REPRODUCIBILITY AND VARIABILITY OF THE RECTAL MUCOSAL PROLIFERATION INDEX USING PROLIFERATING CELL NUCLEAR ANTIGEN IMMUNOHISTOCHEMISTRY

Rectal mucosal proliferation has been shown to be increased in patient s with neoplastic lesions of the large bowel and may serve as a marker of risk for colorectal malignancy. We conducted analyses to determine reliability and components of variability that might suggest optimal analysis strategies for studies of proliferation. Endoscopic pinch bio psies were obtained from 17 adult patients, labeled using proliferatin g cell nuclear antigen, scored using strict rules, and then restored. Labeling index, defined as the proportion of labeled cells in a crypt, was calculated for each crypt, biopsy, subject, and group. There was excellent reproducibility. The technician was able to select previousl y scored crypts 95% of the time. The overall labeling index was identi cal on repeat. There was considerable variability in labeling index am ong crypts from a single biopsy and between biopsies of a single subje ct. Variance component estimates suggested that 20% of the variability of labeling index was due to subject, 30% due to the biopsy within a subject, and 50% due to crypts within a biopsy. There were substantial gains in statistical power by scoring two biopsies rather than one. T here was less gain from further increases in biopsy number. There was little statistical advantage for counting more than 8 crypts/biopsy. D emonstrating a decrease of 25% in the mean labeling index with 90% pow er could require more than 100 subjects/group. We conclude that prolif erating cell nuclear antigen is an extremely reproducible method to de termine proliferation index. There is considerable variability among s ubjects, biopsies, and crypts. Analyses should use models that include random effects for subjects and biopsies to account for these sources of variability. Two to four biopsies with approximately eight scorabl e crypts/biopsy is the optimal number from a statistical standpoint. L arge sample sizes are needed unless there are considerable differences between groups.