Simultaneous cell cycle and phenotypic analysis of primary uveal melanoma by flow cytometry

DNA ploidy and cell cycle measurements of uveal melanoma tissue are regarded as having limited prognostic significance. In contrast, dual-parameter (DNA monoclonal antibody) flow cytometry offers a convenient and rapid way to screen tumour samples for a variety of phenotypic markers, whilst simultaneously measuring DNA ploidy and cell cycle, and therefore has the increased potential to identify clinically relevant indicators of disease progression. The aim of the present study was to identify a simple yet robust method for isolating, preserving and staining cells that could be analysed by flow cytometry.

Using a simple preparation procedure, a panel of membrane-associated antibodies (ICAM-1, W632, HLA-DR) and nuclear or cytoplasmic oncoprotein antibodies (c-erbB-2, c-myc, bcl-2, p53), together with positive (PHM-5) and negative (FITC F(ab')2) controls, were assayed. It was considered important to test the protocol with markers expressed on the cell surface, and in the cytoplasm and nucleus, so as not to be restrictive and thereby exclude an antigen of potential clinical interest. In addition, such panels would also enable the generation of a 'phenotypic profile' for each specimen that may reveal clinically significant trends.

Our results indicate that tissue dissociation followed by brief fixation in 1% paraformaldehyde and permeabilisation in 70% methanol produces a stable single cell suspension, which can subsequently be stained with a wide range of antibodies for the accurate identification of cells in a potentially heterogeneous tumour population.

This technology can rapidly identify sub-populations of cells expressing differing levels of proteins, which may prove to be indicative of disease progression for this aggressive disease.