title: Flow cytometric immunphenotyping of epithelial cancer cells in effusions--technical considerations and pitfalls.
abstract: BACKGROUND
  Data regarding the role of flow cytometry (FCM) in the characterization of malignant effusions are limited to date. In the present study, we optimized the conditions for FCM immunphenotyping of effusions using a four-color analysis and investigated aspects related to the advantages and limitations of this method in this setting.
  
  
  METHODS
  FCM analysis optimization for the study of epithelial cells was undertaken using five carcinoma cell lines, and subsequently applied to malignant pleural and peritoneal effusions using antibodies against epithelial and mesothelial markers (Ber-EP4 and EMA), CD138, and integrin subunits. FCM of frozen versus fresh specimens and the performance of FCM compared to immunhistochemistry were evaluated.
  
  
  RESULTS
  FCM optimization was achieved and applied to clinical specimens, with resulting detection of epithelial markers and adhesion molecules on cancer cells. Frozen clinical specimens and cell lines showed reduced CD138 expression compared to fresh specimens, with conservation of the remaining epitopes. FCM generally showed comparable performance to immunhistochemistry.
  
  
  CONCLUSIONS
  FCM is an effective method for characterization of cancer cells in clinical effusion specimens in both the diagnostic and research setting, and is comparable to immunhistochemistry in terms of sensitivity and specificity, with the additional advantage of providing quantitative data. The majority of epitopes are conserved in frozen cells, but a minority may be lost, suggesting that the thorough testing of each antibody in both conditions is mandatory.
authors.affiliation: Pathology Clinic, Radiumhospitalet-Rikshospitalet Medical Center, University of Oslo, Montebello, N-0310 Oslo, Norway.
authors.forename: Hiep P
authors.initials: HP
authors.lastname: Dong
authors.forename: Arild
authors.initials: A
authors.lastname: Holth
authors.forename: Aasmund
authors.initials: A
authors.lastname: Berner
authors.forename: Ben
authors.initials: B
authors.lastname: Davidson
authors.forename: Björn
authors.initials: B
authors.lastname: Risberg
mesh_terms: D000906:Antibodies; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000032:analysis / Q000276:immunology; D016477:Artifacts; D001202:Ascitic Fluid / Q000276:immunology* / Q000473:pathology; D015415:Biomarkers / Q000032:analysis; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000096:biosynthesis; D002277:Carcinoma / Q000175:diagnosis / Q000276:immunology* / Q000473:pathology; D045744:Cell Line, Tumor; D015925:Cryopreservation / Q000379:methods / Q000592:standards; D004847:Epithelial Cells / Q000276:immunology* / Q000473:pathology; D004848:Epithelium / Q000276:immunology / Q000473:pathology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D008867:Microtomy / Q000379:methods / Q000592:standards; D016066:Pleural Effusion, Malignant / Q000276:immunology* / Q000473:pathology; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D016610:Tissue Embedding / Q000379:methods / Q000592:standards
chemical_list: D000906:Antibodies; D000954:Antigens, Surface; D015415:Biomarkers; D014408:Biomarkers, Tumor