Bansal Sakshi, Sharma Vinit, Gupta Rajesh, Singh Harjeet, Aggarwal Anjali
Department of Anatomy, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Department of GI Surgery, HPB and Liver Transplantation, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Bio Protoc. 2024 Jun 5;14(11):e5010. doi: 10.21769/BioProtoc.5010.
Neutrophils, constituting 50%-70% of circulating leukocytes, play crucial roles in host defense and exhibit anti-tumorigenic properties. An elevated peripheral blood neutrophil-to-lymphocyte ratio is associated with decreased survival rates in cancer patients. In response to exposure to various antigens, neutrophils release neutrophil granular proteins, which combine to form web-like structures known as neutrophil extracellular traps (NETs). Previously, the relative percentage of NETs was found to be increased in resected tumor tissue samples from patients with gastrointestinal malignancies. The presence of NETs in peripheral blood is indicative of underlying pathological conditions. Hence, employing a non-invasive method to detect NETs in peripheral blood, along with other diagnostic tests, shows potential as a valuable tool not just for identifying different inflammatory disorders but also for assessing disease severity and determining patient suitability for surgical resection. While reliable methods exist for identifying NETs in tissue, accurately quantifying them in whole blood remains challenging. Many previous methods are time-consuming and rely on a limited set of markers that are inadequate for fully characterizing NETs. Therefore, we established a unique sensitive smear immunofluorescence assay based on blood smears to identify NETs in only as little as 2 μL of whole blood. To identify the NET complexes that have enhanced specificities, this combines the use of various antibodies against neutrophil-specific CD15, NET-specific myeloperoxidase (MPO), citrullinated histone H3 (Cit H3), and nuclear DNA. This protocol offers an easy, affordable, rapid, and non-invasive method for identifying NETs; thus, it can be utilized as a diagnostic marker and targeted through various therapeutic approaches for treating human malignancies. Key features • Characterization of neutrophil extracellular traps in whole blood smears through immunofluorescence staining. • Affordable and quantitative approach to neutrophil extracellular trap detection.
中性粒细胞占循环白细胞的50%-70%，在宿主防御中起关键作用，并具有抗肿瘤特性。外周血中性粒细胞与淋巴细胞比值升高与癌症患者生存率降低有关。在接触各种抗原后，中性粒细胞会释放中性粒细胞颗粒蛋白，这些蛋白结合形成网状结构，称为中性粒细胞胞外陷阱（NETs）。此前，在胃肠道恶性肿瘤患者的切除肿瘤组织样本中发现NETs的相对百分比增加。外周血中NETs的存在表明存在潜在的病理状况。因此，采用非侵入性方法检测外周血中的NETs，并结合其他诊断测试，不仅有望成为识别不同炎症性疾病的有价值工具，还可用于评估疾病严重程度以及确定患者是否适合手术切除。虽然存在可靠的方法来识别组织中的NETs，但在全血中准确量化它们仍然具有挑战性。许多先前的方法耗时且依赖于一组有限的标志物，不足以全面表征NETs。因此，我们基于血涂片建立了一种独特的灵敏涂片免疫荧光测定法，仅需2μL全血即可识别NETs。为了识别具有增强特异性的NET复合物，该方法结合使用了针对中性粒细胞特异性CD15、NET特异性髓过氧化物酶（MPO）、瓜氨酸化组蛋白H3（Cit H3）和核DNA的各种抗体。该方案提供了一种简单、经济、快速且非侵入性的NETs识别方法；因此，它可作为一种诊断标志物，并通过各种治疗方法用于治疗人类恶性肿瘤。关键特性 • 通过免疫荧光染色表征全血涂片中的中性粒细胞胞外陷阱。 • 一种经济且定量的中性粒细胞胞外陷阱检测方法。
