A sequential procedure for single and multiparameter flow cytometry (FCM) that allows detailed cell proliferation and DNA ploidy studies of human solid tumors is described.

A significant improvement in cell cycle kinetic studies was achieved by the introduction of MoAbs against bromodeoxyuridine (BrdU). The BrdU-based procedure overcomes many of the technical aspects related to S-phase fraction quantitation from DNA histograms based on the use of more or less sophisticated mathematical models.

The major conceptual difference is found between the "in vitro" and "in vivo" procedure. The "in vitro" method yields only S phase size easily, while the "in vivo" technique (together with the possibility of performing a correlated DNA/BrdU analysis) is able to furnish a complete set of kinetic parameters, including DNA syntesis time (Ts) and potential doubling time (Tpot) of the cell population analysed. The analytical capability of multiparameter analysis offers the advantage of using a third immunofluorescence marker for "gating" the analysis. This is extremely important in solid tumor analysis, where many cell types are present in the final suspension. The kinetic parameters acquired in this situation are in fact of little clinical significance if they cannot be related to the sub- populations of specific interest. In the case of epithelials tumors, cytokeratins (CK) can be used as a discriminating parameter able to exclude CK-negative cells from the flow cytometric analysis. Hence, dual parameter FCM allows bidimensional plots to be obtained in which CK-positive cells can be sufficiently differentiated from non epithelial sub populations, thus increasing the clinical significance of the data acquired. A similar analytical strategy based on CK labelling and multicolor FCM can also be set up on BrdU- treated cases.