Impact of delayed PBMC processing on functional and genomic assays

埃利斯波特 外周血单个核细胞 流式细胞术 离体 免疫学 生物 免疫分型 免疫系统 全血 树突状细胞 分子生物学 男科 T细胞 医学 体外 生物化学
作者
P. N. Yi,Luting Zhuo,Julie Lin,Calvin Chang,Audrey Goddard,Oh Kyu Yoon
出处
期刊:Journal of Immunological Methods [Elsevier BV]
卷期号:519: 113514-113514
标识
DOI:10.1016/j.jim.2023.113514
摘要

Peripheral blood mononuclear cells (PBMCs) are commonly isolated from whole blood samples in clinical trials. Isolated PBMCs can be cryopreserved for use in downstream assays such as flow cytometry, single-cell RNA sequencing (scRNA-seq) and enzyme-linked immunosorbent spot (ELISpot) assays to aid understanding of disease biology and treatment effects, and biomarker identification. However, due to logistical practicalities, delays from blood collection to PBMC processing may exceed 24 h, which can potentially affect PBMC function and, ultimately, downstream assay results. Whole blood samples from 20 healthy adults were collected and incubated at 20-25 °C for 2-48 h before PBMC processing. PBMC viability was measured, and flow cytometry immunophenotyping, scRNA-seq and ELISpot were performed following increasing PBMC processing delays. The RosetteSep™ granulocyte depletion kit was used to evaluate the impact of granulocyte contamination following processing delay. Processed scRNA-seq reads were used to identify cell clusters based on marker genes. scRNA-seq data was further used to determine gene expression correlation and pathway activity score in major PBMC cell types (T cells, B cells, natural killer cells, monocytes and dendritic cells) between PBMC preparations subjected to shorter (2-4 h) and longer (8-48 h) processing delays. ELISpot assays evaluated the impact of processing delays on the number of interferon-γ (IFN-γ) secreting cells from ex vivo stimulated PBMCs. PBMC viability was reduced after a 48-h processing delay. Flow cytometry showed that granulocyte contamination of PBMCs increased after 24 h. Cluster analysis of scRNA-seq data identified 23 immune cell type gene expression clusters that were not significantly changed upon granulocyte depletion. Gene expression correlations across the major PBMC cell types were < 0.8 after 24 h of delay compared with 2 or 4 h of delay. Inflammatory, proliferation and signaling pathway activities increased, whereas IFN-γ and metabolic pathway activities decreased with increasing PBMC processing delays. The number of IFN-γ secreting cells trended towards a reduction as PBMC processing delays increased. PBMC processing delays should be minimised when designing clinical trials to reduce outcome variability in downstream assays. Ideally clinical trial sites should have on-site PBMC processing capabilities or be located close to such facilities.
最长约 10秒,即可获得该文献文件

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
PDF的下载单位、IP信息已删除 (2025-6-4)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
暮雨完成签到,获得积分10
刚刚
DOGDAD完成签到,获得积分10
2秒前
英俊的铭应助温柔翰采纳,获得10
2秒前
英姑应助薛建伟采纳,获得10
2秒前
如云完成签到,获得积分20
3秒前
你说的完成签到 ,获得积分10
3秒前
Jasper应助大脸猫4811采纳,获得10
4秒前
hearz完成签到,获得积分10
5秒前
6秒前
P2JY完成签到,获得积分10
6秒前
leiiiiiiii完成签到,获得积分10
6秒前
Bake完成签到 ,获得积分10
8秒前
9秒前
yuan完成签到,获得积分10
9秒前
论文多多完成签到,获得积分10
10秒前
10秒前
Acid完成签到 ,获得积分10
10秒前
1111111111111发布了新的文献求助10
11秒前
linlinyilulvdeng完成签到,获得积分10
11秒前
斯文败类应助历史雨采纳,获得10
12秒前
FashionBoy应助吃个大笼包采纳,获得10
14秒前
海阔天空发布了新的文献求助10
15秒前
薛建伟发布了新的文献求助10
16秒前
高高代珊发布了新的文献求助10
17秒前
害羞的墨镜完成签到,获得积分10
17秒前
lalala发布了新的文献求助10
17秒前
18秒前
guojingjing完成签到,获得积分10
18秒前
打打应助科多兽骑士采纳,获得10
19秒前
angela完成签到,获得积分10
19秒前
19秒前
潇洒的茗茗完成签到 ,获得积分10
21秒前
脂肪小米粥完成签到,获得积分10
22秒前
小幸运完成签到,获得积分10
25秒前
爱吃冻梨完成签到,获得积分10
25秒前
xiang发布了新的文献求助10
25秒前
陈冲冲完成签到,获得积分10
26秒前
changfox完成签到,获得积分10
26秒前
27秒前
27秒前
高分求助中
【提示信息,请勿应助】关于scihub 10000
Les Mantodea de Guyane: Insecta, Polyneoptera [The Mantids of French Guiana] 3000
徐淮辽南地区新元古代叠层石及生物地层 3000
The Mother of All Tableaux: Order, Equivalence, and Geometry in the Large-scale Structure of Optimality Theory 3000
Global Eyelash Assessment scale (GEA) 1000
Picture Books with Same-sex Parented Families: Unintentional Censorship 550
Research on Disturbance Rejection Control Algorithm for Aerial Operation Robots 500
热门求助领域 (近24小时)
化学 材料科学 医学 生物 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 遗传学 基因 物理化学 催化作用 冶金 细胞生物学 免疫学
热门帖子
关注 科研通微信公众号,转发送积分 4038657
求助须知:如何正确求助?哪些是违规求助? 3576306
关于积分的说明 11375198
捐赠科研通 3306108
什么是DOI,文献DOI怎么找? 1819379
邀请新用户注册赠送积分活动 892698
科研通“疑难数据库(出版商)”最低求助积分说明 815066