Alvarez-Arango Santiago, Dispenza Melanie C, Chichester Kristin L, MacGlashan Donald W
Division of Allergy and Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Division of Clinical Pharmacology, Department of Medicine and Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Clin Exp Allergy. 2025 Jan;55(1):75-83. doi: 10.1111/cea.14594. Epub 2024 Nov 17.
Detecting drug-specific IgE (sIgE) is crucial for diagnosing immediate drug-induced hypersensitivity reactions. Basophil activation tests serve as a method to determine the presence of drug-sIgE, highlighting the importance of optimising the assay. Optimisation involves considering multiple factors to ensure sensitisation helps detect an antigen sIgE. The study investigates the complex factors influencing basophil responsiveness thresholds and aims to provide rules-of-thumb guidance for expected results.
Open and occupied FcεRI receptors were analysed by flow cytometry pre- and postdissociation of surface-bound sIgE. Basophils were then sensitised with serial concentrations of penicillin (BPO)-sIgE in serum or buffer and incubated for 1, 4 and 18 h with or without DO and/or IL-3. Basophil sensitivity was evaluated based on FcεRI receptor densities, sIgE/total IgE (tIgE) ratios, responses to BPO(21)-HSA, and DO and/or IL-3 effects, with maximal responses determined using anti-IgE human antibodies. These optimised conditions were tested with peanut-sIgE and cat-sIgE sera.
Basophils from five donors were used. The FcεRI receptor expression initially averaged 155,000/cell (47,000-344,000/cell), with 35% (5%-79%) unoccupied, which postdissociation increased to 98% (82%-100%) unoccupied. Upon sensitisation, the average reloading with BPO-sIgE was 39% (33%-48%). The ED (a measure of cellular sensitivity) was approx. 6000 BPO-sIgE/cell, and the average maximal anti-IgE antibody response was 58% (25%-68%). A 4-h sensitisation at 4°C with IL-3 pretreatment and 70% DO allowed the detection of BPO-sIgE/tIgE ratios as low as 0.02%-0.05% without spontaneous histamine release. Under the same conditions, responses were detected with 0.33% peanut-sIgE and 0.1% cat-sIgE ratios.
This study outlines a method to assess basophil sensitisation, emphasising the minimum sIgE/tIgE ratio needed for basophil responsiveness. It considers factors like FcεRI open/unoccupied FcεRI receptors, sIgE/tIgE ratios and the effect of DO and IL-3. This sets a strong foundation for refining and advancing basophil activation functional assays.
检测药物特异性IgE（sIgE）对于诊断速发型药物性过敏反应至关重要。嗜碱性粒细胞活化试验是确定药物sIgE存在的一种方法，凸显了优化该检测方法的重要性。优化涉及考虑多个因素，以确保致敏有助于检测抗原sIgE。本研究调查了影响嗜碱性粒细胞反应阈值的复杂因素，旨在为预期结果提供经验法则指导。
通过流式细胞术分析表面结合的sIgE解离前后开放和占据的FcεRI受体。然后用血清或缓冲液中系列浓度的青霉素（BPO）-sIgE使嗜碱性粒细胞致敏，并在有或没有地塞米松（DO）和/或白细胞介素-3（IL-3）的情况下孵育1、4和18小时。基于FcεRI受体密度、sIgE/总IgE（tIgE）比值、对BPO(21)-人血清白蛋白（HSA）的反应以及DO和/或IL-3的作用评估嗜碱性粒细胞敏感性，使用抗IgE人抗体确定最大反应。用花生-sIgE和猫-sIgE血清测试这些优化条件。
使用了来自五名供体的嗜碱性粒细胞。FcεRI受体表达最初平均为155,000/细胞（47,000 - 344,000/细胞），其中35%（5% - 79%）未被占据，解离后未被占据的比例增加到98%（82% - 100%）。致敏后，BPO-sIgE的平均重新加载量为39%（33% - 48%）。半数有效剂量（衡量细胞敏感性的指标）约为6000 BPO-sIgE/细胞，平均最大抗IgE抗体反应为58%（25% - 68%）。在4°C下用IL-3预处理和70% DO进行4小时致敏，可检测到低至0.02% - 0.05%的BPO-sIgE/tIgE比值，且无自发组胺释放。在相同条件下，可检测到0.33%花生-sIgE和0.1%猫-sIgE比值的反应。
本研究概述了一种评估嗜碱性粒细胞致敏性的方法，强调了嗜碱性粒细胞反应所需的最低sIgE/tIgE比值。它考虑了诸如FcεRI开放/未占据的FcεRI受体、sIgE/tIgE比值以及DO和IL-3的作用等因素。这为完善和推进嗜碱性粒细胞活化功能检测奠定了坚实基础。
