间接免疫荧光(IIF)的测定法在传统上被用于抗核抗体(ANA)的人血清中的检测。这些抗体的存在可以在全身性自身免疫性风湿病(SARD)帮助诊断。该协议演示了如何有效地进行IIF技术,准确检测这些自身抗体。
对ANA检测风湿病立场声明的美国大学规定使用IIF作为全日空筛选1的金标准方法。虽然IIF是在专家手中一个很好的筛选试验,处理和阅读IIF的技术难点幻灯片 – 如劳动密集型幻灯片处理,人工读数,需要有经验的,训练有素的技术人员和使用黑暗的房间 – 使IIF法难以适应现代化,自动化实验室的工作流。
对高品质的ANA筛查的关键的第一步是仔细滑动处理。这个过程是劳动密集的,并且需要充分理解的过程中,以及关注细节和经验。
幻灯片阅读是通过荧光显微镜在黑暗的房间进行,由受过训练的技术人员谁是熟悉的各种图案,在细胞周期的背景下完成的和间期和分裂细胞的形态。但IIF是第一线筛检工具,可持续农业和农村,了解步骤正确执行此技术是至关重要的。
近年来,数字成像系统已经开发的IIF幻灯片自动读数。这些系统,如NOVA查看自动荧光显微镜,旨在简化日常IIF的工作流程。 NOVA查看获取并存储井的高分辨率数字图像,从而从解释分离图像采集;图像被视为一种解释型的高分辨率电脑显示器。它存储的图像以供将来参考,并通过提供对图像的荧光光强数据支持运营商的解释。它也预先进行分类的结果为正或负,并且提供了模式识别为阳性样品。总之,它省去了暗室,以及自动化和简化的IIF读写,程序纳克/口译工作流程。最重要的是,它增加了阅读器和读数之间的一致性。此外,与使用条形码的幻灯片,抄写错误是通过提供样品的可追溯性和积极的患者识别淘汰。这会导致增加病人的数据完整性和安全性。
此视频的总体目标是展示IIF程序,包括幻灯处理,识别常见的IIF模式,并引进了新的进步,以简化和协调这项技术。
术语抗核抗体(ANA)描述了多种与细胞的细胞核的成分包括DNA,蛋白质和核糖1,2反应的自身抗体。的Hep-2细胞,天然蛋白质阵列与数百抗原,提供了理想的底物为ANA 1的检测。全日空的人血清中检测是一个重要的筛选工具结缔组织疾病,IIF是全日空测试1的参考方法。最近,IIF在HEp-2细胞已被替换与抗原特异性免疫测定法和复用方法的一些实验室。由于担忧假阴性结果和缺乏透明度,临床医生,美国风湿病学院成立了一个特别工作组的结论是,IIF使用HEp-2细胞应该是“金标准”ANA筛查1。
由于主观ANA筛查的性质,HEp-2细胞的质量是在tegral到结果的准确和自信的报告。的大量有丝分裂的细胞的存在,最优细胞形态,足够汇合,以及相关抗原的表达是特别重要的。 IIF模式识别用作在患者以帮助诊断的重要工具。了解各种图案的意义使临床医生和实验室工作人员进行适当的后续测试,以确认诊断。例如,均相ANA模式可以发生在抗dsDNA或染色质的抗体的存在,并可能与全身性红斑狼疮(SLE)3相关联。从另一个方面,它最近已描述了所谓的致密的细斑点图案(DFS),其常见于常规样品的高达12%,主要是被用抗DFS70抗体有关。这些自身抗体,当隔离(无其他疾病特异性ANA)发现未与全身性自身免疫性风湿性疾病相关的<燮> 4-9。从而确证试验抗DFS70抗体可以帮助减少不必要的反射测试,提供了相当大的成本节约,并减轻病人的焦虑。
鉴于IIF是第一行筛选方法来检测自身抗体,它是最重要的,该用户能够理解如何试剂和组织基质的选择可影响结果。由于IIF技术本质上是主观的,重要的是,所用的试剂提供最高质量的结果。
该视频协议的这个目标是使用户熟悉执行IIF法,与ANA相关的常见模式所需的正确步骤,并引进新的进步,可以简化实验室工作流程和规范的结果。
Screening by HEp-2 cells is a critical first step in the diagnosis of patients with SARD. However, IIF methods lack harmonization. Sources of variability include preparation of slides, conjugate specificity, and efficiency of the fluorescent microscope and experience of the reader. Despite these concerns, IIF remains the “gold standard” for ANA testing. HEp-2 cells contain a large variety of autoantigens, and provide the ideal substrate for the detection of these autoantibodies. In some laboratories, IIF has been replaced with solid phase screening methods such as multiplex assay or ELISA. The shortcoming of such tests is that they do not display the full range of antigens to be sufficiently sensitive. As a consequence, true positive patients may be missed which can have deleterious consequences In addition to the issues of treatment delay and wrong diagnosis, additional healthcare costs may occur due to the repetition of confirmatory tests or unnecessary diagnostic investigations. Given the inherent challenges to IIF, it is paramount to perform this technique properly to avoid subjectivity in results.
To ensure accurate interpretation and reporting of results for ANA screening, it is vital to use the highest quality substrate. When selecting a HEp-2 substrate, it is critical that the cells be optimized to express clinically relevant epitopes in their native protein state. Transfected or otherwise modified HEp-2 cell lines may not allow proper antibody-antigen binding. In addition, when several different autoantibodies are present simultaneously, recombinant cells may mask one or more patterns. High number of mitotic cells should also be present in the substrate. Adequate number of mitotic cells allows quick and accurate identification of patterns.
In addition to the attributes of the substrate, the objective of this video protocol is to describe the IIF technique by showing critical steps such as the addition of sample, slide washing, addition of conjugate, cover slipping, and determination of positive and negative results. Results can be compromised if proper technique is not used for each of these steps. Proper washing is important to remove all unbound antibody. Some patients display very high amounts of autoantibodies and in these cases it is important to wash the serum from the wells such that it does not contaminate other wells.
Although IIF has traditionally been a very labor intensive and subjective laboratory method, new technologies such as slide processors, barcoded slides and automated digital microscopy can greatly simplify the workflow, increase the consistency and reduce the sources of variability of interpretation.
The authors have nothing to disclose.
我们感谢卡桑德拉科比用于执行IIF实验和卡罗尔布氏她的专家技术审查。
NOVA Lite HEp-2 IgG (DAPI conjugate) | INOVA Diagnostics | 708102 | |
NOVA View Instrument | INOVA Diagnostics | NV2000 | |
QUANTA Link Workstation | INOVA Diagnostics | LINK010 | |
QUANTA Link Workstation License | INOVA Diagnostics | LINK001 | |
Barcode Scanner | INOVA Diagnostics | LINK019 |