Distinction between HLA class I‐positive and ‐negative cervical tumor subpopulations by multiparameter DNA flow cytometry

Background The study of the molecular‐genetic basis of heterogeneity of HLA class I expression in solid tumors is hampered by the lack of reliable rapid cell‐by‐cell isolation techniques. Hence, we studied the applicability of a flow cytometric approach (Corver et al.: Cytometry 2000;39;96–107). Methods Cells were isolated from five fresh cervical tumors and simultaneously stained for CD45 or vimentin (fluorescein isothiocyanate fluorescence), Keratin (R‐phycoerythrin fluorescence), HLA class I (APC fluorescence), and DNA (propidium iodide fluorescence). A dual‐laser flow cytometer was used for fluorescence analysis. Tissue sections from the corresponding tumors were stained for HLA class I antigens, keratin, vimentin, or CD45. Results Flow cytometry enabled the simultaneous measurement of normal stromal cells (vimentin positive), inflammatory cells (CD45 positive), epithelial cells (keratin positive), and DNA content readily. Normal stromal/inflammatory cells served as intrinsic HLA class I‐positive as well as DNA‐diploid references. Good DNA histogram quality was obtained (average coefficient of variation < 4%). Intratumor keratin positive subpopulations differing in HLA class I expression as well as DNA content could be clearly identified. Losses of allele‐specific HLA class I expression found by immunohistochemistry were also detected by flow cytometry. Conclusions We conclude that multiparameter DNA flow cytometry is a powerful tool to study loss of HLA class I expression in human cervical tumors. The method enables flow‐sorting of discrete tumor and normal cell subpopulations for further molecular genetic analysis. Cytometry 41:73–80, 2000 © 2000 Wiley‐Liss, Inc.