A NOVEL IMAGE CYTOMETRIC METHOD FOR QUANTITATION OF IMMUNOHISTOCHEMICAL STAINING OF CYTOPLASMIC ANTIGENS

Evaluation of molecular markers by immunohistochemical labelling of ti ssue sections has traditionally been performed by qualitative assessme nt by trained pathologists. For those markers with a staining componen t present outside of the nucleus, there has been no image histometric method available to reliably and consistently define cell interfaces w ithin the tissue. We present a new method of approximating cellular bo undaries to define cellular regions within which quantitative measurem ents of staining intensity may be made. The method is based upon Voron oi tessellation of a defined region of interest (ROI), and requires on ly the position of the nuclear centroids within the ROI. Here we descr ibe the VORSTAIN software which has been developed based on the Oncome trics 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 b etween 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 stainin g densities for each slide correlated well with subjective scoring per formed by two independent pathologists. Using VORSTAIN, significant va riation of staining intensities between regions within the same slide was measured for some sections, indicating a large degree of heterogen eity within the tumours. The ability to accurately quantitate the degr ee of heterogeneity of molecular marker expression within tumours may be a valuable tool in prognostication.