USES AND LIMITATIONS OF ANCILLARY TECHNIQUES
APPLIED TO CYTOPATHOLOGY

Jeffrey S. Ross, M.D.

TECHNICAL FACTORS INFLUENCING THE ACCURACY OF CYTOMETRIC DNA CONTENT ANALYSIS The factors known to significantly influence the sensitivity and specificity of both flow and image cytometry for the determination of tumor DNA ploidy are summarized in Table 9 and include: specimen volume; storage conditions prior to testing; type of fixative used; cytometric stain employed; proper use of diploid standards and controls; the impact of internal versus external control cell populations; operator expertise; histogram interpretation; the definition of aneuploidy for the method and cell sample utilized; the ability of the system to resolve near-diploid aneuploid tumor cell populations; the ability of the system to separate tetraploid aneuploid tumor populations from the G2M peak of the diploid cell population.; the impact of disaggregation techniques for solid tumors when flow cytometry is utilized (generally not a significant factor for cytopathology fluids and aspirates); the selective loss of aneuploid cells during the obtaining and processing of the specimen for DNA analysis; and the heterogeneity of ploidy patterns within the same tumor. Table 7: Technical Issues of Importance in Quantitative Determination of Total DNA Content in Cytologic Specimens Specimen volume Storage conditions prior to testing Type of fixatives used Cytometric stain employed Diploid standards, controls and the impact of internal versus external control cell populations Operator expertise Histogram interpretation and the definition of aneuploidy Resolution of near diploid aneuploid tumor cell populations Impact of disaggregation when necessary for flow cytometry Selective loss of aneuploid cells during processing Heterogeneity of ploidy patterns within the same tumor