USES AND LIMITATIONS OF ANCILLARY TECHNIQUES
APPLIED TO CYTOPATHOLOGY

Jeffrey S. Ross, M.D.

FLOW VERSUS IMAGE CYTOMETRY IN DNA PLOIDY ANALYSIS OF CYTOPATHOLOGIC SPECIMENS Advantages and Disadvantages of Flow Cytometry The advantages of flow cytometry for the determination of DNA content in cytologic specimens (Table 8) include: the ability to obtain high speed, rapid results; the preparation; and the ability to perform two color immunocytochemistry to fact that cytologic specimens are generally disaggregated when collected precluding the errors introduced by disaggregation techniques; the relatively high cell count analyzed by flow cytometry allows resolution of near diploid aneuploid peaks; and the use of two color techniques that allow inclusion of epithelial cells via cytoplasmic markers (cytokeratin, CEA, EMA) or exclusion of lymphoid cells by lymphoid markers. The disadvantages of flow cytometry include the requirement for large cell volume for proper histogram development; the lack of simultaneous morphologic evaluation of the specimen during analysis; and the potential masking of aneuploid cell populations by large numbers of inflammatory and non-neoplastic cells when the tumor cell population is small and not controlled by two-color techniques or cytologic screening of the suspension prior to flow cytometric evaluation. Table 8: Advantages and Disadvantages of Flow Cytometry for DNA Content Analysis in Cytologic Specimens Advantages High speed rapid results Specimens are generally pre-disaggregated High cell count allows resolution of near diploid aneuploid peaks Two color techniques allows inclusions of desired and exclusion of undesired cells in histogram Disadvantages Requires large cell volume for histogram Lack of simultaneous morphologic evaluation during analysis Potential masking of aneuploid cells by large numbers of inflammatory and non-neoplastic cells Advantages and Disadvantages of Image Cytometry The advantages for image analysis determination of DNA content in cytologic specimens (Table 9) include: the fact that it can be performed on small samples such as hypocellular fine needle aspirates; can be performed on smears and touch imprints as well as liquid cell suspensions after cytospin or "thin-prep" processing; enhanced sensitivity for aneuploidy via the simultaneous morphologic assessment of the sample during histogram preparation to selectively identify tumor cells during histogram selectively include or exclude cells from the histogram based on antigen expression. The disadvantages of image analysis DNA content measurement include: the fact that the technique is more time consuming; the histogram generally features a lower total cell count may fail to resolve near diploid aneuploid populations; and that the system operation requires a skilled morphologist. Table 9: Advantages and Disadvantages of Image Cytometry for DNA Content Analysis in Cytologic Specimens Advantages Can be performed on small samples as hypocellular aspirates Can be performed on smears and touch imprints as well as cell suspensions, cytospins and thin-prep slides Enhanced sensitivity for aneuploidy via simultaneous morphologic assessment of sample during histogram preparation Ability to perform two color techniques allows inclusions of desired and exclusion of undesired cells in histogram Disadvantages Technique is more time consuming Histogram features a lower total cell count and may fail to resolve near diploid aneuploid populations System requires a skilled morphologist Comparative studies have highlighted an excellent overall comparison of the two methodologies with approximately 95% of samples showing similar diploid or aneuploid histograms by image analysis and flow cytometry.93  Although new methods of pre-test cytologic screening of the sample to confirm intact tumor cells and two-color procedures have improved the sensitivity of flow cytometry for the identification of aneuploidy,94  the selective nature of image analysis has led to the conclusion that it is the more sensitive technique.95  In a recent study of breast carcinomas a significant number of cases classified by flow cytometry were found to be non-diploid on image analysis.96