Automated fluorescent miscroscopic image analysis of PTBP1 expression in glioma.
Automated fluorescent miscroscopic image analysis of PTBP1 expression in glioma.
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DOI：
10.1371/journal.pone.0170991
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Otero JJ
中科院分区：
文献类型：
--
作者：
Kaya B;Goceri E;Becker A;Elder B;Puduvalli V;Winter J;Gurcan M;Otero JJ
由于存在若干技术障碍，其中包括自发荧光，多重免疫荧光检测尚未应用于诊断神经病理学。本研究介绍了一种能够通过使用自动数字图像分析克服荧光显微镜在诊断神经病理学中的视觉挑战的方法，其长期目标是为实体组织神经病理学的多重生物标志物提供无偏定量分析。在本研究中，我们验证了多聚嘧啶区结合蛋白1（PTBP1）这一胶质瘤的潜在生物标志物，并测试了免疫荧光显微镜结合自动无偏图像分析在多大程度上能够使PTBP1作为生物标志物用于区分具有诊断挑战性的手术活检标本。作为范例，我们利用了被诊断为反应性脑改变（假性进展）和复发性胶质母细胞瘤（真性进展）患者的二次切除标本。我们的图像分析流程能够去除背景自发荧光，并能够对4',6 - 二脒基 - 2 - 苯基吲哚（DAPI） - PTBP1阳性细胞、PTBP1阳性细胞核以及细胞中PTBP1信号的平均强度值进行定量。由于神经病理学专家之间的不一致率过高，传统的病理学解读无法区分这些组别。我们的数据表明，与仅显示反应性胶质增生的二次手术切除标本相比，复发性胶质母细胞瘤显示出更多的DAPI - PTBP1阳性细胞以及更高的PTBP1信号平均强度值。我们的工作展示了利用自动图像分析克服在诊断神经病理学中应用荧光显微镜所面临挑战的潜力。
Multiplexed immunofluorescent testing has not entered into diagnostic neuropathology due to the presence of several technical barriers, amongst which includes autofluorescence. This study presents the implementation of a methodology capable of overcoming the visual challenges of fluorescent microscopy for diagnostic neuropathology by using automated digital image analysis, with long term goal of providing unbiased quantitative analyses of multiplexed biomarkers for solid tissue neuropathology. In this study, we validated PTBP1, a putative biomarker for glioma, and tested the extent to which immunofluorescent microscopy combined with automated and unbiased image analysis would permit the utility of PTBP1 as a biomarker to distinguish diagnostically challenging surgical biopsies. As a paradigm, we utilized second resections from patients diagnosed either with reactive brain changes (pseudoprogression) and recurrent glioblastoma (true progression). Our image analysis workflow was capable of removing background autofluorescence and permitted quantification of DAPI-PTBP1 positive cells. PTBP1-positive nuclei, and the mean intensity value of PTBP1 signal in cells. Traditional pathological interpretation was unable to distinguish between groups due to unacceptably high discordance rates amongst expert neuropathologists. Our data demonstrated that recurrent glioblastoma showed more DAPI-PTBP1 positive cells and a higher mean intensity value of PTBP1 signal compared to resections from second surgeries that showed only reactive gliosis. Our work demonstrates the potential of utilizing automated image analysis to overcome the challenges of implementing fluorescent microscopy in diagnostic neuropathology.