QuPath在低细胞组织（如椎间盘）免疫组织化学染色切片半自动定量分析中的应用与验证

IF 3.4 3区医学 Q1 ORTHOPEDICS

JOR Spine Pub Date : 2025-03-06 DOI:10.1002/jsp2.70054

Andrea Nüesch, Maria Paola Ferri, Christine L. Le Maitre

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引用次数: 0

摘要

免疫组织化学（IHC）是一种广泛应用于组织样品中蛋白质定位和半定量的方法。传统的免疫组化分析通常依赖于在指定区域内手动计数200个细胞，这是一个耗时且主观的过程，可能会损害再现性和准确性。数字扫描和生物图像分析工具的进步，如开源软件QuPath，实现了半自动化细胞计数，减少了主观性，提高了效率。本项目开发了一个基于qupath的脚本和详细指南，用于半自动细胞计数，特别是对于低细胞组织，如椎间盘和软骨。方法与结果手工计数与半自动定量之间无显著差异（p = 0.783, p = 0.386），而II型胶原染色方法（r = 0.9602, p < 0.0001）与N-cadherin染色方法（r = 0.9044, p = 0.0001）之间有很强的相关性。此外，采用半自动化的定量方法，3个不同学术等级和免疫结构分析经验的评分者之间具有很强的相关性（类内相关系数(ICC) = 0.853）。该方法确保了高再现性和准确性，减少了评分者和实验室之间的可变性。这种半自动化的方法特别适合于具有高细胞外基质与细胞比例和低细胞密度的组织。通过最大限度地减少主观性和评估时间，它为手动计数提供了一个健壮的替代方案，使其成为对再现性和标准化至关重要的应用程序的理想选择。虽然该方法在低细胞组织中有效，但其在其他组织类型中的应用仍有待进一步探索。这些发现强调了QuPath在简化IHC分析和增强实验室间可比性方面的潜力。

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Application and Validation of Semiautomatic Quantification of Immunohistochemically Stained Sections for Low Cellular Tissue Such as Intervertebral Disc Using QuPath

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Application and Validation of Semiautomatic Quantification of Immunohistochemically Stained Sections for Low Cellular Tissue Such as Intervertebral Disc Using QuPath

Background

Immunohistochemistry (IHC) is a widely used method for localizing and semi-quantifying proteins in tissue samples. Traditional IHC analysis often relies on manually counting 200 cells within a designated area, a time-intensive and subjective process that can compromise reproducibility and accuracy. Advances in digital scanning and bioimage analysis tools, such as the open-source software QuPath, enable semi-automated cell counting, reducing subjectivity and increasing efficiency.

Aims

This project developed a QuPath-based script and detailed guide for semi-automatic cell counting, specifically for tissues with low cellularity, such as intervertebral discs and cartilage.

Methods and Results

The methodology was validated by demonstrating no significant differences between the manual counting and the semi-automatic quantification (p = 0.783, p = 0.386) while showing a strong correlation between methods for both collagen type II staining (r = 0.9602, p < 0.0001) and N-cadherin staining (r = 0.9044, p = 0.0001). Furthermore, a strong correlation (intraclass correlation coefficient (ICC) single raters = 0.853) between 3 individual raters with varying academic ranks and experiences in IHC analysis was shown using the semi-automatic quantification method.

Discusssion

The approach ensures high reproducibility and accuracy, with reduced variability between raters and laboratories. This semi-automated method is particularly suited for tissues with a high extracellular matrix to cell ratio and low cellularity. By minimizing subjectivity and evaluation time, it provides a robust alternative to manual counting, making it ideal for applications where reproducibility and standardization are critical. While the methodology was effective in low-cellularity tissues, its application in other tissue types warrants further exploration.