Beckman Coulter Inc, F-13009 Marseille, France; Aix Marseille Univ, IRD, APHM, MEPHI, Marseille, France; Aix Marseille Univ, IHU Méditerranée Infection, Marseille, France.
Aix-Marseille Univ, APHM Service de Pédiatrie Multidisciplinaire, Hôpital Timone Enfants, F-13005 Marseille, France; Cabinet de Pédiatrie, 4 rue Sainte, Marseille, France.
J Immunol Methods. 2020 Jun-Jul;481-482:112793. doi: 10.1016/j.jim.2020.112793. Epub 2020 May 6.
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.
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.
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 °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.
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 intra-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 5 μL of blood per point could be used.
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.
许多研究已经证明了嗜碱性粒细胞激活试验（BAT）的能力，但各种参数，如缺乏标准化以及耗时和劳动密集型的工作流程，仍然阻碍了该技术充分发挥其能力。当需要考虑儿科患者时，另一个限制因素与血液量消耗有关。
本研究旨在开发和描述一种简化和标准化的全血基础 BAT 原型程序，并进一步评估将开发的测定自动化和微型化为 96 孔板格式的可行性。
使用干燥和室温稳定的试剂技术来简化和标准化 BAT。在优化条件下，将非过敏和过敏供体的 EDTA 抗凝全血样本（<24 小时）与含钙缓冲液一起加入到即用型干试剂管或 96 孔板（阴性对照、阳性对照和过敏原测试）中，其中包含 5 种颜色补偿免抗体面板（CD45-KrO/CD3-PC7/CRTH2-A647/CD203c-PE/CD63-PB）。混合并在 37°C 孵育 15 分钟后，裂解红细胞，无需进一步洗涤步骤即可通过流式细胞术分析样品。虽然精确控制孵育时间以最小化测定变异性非常重要，但在此，选择 15 分钟孵育时间是因为它为嗜碱性粒细胞激活的幅度和染色质量提供了一个合适的折衷。Biomek NXP 机器人平台（Beckman Coulter）用于自动化，监测 CD203c 和 CD63 水平以表征嗜碱性粒细胞反应性。
该简化的 BAT 方案无需洗涤、无需补偿，仅需完成 4 个移液步骤。评估测定性能特征表明，该测定具有广泛的适用性、令人满意的重复性和高度的标准化，表明内源性和操作者间变异性（CV<10%）非常低。利用这些技术基础，然后证明这种新的 BAT 程序可以很容易地转换为 96 孔板格式，从而受益于微型化格式和完全自动化功能。当考虑 8 个稀释点来描述给定全血样本的体外嗜碱性粒细胞反应性时，我们发现每个点只需使用 5μL 的血液。
提出了一种基于全血的简化 BAT 程序。它依赖于干燥的抗体制剂技术，仅需完成几个手动步骤。当需要减少样本量、增加通量或无人值守的样本制备时，该程序也可以转换为 96 孔板格式，完全自动化和微型化。
