preparation of whole blood samples for flow cytometry and cytokine analysis to determine antigen-specific host immunity to fungal and viral pathogens

<meta charset="utf8"></head><body>用于分析真菌和病毒免疫的优化全血免疫检测

Quantification and characterization of antigen-specific immunity, especially specific T-cell responses, is pivotal for immunobiology and vaccination research, as well as some diagnostic tests. Traditionally, antigen-specific immunoassays commonly relied on isolated peripheral blood mononuclear cells (PBMCs). However, isolation of these cells is time-consuming and resource-intensive and often requires relatively large blood volumes. Additionally, to prevent granulocyte activation and subsequent T-cell disturbance during pre-analytic storage1 rapid processing of the samples is paramount, which is often not feasible in clinical practice. These limitations hamper the practicability of antigen-specific immunoassays in high-throughput research scenarios and clinical routines. Therefore, the development of easy-to-use and potentially automatable whole blood-based approaches in recent years has opened new areas of immunoassay applications. However, current commercially available systems usually lack optimal co-stimulatory environments for T-cells and are susceptible to pre-analytic delays. For instance, a widely used whole blood-based IFN-&#947; release assay has a 19% positive to negative reversion rate after 6 h of pre-analytic blood storage2. Optimized protocols with dual anti-CD28 and anti-CD49d co-stimulation have been developed to overcome these limitations3,4,5,6.
The protocol presented here allows for accurate and reproducible quantification and characterization of antigen-specific T-cells, assessment of antigen-induced cytokine responses, and other (flow cytometric or transcriptional) functional immune markers from minimal blood volume, i.e., 500 &#181;L of blood per stimulation tube. Further advantages of this protocol include low hands-on time, high resilience to pre-analytic confounders, and preservation of functional inter-cellular interactions in a relatively physiological ex vivo environment. The comparability of whole blood-based flow cytometric antigen-specific&#160;T-cell characterization with data generated from traditional PBMC-based assays has been previously shown in the context of mold-specific T-cell quantification6. Furthermore, direct stimulation of the subjects&#39; blood abrogates the need for supplementation with autologous, allogenic, or even xenogeneic serum that is commonly required for optimal PBMC stimulation. Omission of cell isolation also reduces shear and temperature stress, thereby improving cell viability. Most importantly, whole blood-based assays preserve granulocyte populations that are lost during gradient centrifugation for isolation of PBMCs7. Thereby, this assay setup preserves and captures functional interaction loops between granulocytes and mononuclear cells4.
Of note, this protocol requires only minimal modifications to accommodate different readout modalities and even allows for dual analysis of cytokine release and transcriptional responses from the same stimulation tube. Specifically, while cytokines are analyzed from the culture supernatant after stimulation, the cell pellet can be used for RNA isolation with subsequent transcriptomic analysis. The general workflow for the various readout modalities is summarized in Figure 1.
In recent years, an increasing number of whole blood-based assays has been developed for pathogen-reactive immune monitoring in research and clinical settings, e.g., for Mycobacterium tuberculosis8,9, Bordetella pertussis3, Orientia tsutsugamushi10, and SARS-CoV-25,11,12. For instance, a previously established system has been used for multiple antigens, including M. tuberculosis, Influenza A virus, and SARS-CoV-2, but does not use co-stimulatory factors optimized for T-helper (Th) cell stimulation13,14,15. Even though the blood volume required for these assays is already significantly lower than that used for traditional PBMC-based assays or commercially available whole blood stimulation kits, an even smaller sample volume might be warranted for applications in pediatrics, neonatology, patients in the intensive care unit (ICU), and preclinical research in small animal models. For instance, even terminal blood sampling from mice (e.g., by cardiac puncture) commonly yields a maximum of 0.7-1 mL of blood. Thus, the possibility to further downscale previously established whole blood-based immunoassay protocols4,6 for precise quantification and characterization of antigen-reactive T-cell responses from 250 &#181;L of blood volume per stimulation tube has been evaluated as part of this protocol.

<meta charset="utf8"></head><body>作者聚焦：使用全血检测推进重症监护患者的免疫监测

具有双重共刺激的全血测定，用于宿主对真菌和病毒病原体免疫的抗原特异性分析

制备用于流式细胞术和细胞因子分析的全血样品，以确定抗原特异性宿主对真菌和病毒病原体的免疫力

preparation-whole-blood-samples-for-flow-cytometry-cytokine-analysis

Research

JoVE Journal

Immunology and Infection

Preparation of Whole Blood Samples for Flow Cytometry and Cytokine Analysis to Determine Antigen-Specific Host Immunity to Fungal and Viral Pathogens

295 次观看. 要开始流式细胞术的样品制备，请从培养箱中取出含有 Brefeldin A 的全血样品的刺激管。向每个含有布雷菲德菌素 A.的刺激管中加入 500 微升 0.5 摩尔 EDTA 溶液，将样品转移到新的 15 毫升离心管中。然后将 1 毫升红细胞裂解缓冲液移液到每个刺激管中冲洗。将缓冲液和细胞悬液转移到同一个离心管中。将试管以 600 g 离心 7 分钟，然后小心吸出上?...

视频: 制备用于流式细胞术和细胞因子分析的全血样品，以确定抗原特异性宿主对真菌和病毒病原体的免疫力

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 &#181;L) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

Whole blood-based immunoassays provide a facile and resource-efficient tool to analyze antigen-specific immunity for diagnostic and research purposes. This article provides an optimized whole blood-based protocol with dual co-stimulation for comprehensive analysis of host immunity to fungal and viral pathogens, including a low-volume version for pediatric patients and small animals.

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...