A novel method for automated assessment of megakaryocyte differentiation and proplatelet formation

Transfusion of platelet concentrates represents an important treatment for various bleeding complications. However, the short half-life and frequent contaminations with bacteria restrict the availability of platelet concentrates and raise a clear demand for platelets generated <i>ex vivo</i>. Therefore, <i>in vitro</i> platelet generation from megakaryocytes represents an important research topic. A vital step for this process represents accurate analysis of thrombopoiesis and proplatelet formation, which is usually conducted manually. We aimed to develop a novel method for automated classification and analysis of proplatelet-forming megakaryocytes <i>in vitro</i>. After fluorescent labelling of surface and nucleus, MKs were automatically categorized and analysed with a novel pipeline of the open source software <i>CellProfiler</i>. Our new workflow is able to detect and quantify four subtypes of megakaryocytes undergoing thrombopoiesis: proplatelet-forming, spreading, pseudopodia-forming and terminally differentiated, anucleated megakaryocytes. Furthermore, we were able to characterize the inhibitory effect of dasatinib on thrombopoiesis in more detail. Our new workflow enabled rapid, unbiased, quantitative and qualitative in-depth analysis of proplatelet formation based on morphological characteristics. Clinicians and basic researchers alike will benefit from this novel technique that allows reliable and unbiased quantification of proplatelet formation. It thereby provides a valuable tool for the development of methods to generate platelets <i>ex vivo</i> and to detect effects of drugs on megakaryocyte differentiation.

血小板浓缩物输注是治疗各类出血并发症的重要手段。然而，血小板浓缩物半衰期短且易频发细菌污染，限制了其临床可及性，同时催生了体外（ex vivo）制备血小板的明确需求。因此，从巨核细胞（megakaryocytes）进行体外（in vitro）血小板生成已成为重要的研究课题。该流程的核心环节是对血小板生成（thrombopoiesis）与前血小板形成（proplatelet formation）进行精准分析，而此类分析以往多依赖人工完成。本研究旨在开发一种可自动分类并分析体外前血小板形成巨核细胞的全新方法。对巨核细胞的表面与细胞核完成荧光标记后，我们借助开源软件CellProfiler搭建的新型分析流程，实现了巨核细胞的自动分类与分析。我们的新型分析流程可检测并量化四种处于血小板生成阶段的巨核细胞亚型：形成前血小板的巨核细胞、铺展型巨核细胞、伪足形成型巨核细胞，以及终末分化的无核巨核细胞。此外，我们还详细阐明了达沙替尼（dasatinib）对血小板生成的抑制效应。我们的新型分析流程可基于细胞形态学特征，快速、无偏地完成前血小板形成的定性与定量深度分析。临床医师与基础研究人员均可从该技术中获益：该技术可可靠且无偏地量化前血小板形成过程，从而为体外（ex vivo）制备血小板的方法开发，以及检测药物对巨核细胞分化的影响提供了极具价值的研究工具。