Interrelation of formalin fixation, chromatin compactness and DNA values as measured by flow and image cytometry

The severity and consistency of the effect of formalin fixation on the quantitation of DNA by flow cytometry (FCM) and image cytometry (ICM) were studied. As compared to ethanol, formalin fixation substantially decreased the propidium iodide fluorescence from mouse hepatocyte nuclei analyzed by FCM; it was also associated with an altered 4n-to-2n signal ratio and with false aneuploid peaks by FCM, but not by ICM (microspectrophotometry). ICM, on the other hand, suffered from a dependence of the DNA signal on nuclear size, which was not seen with FCM. The DNA signal variation was related to variations in the chromatin state, as shown by differences between monocytes and lymphocytes, and between RAJI cells fixed under various ionic strengths. The dependence of the DNA signal on the chromatin state indicates a need for caution in interpreting aneuploidy in formalin-fixed cells. For FCM, pseudoaneuploidy appears avoidable by using a Feulgen fluorescence staining technique. New imaging modes may be necessary to solve the problem of cell size dependence for ICM DNA determination.