Abstract
BackgroundDetermining the correct number of positive immune cells in immunohistological sections of colorectal cancer and other tumor entities is emerging as an important clinical predictor and therapy selector for an individual patient. This task is usually obstructed by cell conglomerates of various sizes. We here show that at least in colorectal cancer the inclusion of immune cell conglomerates is indispensable for estimating reliable patient cell counts. Integrating virtual microscopy and image processing principally allows the high-throughput evaluation of complete tissue slides.Methodology/Principal findingsFor such large-scale systems we demonstrate a robust quantitative image processing algorithm for the reproducible quantification of cell conglomerates on CD3 positive T cells in colorectal cancer. While isolated cells (28 to 80 µm2) are counted directly, the number of cells contained in a conglomerate is estimated by dividing the area of the conglomerate in thin tissues sections (≤6 µm) by the median area covered by an isolated T cell which we determined as 58 µm2. We applied our algorithm to large numbers of CD3 positive T cell conglomerates and compared the results to cell counts obtained manually by two independent observers. While especially for high cell counts, the manual counting showed a deviation of up to 400 cells/mm2 (41% variation), algorithm-determined T cell numbers generally lay in between the manually observed cell numbers but with perfect reproducibility.ConclusionIn summary, we recommend our approach as an objective and robust strategy for quantifying immune cell densities in immunohistological sections which can be directly implemented into automated full slide image processing systems.
Highlights
In situ immunohistochemical staining of tumor-infiltrating immune cells against the immune cell surface molecules CD3, CD8, CD45RO and Granzyme B in large cohorts of human colorectal cancers [1,2,3] supports the hypothesis that the adaptive immune response influences the behavior of human tumors
It is important to note that the observed immune cell densities were better predictors of prognosis than the classical TNM classification
[4,5,6], initiating a debate on the feasibility of individualized prognosis prediction based on immune cell densities
Summary
In situ immunohistochemical staining of tumor-infiltrating immune cells against the immune cell surface molecules CD3, CD8, CD45RO and Granzyme B in large cohorts of human colorectal cancers [1,2,3] supports the hypothesis that the adaptive immune response influences the behavior of human tumors. For independent observers it is frequently rather difficult to reproduce quantities of cell densities roughly estimated by others [13,14] This is especially the case with the difficult estimation of the number of cells contained in complex cell conglomerates (see figure 1), leading to incomplete evaluations of slides [15]. Determining the correct number of positive immune cells in immunohistological sections of colorectal cancer and other tumor entities is emerging as an important clinical predictor and therapy selector for an individual patient. This task is usually obstructed by cell conglomerates of various sizes. Integrating virtual microscopy and image processing principally allows the high-throughput evaluation of complete tissue slides
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