{"title":"In Vitro Monocyte/Macrophage Phagocytosis Assay for the Prediction of Drug-Induced Thrombocytopenia","authors":"Padma Kumar Narayanan, Nianyu Li","doi":"10.1002/cptx.68","DOIUrl":null,"url":null,"abstract":"<p>Phagocytosis of platelets by monocytes and macrophages is a primary mechanism of platelet clearance <i>in vivo</i> and has been increasingly implicated in playing an important role in thrombocytopenia mediated by monoclonal antibodies intended for therapeutic purposes. In the present article, we describe an <i>in vitro</i> flow cytometry assay to assess the effect of antibody-mediated platelet phagocytosis by monocytes. Freshly isolated platelets were labeled with a fluorescent probe, 5-chloromethylfluorescein diacetate (CMFDA) and then co-cultured with isolated peripheral blood mononuclear cells (PBMCs) from the same donor in the presence of increasing concentrations of a monoclonal antibody drug. After incubation, an increase in CMFDA fluorescence intensity of CD14 positive monocytes was evaluated by flow cytometry as an assessment for drug-mediated platelet phagocytosis by monocytes. The assay has been evaluated using both human and cynomolgus monkey cells for the prediction of drug-induced thrombocytopenia. © 2019 by John Wiley & Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"79 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2019-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.68","citationCount":"5","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols in toxicology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cptx.68","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 5
Abstract
Phagocytosis of platelets by monocytes and macrophages is a primary mechanism of platelet clearance in vivo and has been increasingly implicated in playing an important role in thrombocytopenia mediated by monoclonal antibodies intended for therapeutic purposes. In the present article, we describe an in vitro flow cytometry assay to assess the effect of antibody-mediated platelet phagocytosis by monocytes. Freshly isolated platelets were labeled with a fluorescent probe, 5-chloromethylfluorescein diacetate (CMFDA) and then co-cultured with isolated peripheral blood mononuclear cells (PBMCs) from the same donor in the presence of increasing concentrations of a monoclonal antibody drug. After incubation, an increase in CMFDA fluorescence intensity of CD14 positive monocytes was evaluated by flow cytometry as an assessment for drug-mediated platelet phagocytosis by monocytes. The assay has been evaluated using both human and cynomolgus monkey cells for the prediction of drug-induced thrombocytopenia. © 2019 by John Wiley & Sons, Inc.
体外单核细胞/巨噬细胞吞噬试验预测药物性血小板减少症
单核细胞和巨噬细胞吞噬血小板是体内血小板清除的主要机制,并且越来越多地涉及在治疗目的的单克隆抗体介导的血小板减少症中发挥重要作用。在这篇文章中,我们描述了一种体外流式细胞术测定,以评估抗体介导的单核细胞吞噬血小板的作用。用荧光探针标记新分离的血小板,5-氯甲基荧光素二乙酸酯(CMFDA),然后在增加单克隆抗体药物浓度的情况下,与来自同一供者的分离的外周血单核细胞(PBMCs)共培养。孵育后,流式细胞术评估CD14阳性单核细胞CMFDA荧光强度的增加,以评估单核细胞对药物介导的血小板吞噬作用。该检测方法已经用人类和食蟹猴细胞对药物性血小板减少症的预测进行了评估。©2019 by John Wiley &儿子,Inc。
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