关于提高激光扫描细胞术对人实体瘤固定细胞悬液进行定量多参数免疫荧光测量可重复性的指南。

Shackney Stanley, Emlet David R, Pollice Agnese, Smith Charles, Brown Kathryn, Kociban Deborah

Laboratory of Cancer Cell Biology and Genetics, Department of Human Oncology, Allegheny Singer Research Institute, Allegheny General Hospital, Pittsburgh, Pennsylvania 15212, USA.

Cytometry B Clin Cytom. 2006 Jan;70(1):10-9. doi: 10.1002/cyto.b.20084.

BACKGROUND

Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements.

METHODS AND RESULTS

We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided.

CONCLUSIONS

Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications.

背景

激光扫描细胞术（LSC）是一项多功能技术，它能够对单个细胞进行多项测量，并将每个细胞的测量结果与其他细胞特征逐一关联起来。能够对人类实体瘤中的单个细胞进行可重复、定量且相互关联的基于细胞的免疫荧光研究，将是非常理想的。然而，由于存在大量细胞聚集体和其他混杂因素，此类研究可能具有挑战性。已经开发出一些技术来处理LSC收集的数据集中的细胞聚集体。在解决其他可能影响此类基于细胞的免疫荧光测量可重复性的关键技术和方法学问题方面也积累了经验。

方法与结果

我们描述了细胞样本采集、细胞固定和染色的实际操作、通过LSC进行多参数免疫荧光测量的方案、对照和参考样本的使用，以及我们发现对提高在人类肿瘤样本中获得的LSC数据的准确性和可重复性有用的数据分析方法。我们提供了LSC在检查基于细胞分析的定量方面潜在优势的示例。基于细胞的多参数免疫荧光测量质量的提高使得从相对少量的细胞中提取有用信息成为可能。这反过来又允许在每个肿瘤样本上进行多个多色检测。通过重叠测量提供的不同检测之间的联系，可以在人类实体瘤的临床样本中开发出越来越广泛的多种蛋白质细胞内表达谱。文中提供了此类关联检测的示例。