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Distinction between HLA class I-positive and -negative cervical tumor subpopulations by multiparameter DNA flow
W E Corver1, L A Koopman, A Mulder
1Department of Pathology, Leiden University Medical Center, The Netherlands. W.E.Corver@Pathology.MedFac.LeidenUniv.nl
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.