a Department of Internal Medicine 1 - Gastroenterology, Pneumology and Endocrinology , Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Universitätsklinikum Erlangen , Erlangen , Germany.
Autoimmunity. 2018 Sep;51(6):288-296. doi: 10.1080/08916934.2018.1527317.
Anti-neutrophil-cytoplasmic antibodies (ANCA) are auto-antibodies directed against components of neutrophil granulocytes and may be found in various inflammatory conditions, like small-vessel vasculitis or ulcerative colitis (UC). Routine ANCA screening is performed on ethanol-fixed neutrophils using indirect immunofluorescence technique. Yet, how neutrophil granule proteins become available to immunologic presentation is a matter of debate. In recent years, various studies have shown that neutrophils are able to extrude their chromatin decorated with granular proteins as neutrophil extracelullar traps (NETs).
We hypothesized that (I) ANCA immunoreactivity may be found on NETs and (II) NETs may serve as a useful tool in a novel approach for ANCA detection.
Sera from patients suffering from either ANCA-associated vasculitis (n = 10), UC (n = 30) or sera from patients without diagnosed ANCA-associated diseases (n = 20), respectively, were subjected to indirect immunofluorescence and a newly developed method to detect ANCA by flow cytometry employing microbead technology.
ANCA-related immunofluorescence was readily detectable on ethanol-fixed NETs, establishing NETs as a structure carrying ANCA target antigens. Moreover, we observed that neutrophils form NETs in response to microbeads and stick to the surface of these beads. Using these NET-coated microbeads in flow cytometry, we were capable of reliably detecting p-ANCA, c-ANCA, and a-ANCA in tested patient sera. UC-related complex DNase-1-sensitive ANCA (NET-ANCA) antigens were also detected on NET-coated microbeads.
NET-coated microbeads may be commercially developed as a novel tool for automated ANCA screening assays using flow cytometry.
抗中性粒细胞胞浆抗体（ANCA）是针对中性粒细胞颗粒成分的自身抗体，可存在于各种炎症性疾病中，如小血管血管炎或溃疡性结肠炎（UC）。常规使用间接免疫荧光技术在乙醇固定的中性粒细胞上进行 ANCA 筛查。然而，中性粒细胞颗粒蛋白如何可被免疫呈递仍存在争议。近年来，多项研究表明，中性粒细胞能够将其染色质挤出，这些染色质上装饰着颗粒蛋白，形成中性粒细胞胞外陷阱（NETs）。
我们假设（I）ANCA 免疫反应性可能存在于 NETs 上，以及（II）NETs 可能成为用于检测 ANCA 的新方法的有用工具。
分别来自患有 ANCA 相关性血管炎（n=10）、UC（n=30）或未诊断为 ANCA 相关性疾病的患者（n=20）的血清，分别进行间接免疫荧光和使用微珠技术的流式细胞术检测 ANCA 的新方法。
在乙醇固定的 NETs 上可轻易检测到与 ANCA 相关的免疫荧光，证实 NETs 是携带 ANCA 靶抗原的结构。此外，我们观察到中性粒细胞在微珠的刺激下形成 NETs，并黏附在这些微珠的表面。使用这些涂覆有 NET 的微珠在流式细胞术中，我们能够可靠地检测到测试患者血清中的 p-ANCA、c-ANCA 和 a-ANCA。还在涂覆有 NET 的微珠上检测到 UC 相关的复杂 DNA 酶-1 敏感的 ANCA（NET-ANCA）抗原。
NET 涂覆的微珠可能被开发为使用流式细胞术进行自动化 ANCA 筛选检测的新型工具。
