Department of Pathology and Oncology, Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA.
Cytometry B Clin Cytom. 2010 Jul;78(4):211-30. doi: 10.1002/cyto.b.20525.
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by a somatic mutation in the PIGA gene, leading to a deficiency of proteins linked to the cell membrane via glycophosphatidylinositol (GPI) anchors. While flow cytometry is the method of choice for identifying cells deficient in GPI-linked proteins and is, therefore, necessary for the diagnosis of PNH, to date there has not been an attempt to standardize the methodology used to identify these cells.
In this document, we present a consensus effort that describes flow cytometric procedures for detecting PNH cells.
We discuss clinical indications and offer recommendations on data interpretation and reporting but mostly focus on analytical procedures important for analysis. We distinguish between routine analysis (defined as identifying an abnormal population of 1% or more) and high-sensitivity analysis (in which as few as 0.01% PNH cells are detected). Antibody panels and gating strategies necessary for both procedures are presented in detail. We discuss methods for assessing PNH populations in both white blood cells and red blood cells and the relative advantages of measuring each. We present steps needed to validate the more elaborate high-sensitivity techniques, including the need for careful titration of reagents and determination of background rates in normal populations, and discuss technical pitfalls that might affect interpretation.
This document should both enable laboratories interested in beginning PNH testing to establish a valid procedure and allow experienced laboratories to improve their techniques.
阵发性睡眠性血红蛋白尿症（PNH）是一种罕见的造血干细胞疾病，其特征是 PIGA 基因突变，导致通过糖基磷脂酰肌醇（GPI）锚定连接到细胞膜的蛋白缺失。虽然流式细胞术是识别 GPI 连接蛋白缺陷细胞的首选方法，因此是 PNH 诊断所必需的，但迄今为止，尚未尝试标准化用于识别这些细胞的方法学。
在本文档中，我们提出了一项共识努力，描述了用于检测 PNH 细胞的流式细胞术程序。
我们讨论了临床指征，并就数据解释和报告提出了建议，但主要侧重于分析对于分析很重要的程序。我们区分了常规分析（定义为识别出 1%或更多异常细胞群）和高灵敏度分析（其中检测到的 PNH 细胞少至 0.01%）。详细介绍了这两种程序所需的抗体面板和门控策略。我们讨论了在白细胞和红细胞中评估 PNH 群体的方法，以及测量每种方法的相对优势。我们介绍了验证更复杂的高灵敏度技术所需的步骤，包括仔细滴定试剂和确定正常人群中的背景率的需要，并讨论了可能影响解释的技术难题。
本文档既应使有兴趣开始进行 PNH 检测的实验室能够建立有效的程序，又应使有经验的实验室能够改进其技术。
