Nguyen Xuan Duc, Dugrillon Alex, Beck Christian, Kerowgan Mohammad, Klüter Harald
Institute of Transfusion Medicine and Immunology, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Red Cross Blood Service of Baden-Württemberg-Hessen, 68167 Mannheim, Germany.
Br J Haematol. 2004 Dec;127(5):552-60. doi: 10.1111/j.1365-2141.2004.05233.x.
Glycoprotein (GP)-specific platelet antibodies can cause allo-immune and auto-immune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Here, we describe an improved method based on simultaneous detection of various platelet-specific immunoglobulin G (IgG) and IgM antibodies. Bead populations with distinct fluorescence intensities, coated with monoclonal antibodies specific for mouse heavy chain isotypes, were used for the simultaneous immobilization of platelet-GP [IIb/IIIa, Ib/IX, human leucocyte antigen (HLA) class I, or Ia/IIa, CD32, GPIV or CD109, Ib/IX, HLA class I]. In order to detect human IgG and IgM antibodies simultaneously, phycoerythrin- and fluorescein isotiocyanate-conjugated goat anti-human IgG and IgM were added. On this basis, the abundance of six distinct antibodies (three anti-GP, each with subclasses IgG and IgM) were simultaneously analysed without cross-reaction by flow cytometry. For evaluation, sera and platelets from 169 patients with platelet-binding and/or platelet-associated antibodies were investigated. The monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay was performed in parallel as reference test. The simultaneous analysis of platelet-specific antibodies (SASPA) assay was able to detect all platelet-specific IgG and IgM that were also recognized by MAIPA with a comparable sensitivity. SASPA proved to be a rapid and reliable assay that required less platelets than other methods. This method has the potential to pave the way for new investigations of platelet-specific antibodies.
糖蛋白（GP）特异性血小板抗体可导致同种免疫性和自身免疫性血小板减少症。相关抗体的特异性检测是诊断和治疗的前提条件。在此，我们描述了一种基于同时检测各种血小板特异性免疫球蛋白G（IgG）和IgM抗体的改进方法。使用涂有小鼠重链同种型特异性单克隆抗体的具有不同荧光强度的微珠群体，用于同时固定血小板GP[IIb/IIIa、Ib/IX、人类白细胞抗原（HLA）I类或Ia/IIa、CD32、GPIV或CD109、Ib/IX、HLA I类]。为了同时检测人IgG和IgM抗体，加入了藻红蛋白和异硫氰酸荧光素偶联的山羊抗人IgG和IgM。在此基础上，通过流式细胞术同时分析六种不同抗体（三种抗GP，每种都有IgG和IgM亚类）的丰度，且无交叉反应。为了进行评估，研究了169例具有血小板结合和/或血小板相关抗体患者的血清和血小板。同时进行单克隆抗体特异性血小板抗原固定（MAIPA）试验作为参考试验。血小板特异性抗体同步分析（SASPA）试验能够检测出所有也被MAIPA识别的血小板特异性IgG和IgM，灵敏度相当。SASPA被证明是一种快速可靠的试验，所需血小板比其他方法少。该方法有可能为血小板特异性抗体的新研究铺平道路。
