High-throughput microsphiltration to assess red blood cell deformability and screen for malaria transmission-blocking drugs
High-throughput microsphiltration to assess red blood cell deformability and screen for malaria transmission-blocking drugs
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DOI：
10.1038/nprot.2018.035
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发表时间：
2018-06-01
期刊：
影响因子：
14.8
通讯作者：
Buffet, Pierre
中科院分区：
文献类型：
--
作者：
Duez, Julien;Carucci, Mario;Buffet, Pierre
刚性红细胞在脾脏中的机械滞留是主要血液学疾病如遗传性球形红细胞增多症、镰状细胞病和疟疾的核心。在这里，我们描述了使用微球过滤（微球过滤），以评估红细胞变形性在数百至数千个样品中平行，通过微球层过滤他们在384孔板适合于发现化合物，恶性疟原虫配子体，目的是中断疟疾传播。将暴露于化合物的配子母细胞加载到微量离心板中，过滤，然后转移到成像板中进行分析。高含量成像检测过滤器上游和下游的活配子体，并量化脾脏样滞留。该筛选试验需要3-4天。与目前用于评估红细胞（RBC）变形能力的方法不同，微离心能够进行高通量药理学筛选（在几个月内测试数万种化合物），并涉及诱导生理相关哑铃形变形的细胞机械挑战。因此，它直接评估RBC在体内穿过内皮间脾缝的能力。该方法在输血质量控制和血液病红细胞表型标志物测定中具有潜在的应用价值。
The mechanical retention of rigid erythrocytes in the spleen is central in major hematological diseases such as hereditary spherocytosis, sickle-cell disease and malaria. Here, we describe the use of microsphiltration (microsphere filtration) to assess erythrocyte deformability in hundreds to thousands of samples in parallel, by filtering them through microsphere layers in 384-well plates adapted for the discovery of compounds that stiffen Plasmodium falciparum gametocytes, with the aim of interrupting malaria transmission. Compound-exposed gametocytes are loaded into microsphiltration plates, filtered and then transferred to imaging plates for analysis. High-content imaging detects viable gametocytes upstream and downstream from filters and quantifies spleen-like retention. This screening assay takes 3-4 d. Unlike currently available methods used to assess red blood cell (RBC) deformability, microsphiltration enables high-throughput pharmacological screening (tens of thousands of compounds tested in a matter of months) and involves a cell mechanical challenge that induces a physiologically relevant dumbbell-shape deformation. It therefore directly assesses the ability of RBCs to cross inter-endothelial splenic slits in vivo. This protocol has potential applications in quality control for transfusion and in determination of phenotypic markers of erythrocytes in hematological diseases.