评估肠道活检组织保存方法以促进大规模黏膜微生物群研究。
Evaluation of intestinal biopsy tissue preservation methods to facilitate large-scale mucosal microbiota research.
作者信息
Wyatt Nicola J, Watson Hannah, Young Gregory R, Doona Mary, Tilling Ned, Allerton Dean, Masi Andrea C, Ahmad Tariq, Doyle Jennifer A, Frith Katherine, Hart Ailsa, Hildreth Victoria, Irving Peter M, Jones Claire, Kennedy Nicholas A, Lawrence Sarah, Lees Charlie W, Lees Robert, Liddle Trevor, Lindsay James O, Marchesi Julian R, Parkes Miles, Powell Nick, Prescott Natalie J, Raine Tim, Satsangi Jack, Whelan Kevin, Wood Ruth, King Andrew, Jostins-Dean Luke, Speight R Alexander, McGregor Naomi, Stewart Christopher J, Lamb Christopher A
机构信息
Translational & Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom; Department of Gastroenterology, The Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom.
Translational & Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom.
出版信息
EBioMedicine. 2025 Feb;112:105550. doi: 10.1016/j.ebiom.2024.105550. Epub 2024 Dec 31.
BACKGROUND
Large-scale multicentre studies are needed to understand complex relationships between the gut microbiota, health and disease. Interrogating the mucosal microbiota may identify important biology not captured by stool analysis. Gold standard tissue cryopreservation ('flash freezing') limits large-scale study feasibility. We aimed to compare gut microbiota in gold standard and pragmatic mucosal biopsy storage conditions.
METHODS
We collected endoscopic recto-sigmoid biopsies from 20 adults with inflammatory bowel disease. Biopsies were preserved using three methods: (i) flash freezing (most proximal and distal biopsy sites); (ii) nucleic acid preservative reagents (QIAGEN Allprotect®, Invitrogen RNAlater™, and Zymo DNA/RNA Shield™); and (iii) formalin fixation with paraffin embedding (FFPE), which is used to preserve tissue for clinical histopathology within healthcare settings. Microbiota were sequenced on the MiSeq platform (V4 region, 16S rRNA gene).
FINDINGS
Tissue microbiota were consistent between most proximal and distal tissue suggesting any within-patient variation observed reflected storage condition, not biopsy location. There was no significant difference in alpha-diversity or microbial community profiles of reagent-preserved versus gold standard tissue. FFPE community structure was significantly dissimilar to other tissue samples, driven by differential relative abundance of obligate gut anaerobes; Faecalibacterium, Anaerostipes and Lachnospiraceae. Despite these differences, tissue microbiota grouped by participant regardless of preservation and storage conditions.
INTERPRETATION
Preservative reagents offer a convenient alternative to flash freezing tissue in prospective large-scale mucosal microbiota studies. Whilst less comparable, FFPE provides potential for retrospective microbiota studies using historical samples.
FUNDING
Medical Research Council (MR/T032162/1) and The Leona M. and Harry B. Helmsley Charitable Trust (G-2002-04255).
背景
需要开展大规模多中心研究来了解肠道微生物群、健康与疾病之间的复杂关系。对黏膜微生物群进行研究可能会发现粪便分析未捕捉到的重要生物学信息。金标准组织冷冻保存法（“速冻”）限制了大规模研究的可行性。我们旨在比较金标准保存条件和实际黏膜活检保存条件下的肠道微生物群。
方法
我们收集了20名患有炎症性肠病的成年人的乙状结肠直肠内镜活检组织。活检组织采用三种方法保存：（i）速冻（最近端和最远端活检部位）；（ii）核酸保存试剂（QIAGEN Allprotect®、Invitrogen RNAlater™和Zymo DNA/RNA Shield™）；（iii）福尔马林固定石蜡包埋（FFPE），这是医疗环境中用于保存组织以进行临床组织病理学检查的方法。在MiSeq平台上对微生物群进行测序（V4区域，16S rRNA基因）。
研究结果
最近端和最远端组织之间的组织微生物群是一致的，这表明观察到的患者体内任何差异反映的是保存条件，而非活检部位。试剂保存组织与金标准组织在α多样性或微生物群落谱方面没有显著差异。FFPE的群落结构与其他组织样本显著不同，这是由专性肠道厌氧菌（粪杆菌属、厌氧短杆菌属和毛螺菌科）相对丰度的差异驱动的。尽管存在这些差异，但无论保存和储存条件如何，组织微生物群都按参与者进行分组。
解读
在前瞻性大规模黏膜微生物群研究中，保存试剂为速冻组织提供了一种方便的替代方法。虽然可比性较差，但FFPE为利用历史样本进行回顾性微生物群研究提供了可能性。
资金来源
医学研究理事会（MR/T032162/1）和利昂娜·M.与哈里·B.赫尔姆斯利慈善信托基金（G-2002-04255）。
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