Application of the crypt-isolation technique to flow-cytometric analysis of DNA content in colorectal neoplasms

Abstract

Conventional flow-cytometric analysis of colorectal tumor DNA content includes both tumor and stromal cell DNA. To improve the method, we evaluated the DNA content of purified epithelial cells using the crypt-isolation technique. Normal and neoplastic crypts were isolated from fresh resected colorectum nonenzymatically. Isolated crypts were digested into single nuclei suspensions and examined by flow cytometry (FCM). Internal controls from normal crypts were used to define diploidy. The neoplastic nuclei in the same individual and mixtures of both normal and neoplastic nuclei in various ratios were analyzed. Tumors having both diploid and aneuploid stemlines were found more frequently than that reported using conventional FCM. Near-diploid DNA stemlines with DNA indices ranging between 0.90 and 1.10 were detectable. The cases of advanced cancer in which the DNA index was between 0.90 and 0.95 were associated significantly with Dukes' stage C. This is the first reported analysis of the DNA content of "pure" neoplastic colorectal epithelia using FCM. Multiploid and near-diploid stemlines were more accurately analyzed than that using conventional FCM.