Quantification of proliferating cell nuclear antigen in large intestinal crypt by computer-assisted image analysis

Abstract

Measurements of proliferating cell nuclear antigen (PCNA) labeling of the large intestinal crypts scored by experienced observers were compared with those generated by computer-assisted image analysis (CAIA). (CAIA was performed at Pathology Expertise, Inc., Newton, MA, by A. V. Sotnikov.) Serial sections (3 microns) of the rectal biopsy specimens from 32 patients were immunostained for PCNA and then counterstained with hematoxylin. The same set of slides and rules for the location/acquisition of complete crypts was used to assess a minimum of ten complete crypts/patient. Each crypt was subdivided longitudinally into five equal compartments. With CAIA, the images were stored digitally, and once the color references were set, the areas occupied by labeled and counterstained nuclei were quantified automatically. The labeling index (LI) was calculated from the PCNA-labeled nuclei area/total nuclei area in CAIA and from the number of labeled cells/total number of cells in visual scoring. The LI of whole crypts averaged 1.04 +/- 0.18 by CAIA and 3.91 +/- 0.46 by the visual method, and the Spearman correlation (rs) between the two methods was 0.89. The different modes of evaluation and color reference selection are likely to have contributed to the differences in the LI ranges observed in the two methods. The high correlation between PCNA quantification by CAIA and visual scoring by experienced technicians indicates that CAIA can reliably rank individual subjects. Thus, measurement of PCNA labeling by CAIA is a practical alternative for evaluating colorectal epithelial cell proliferation.