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.
Corver
W E
WE
Department of Pathology, Leiden University Medical Center, The Netherlands. W.E.Corver@Pathology.MedFac.LeidenUniv.nl
Koopman
L A
LA
Mulder
A
A
Cornelisse
C J
CJ
Fleuren
G J
GJ
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm / Q000032:analysis; D015536:Down-Regulation / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D018740:Genetic Heterogeneity; D015234:HLA-A Antigens / Q000032:analysis; D059870:HLA-A11 Antigen; D059871:HLA-A24 Antigen; D015235:HLA-B Antigens / Q000032:analysis; D015395:Histocompatibility Antigens Class I / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007633:Keratins / Q000032:analysis; D017493:Leukocyte Common Antigens / Q000032:analysis; D011003:Ploidies; D002583:Uterine Cervical Neoplasms / Q000737:chemistry / Q000276:immunology / Q000473:pathology
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D015234:HLA-A Antigens; D059870:HLA-A11 Antigen; D059871:HLA-A24 Antigen; D015235:HLA-B Antigens; C042388:HLA-Bw4 antigen; D015395:Histocompatibility Antigens Class I; D007633:Keratins; D017493:Leukocyte Common Antigens