A novel image cytometric method for quantitation of immunohistochemical staining of cytoplasmic antigens

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.