Cytotoxicity tests are important for the screening and evaluation of biocompatibility of artificial organs. Morphologic changes of cells that were contacted biomaterials or biomaterial extracts indicate their toxicity. However, information on cytotoxic effects is still obtained by subjective visual inspection of microscopic samples. In this article, a novel computer assisted method is introduced. The automatic analysis of digitized micrographs is achieved in several stages: segmentation, separation, classification, and measurement. The segmentation of the image is provided by a new local adaptive thresholding technique, which adapts the threshold window sizes onto local gray level distribution and yields optimal window sizes. The actual threshold is obtained by maximizing interclass variances and minimizing intraclass variance. For the separation of connected cells, the binarized samples are cleaned from “false” markers by morphologic filtering. The subsequent separation is a two phase approach. Information levels are generated top-down by successively applying an enhanced erosion operator, which yields markers and filters noise usually evolving from multiple erosions. The converse bottom-up integration of the eroded markers is performed by successively applying an enhanced dilation operator, which reconstructs the cells and prevents merging of already separated objects. The subsequent measuring provides quantitative parameters of the distribution of size and compactness of the cells contained within the sample. The method was evaluated by L-929 fibroblasts that were in contact with 0%, 5%, and 10% concentrations of ethanol. For each concentration, 268 images of the cell populations were captured. The obtained quantitative parameters are highly correlated to the common verbal description of morphologic changes. Therefore, the proposed automatic method has several advantages compared with subjective examinations. The results allow an objective comparison of the quantification of phenomena; the subjective influence of the observer is eliminated; and the laboratory staff is relieved of time consuming routine work. ASAIO Journal 1999; 45:264–271.
Copyright © 1999 by the American Society for Artificial Internal Organs