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Published on: June 19, 2013
A novel image cytometric method for quantitation of immunohistochemical staining of cytoplasmic antigens
M Guillaud1, J B Matthews, A Harrison
1Cancer Imaging Department, BC Cancer Research Centre, Vancouver, Canada.
Insights
A new VORSTAIN software method uses Voronoi tessellation to quantify molecular marker staining intensity in tissues. This automated image cytometry approach improves consistency and reveals tumor heterogeneity, aiding prognostication.
Area of Science:
- Computational pathology
- Biomedical image analysis
- Cancer research
Background:
- Traditional immunohistochemical evaluation relies on subjective pathologist assessment.
- Quantitative analysis of non-nuclear staining is limited by challenges in defining cellular boundaries.
- Automated image cytometry offers potential for objective and consistent molecular marker evaluation.
Purpose of the Study:
- To introduce VORSTAIN software for quantitative assessment of molecular marker expression.
- To develop a novel image histometric method for defining cellular regions and measuring staining intensity.
- To evaluate the accuracy and reproducibility of VORSTAIN compared to traditional methods.
Main Methods:
- Development of VORSTAIN software based on the Oncometrics CytoSavant Automated Image Cytometry System.
- Application of Voronoi tessellation to approximate cellular boundaries using nuclear centroid positions.
- Quantitative measurement of staining intensity within defined cellular regions in human breast cancer tissue sections.
Main Results:
- VORSTAIN demonstrated low intra-observer (1.5-2.6%) and inter-observer (1.8-4.4%) variation.
- Quantitative staining density analysis correlated well with subjective pathologist scoring.
- The software detected significant intratumoral heterogeneity in molecular marker expression.
Conclusions:
- VORSTAIN provides a reliable and consistent method for quantitative immunohistochemical analysis.
- The software accurately quantifies molecular marker expression and reveals tumor heterogeneity.
- Accurate measurement of tumor heterogeneity using VORSTAIN may enhance cancer prognostication.
Abstract:
Evaluation of molecular markers by immunohistochemical labelling of tissue sections has traditionally been performed by qualitative assessment by trained pathologists. For those markers with a staining component present outside of the nucleus, there has been no image histometric method available to reliably and consistently define cell interfaces within the tissue. We present a new method of approximating cellular boundaries to define cellular regions within which quantitative measurements of staining intensity may be made. The method is based upon Voronoi tessellation of a defined region of interest (ROI), and requires only the position of the nuclear centroids within the ROI. Here we describe the VORSTAIN software which has been developed based on the Oncometrics CytoSavant Automated Image Cytometry System. To demonstrate this technique, human breast cancer sections immunohistochemically stained for bcl-2 protein and counter-stained with nuclear methyl green stain were evaluated. Intra-observer variation in the measured values was between 1.5-2.6% and inter-observer variation was between 1.8-4.4%. The primary source of variability was due to difficulties in interpreting the exact position of the nuclear centroids. Analysis of mean staining densities for each slide correlated well with subjective scoring performed by two independent pathologists. Using VORSTAIN, significant variation of staining intensities between regions within the same slide was measured for some sections, indicating a large degree of heterogeneity within the tumours. The ability to accurately quantitate the degree of heterogeneity of molecular marker expression within tumours may be a valuable tool in prognostication.
