Image analysis cytometry (IAC) combines some aspects of traditional visual morphology of cell nuclei with nuclear DNA analysis as done in FCM but using nonfluorescent visible nuclear stains. The instrument’s operator finds cancer cells on a tissue slide or smear and instructs the equipment’s computer to search for a certain number of tumor nuclei using instructions on what the nuclei should look like in terms of size, shape, chromatin density or distribution, and nucleoli. This helps differentiate tumor nuclei from nontumor nuclei and permits the instrument to search later on its own. The slide can then be stained with a nuclear material stain such as Feulgen (similar to flow cytometry nonspecific nuclear staining but with a different type of stain). The instrument then finds nuclei that fit the parameters given to it and analyzes the intensity of the nuclear staining reaction (again, similar to FCM). The instrument uses the sum of the nuclear density readings to calculate the average amount of DNA and displays this information as a bar-graph histogram (as in FCM) showing nuclear density composition compared to normal diploid control cell nuclei. IAC is most often performed on smears made from fresh cellular material but can be done on smears prepared from fixed tissue or even on regular formalin-fixed paraffin-embedded microscopic slides. However, tissue slide sections (rather than smears) present more difficulty due to overlapping nuclei and tissue background.

The major advantages of IAC over FCM is that the cell selection process is more likely to analyze tumor cells only. The major advantage of FCM is less variation in the DNA peak composition (height and width) due to better counting statistics generated from thousands of cell nuclei rather than the 100-200 cells (range, 50-250 cells) usually counted in IAC. Therefore, differences in diploid-aneuploid results between FC and IAC have ranges from 9%–24%. This most commonly occurs when the number of tumor cells is very small, such as often occurs in effusions or (to a lesser extent) in fine-needle aspirate smears. Under these circumstances, IAC tends to detect malignant cells somewhat (but not always) more often than FCM. On the other hand, the relatively small numbers of cells analyzed in IAC make S-phase peak analysis very difficult or impossible, necessitating other ways to obtain cell proliferation activity information (such as monoclonal antibody stain for proliferating cell nuclear antigen or for Ki-67 antigen).