Müller Jens, Hamedani Nasim Shahidi, McRae Hannah L, Rühl Heiko, Oldenburg Johannes, Pötzsch Bernd
Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn 53127, Germany.
ACS Omega. 2022 Aug 24;7(35):30801-30806. doi: 10.1021/acsomega.2c02077. eCollection 2022 Sep 6.
A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) is a metalloprotease that regulates the size of circulating von Willebrand factor (vWF) multimers. Severe lack of ADAMTS-13 activity [<10% of normal (0.1 IU/mL)] leads to thrombotic thrombocytopenic purpura (TTP), a specific type of thrombotic microangiopathy (TMA). Timely determination of plasma ADAMTS-13 activity is essential to discriminate TTP from other types of TMA with respect to adequate treatment. Identification of the minimal substrate motif for ADAMTS-13 within the A2 domain of vWF (vWF73) as well as the generation of monoclonal antibodies (mAbs) that specifically recognize the ADAMTS-13 cleavage site enabled the development of a variety of methods for determination of plasma ADAMTS-13 activity. In order to further extend the range of analytical platforms applicable for quantitative determination of plasma ADAMTS-13 activity, a specific, vWF/mAb-based assay with flow cytometric readout was developed and validated. Basic assay characteristics include a total assay time of 80 to 90 min, a near linear dynamic range from 0.005 (lower limit of quantification) to 0.2 IU/mL, and intra- and interassay coefficients of variation below 5 and 30% at input plasma ADAMTS-13 activities of 0.015 and ≤0.050 IU/mL, respectively. When compared to the results obtained with a commercially available quantitative ADAMTS-13 activity ELISA, analysis of 18 plasma samples obtained from patients with suspected TTP revealed full agreement of results with respect to the clinical 0.1 IU/mL TTP threshold. Based on these data, it is assumed that the described assay principle can be successfully transferred to virtually all laboratories that have a flow cytometer available.
含Ⅰ型血小板反应蛋白基序的解聚素和金属蛋白酶13（ADAMTS - 13）是一种金属蛋白酶，可调节循环中血管性血友病因子（vWF）多聚体的大小。ADAMTS - 13活性严重缺乏[<正常水平的10%（0.1 IU/mL）]会导致血栓性血小板减少性紫癜（TTP），这是一种特定类型的血栓性微血管病（TMA）。及时测定血浆ADAMTS - 13活性对于在进行适当治疗时鉴别TTP与其他类型的TMA至关重要。确定vWF A2结构域内ADAMTS - 13的最小底物基序（vWF73）以及产生特异性识别ADAMTS - 13切割位点的单克隆抗体（mAb），使得能够开发出多种测定血浆ADAMTS - 13活性的方法。为了进一步扩大适用于定量测定血浆ADAMTS - 13活性的分析平台范围，开发并验证了一种基于vWF/mAb且采用流式细胞术读数的特异性检测方法。该检测方法的基本特性包括：总检测时间为80至90分钟，接近线性的动态范围为0.005（定量下限）至0.2 IU/mL，在输入血浆ADAMTS - 13活性分别为0.015和≤0.050 IU/mL时，批内和批间变异系数分别低于5%和30%。与使用市售定量ADAMTS - 13活性ELISA获得的结果相比，对18份疑似TTP患者的血浆样本进行分析发现，在临床TTP阈值0.1 IU/mL方面，结果完全一致。基于这些数据，假定所描述的检测原理能够成功应用于几乎所有配备流式细胞仪的实验室。
