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Streamlining basophil activation testing to enable assay miniaturization and automation of sample preparation.
Journal of Immunological Methods ( IF 2.2 ) Pub Date : 2020-05-06 , DOI: 10.1016/j.jim.2020.112793
Rihane Arif-Lusson 1 , Chantal Agabriel 2 , Ania Carsin 3 , Isabelle Cabon 4 , Hélène Sénéchal 5 , Pascal Poncet 6 , Joana Vitte 7 , Jean-Marc Busnel 8
Affiliation  

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 °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 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.

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.



中文翻译:

简化嗜碱性粒细胞活化测试,以实现测定的小型化和样品制备的自动化。

背景

许多研究已经证明了嗜碱性粒细胞活化测试(BAT)的功能,但是各种参数(例如缺乏标准化)以及耗时且费力的工作流程继续阻碍该领域充分利用该技术的功能。当必须考虑小儿患者时,额外的限制与血容量的消耗有关。

目标

这项工作旨在开发和表征简化的,标准化的基于全血的BAT原型程序,并进一步评估将开发的测定自动化和小型化为96孔板形式的可行性。

方法

使用干燥和室温稳定的试剂技术来简化和标准化BAT。在最佳条件下,将非过敏和过敏供体(<24小时龄)的EDTA抗凝全血样品与含钙缓冲液一起添加到即用型干试剂管或96孔板(阴性对照,阳性对照和过敏原)中测试)包含5种无颜色补偿的抗体组(CD45-KrO / CD3-PC7 / CRTH2-A647 / CD203c-PE / CD63-PB)。混合并在37°C下孵育15分钟后,裂解红细胞,无需进一步洗涤即可通过流式细胞仪分析样品。尽管精确控制孵育时间以最大程度地减少化验变异性很重要,但在此,选择15分钟的孵育时间,因为它为嗜碱性粒细胞的活化程度和染色质量提供了合适的折衷方法。使用Biomek NXP机器人平台(贝克曼库尔特)进行自动化,同时监测CD203c和CD63的水平以表征嗜碱性粒细胞的反应性。

结果

这种精简的BAT协议无需清洗,无需补偿,仅需完成4个移液步骤即可。分析性能特征的评估显示出广泛的适用性,令人满意的重复性和高度的标准化,如非常低的分析内和操作者间差异(CVs <10%)所证明。利用这些技术基础,然后证明了这种新的BAT程序可以轻松地转换为96孔板格式,从而受益于小型化格式和全自动功能。当考虑8个稀释点来表征给定全血样品的离体嗜碱性粒细胞反应性时,我们发现每个点可使用的血液量仅为5μL。

结论

提出了基于全血的BAT简化程序。它依靠干抗体配制技术,仅需完成几个手动步骤即可。当需要减少样品体积,增加通量或进行无人值守样品制备时,该程序也可以96孔板格式进行转置,实现全自动且小型化。

更新日期:2020-05-06
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