A rapid spectrophotometric screening assay for paroxysmal nocturnal hemoglobinuria.
A rapid spectrophotometric screening assay for paroxysmal nocturnal hemoglobinuria.
复制标题
阵发性睡眠性血红蛋白尿的快速分光光度筛查测定。
DOI：
10.1159/000057638
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发表时间：
2002
影响因子：
2.4
通讯作者：
Brodsky,RobertA
中科院分区：
文献类型：
--
作者：
Mukhina,GalinaL;Buckley,Thomas;Brodsky,RobertA
阵发性睡眠性血红蛋白尿症 (PNH) 是一种克隆性造血干细胞疾病，可导致补体介导的溶血性贫血和血栓形成倾向[1]。 PNH 干细胞及其后代的遗传标志是 x 连锁基因 PIGA 的体细胞突变 [2]。因此，细胞无法合成糖基磷脂酰肌醇 (GPI) 锚，该锚用于将多种蛋白质束缚到质膜上。因此，许多 GPI 锚定蛋白，包括补体调节蛋白 CD59 和 CD55，在 PNH 血细胞上缺乏。在发达国家，传统的 PNH 筛查试验（例如 Ham 试验和蔗糖溶血试验）已被更灵敏和特异的流式细胞术试验所取代，这些试验利用荧光标记的单克隆抗体来检测 GPI 锚定蛋白的缺陷 [3, 4]。我们最近描述了一种检测 PNH 的改进方法，基于我们的发现，即 GPI 锚定蛋白充当气溶素 [5] 的受体，气溶素是一种能够通过形成膜通道裂解细胞的细菌毒素。由于 PNH 细胞缺乏 GPI 锚定蛋白，因此它们不会结合气溶素，并且对这种毒素具有独特的抵抗力 [6]。因此，气溶素和荧光标记的无活性气溶素原变体 (FLAER) 可用于流式细胞术检测 PNH 细胞，比单个单克隆抗体具有更高的灵敏度和特异性 [7]。尽管使用 FLAER 或单克隆抗体的流式细胞术已成为发达国家 PNH 检测的标准，但它需要昂贵的设备和重要的技术专业知识，而这些设备和技术知识尚未广泛普及，特别是在 PNH 更常见的发展中国家。我们现在描述了一种快速且廉价的 PNH 筛查方法，仅需要读板器和气溶素。
Paroxysmal nocturnalhemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder that leads to a complement-mediated hemolytic anemia and a propensity to thrombosis [1]. The genetic hallmark of PNH stem cells and their progeny is a somatic mutation in the x-linked gene, PIGA [2]. As a result, the cells are unable to synthesize glycosylphosphatidylinositol (GPI) anchors, which are used to tether a wide array of proteins to the plasma membrane. Consequently, many GPI-anchored proteins, including the complement-regulatory proteins CD59 and CD55, are deficient on PNH blood cells.In developed countries, traditional screening assays for PNH, such as the Ham test and sucrose hemolysis test, have been replaced by more sensitive and specific flow cytometric assays that utilize fluorescently labeled monoclonal antibodies to detect a deficiency of GPI-anchored proteins [3, 4]. We recently described an improved method to detect PNH, based on our discovery that GPI-anchored proteins serve as receptors for aerolysin [5], a bacterial toxin capable of lysing cells by forming membrane channels. Because they lack GPI-anchored proteins, PNH cells do not bind aerolysin and they are uniquely resistant to the toxin [6]. As a result, aerolysin and a fluorescently labeled inactive proaerolysin variant (FLAER) can be used in flow cytometric assays to detect PNH cells with greater sensitivity and specificity than individual monoclonal antibodies [7]. Although flow cytometry with FLAER or with monoclonal antibodies has become the standard for PNH detection in the developed world, it requires expensive equipment and significant technical expertise that is not widely available, especially in developing countries where PNH is more common. We now characterize a rapid and inexpensive screening assay for PNH that requires only a plate reader and aerolysin.