo diagnostično vrednost. MAT je imel pri izbrani meji za pozitiven rezultat 5,2 % aktiviranih celic LAD2 97,7% specifičnost in 20,5% občutljivost. V drugem delu raziskave smo MAT opravili pri skupini bolnikov z neodzivnimi bazofilci na stimulacijo s pozitivno kontrolo anti-FcεRI pri BAT. Ti bolniki predstavljajo približno 10 % testiranj, njihove diagnoze pa z obstoječimi laboratorijskimi metodami ni mogoče potrditi. Vsi bolniki so imeli klinično in laboratorijsko potrjeno alergijo za strup čebele in/ali ose. Uporabnost MAT pri bolnikih z neodzivnimi bazofilci pri BAT v literaturi še ni bila sistemsko opredeljena. Naši rezultati so podali pozitiven rezultat MAT pri manjšem deležu bolnikov (14 %), na podlagi česar ugotavljamo, da bi MAT lahko prispeval k boljši diagnozi te skupine bolnikov. V zaključku ugotavljamo, da je bila občutljivost testa MAT v naših raziskavi nizka, kar je nasprotno od rezultatov že objavljenih raziskav in je lahko posledica proučevanja drugih alergenov ter uporabe različnih protokolov za izvedbo MAT. Menimo, da bi lahko naše rezultate izboljšali z optimizacijo protokola za izvedbo MAT. Naši rezultati izkazujejo uporabnost MAT v primeru bolnikov z neodzivnimi bazofilci pri testu BAT.

• Language: Slovenian
• Keywords: mastociti, LAD2, alergija, kožekrilci, test aktivacije mastocitov
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-137215
• Publication date in RUL: 07.06.2022

Secondary language

• Language: English
• Title: Diagnostic value of testing allergenic activity via activation of sensitized mast cells in allergy to bee or wasp venom

Abstract

Hymenoptera venom allergy (HVA) is one of the most common reasons for strong allergic reactions besides food allergens and drugs. Laboratory diagnostics play an important role in the diagnosis of allergy and focus on its IgE mechanism. The mast cell activation test (MAT) is a novel in vitro cell test, where the allergenic activity of IgE antibodies is evaluated via the activation of sensitized mast cells. The use of MAT in HVA is yet to be well described in literature. The aim of the study was to determine the diagnostic parameters of MAT in the diagnosis of HVA and evaluate its use in the case of the basophil activation test (BAT) non-responders. In the first part of the study we included a group of patients with clinically and laboratory confirmed allergy to bee or wasp venom. All the patients had a clear anamnesis and positive results of skin prick tests, specific IgE antibodies (sIgE) and BAT to bee or wasp venom. A group of non-sensitized healthy controls was included for comparison. MAT was performed with the use of LAD2 cell line and plasma samples of patients and controls. Activation of LAD2 cells was assessed with the use of the CD63 marker by means of flow cytometry. Results of the MAT from both groups were used to establish the optimal cut-off value of 5.2% activated LAD2 cells. At the given cut-off value the MAT showed a specificity of 97.7% and a sensitivity of 20.5%. In the second part of the study we included a group of patients with non responsive basophils upon stimulation with anti-FcεRI in the BAT. This occurs in up to 10% of cases and makes the current laboratory methods unable to confirm the diagnosis. All of the patients had clinically and laboratory confirmed allergy to bee and/or wasp venom. To our knowledge this is the first study to systematically evaluate the use of MAT in BAT non-responding patients. Our data showed some positive results (14%); therefore MAT could contribute to a better diagnosis of such patients. We conclude that our results show a low sensitivity of the MAT which is contrary to the data published in literature. This could be due to the difference in studied allergens and different MAT protocols. We believe that our data could be improved with the optimization of the MAT protocol. However, to some extent our results show that the MAT can contribute to a better diagnosis of patients with non responsive basophils in the BAT.

• Keywords: mast cells, LAD2, IgE, allergy, hymenoptera, mast cell activation test