title: Software compensation improves the analysis of heterogeneous tumor samples stained for multiparameter DNA flow cytometry.
abstract: BACKGROUND
  High concentrations of propidium iodide (PI), in combination with fluorescein isothiocyanate (FITC) and R-phycoerythrin (RPE) used for multiparameter DNA flow cytometry (FCM), cause spectral cross-talk into the green fluorescence channel (FL1). We have evaluated the use of post-acquisition software compensation (N-Color Compensation) in order to correct this spectral cross-talk caused by PI.
  
  
  METHODS
  Cell mixtures were prepared consisting of keratin 8/18 FITC labeled, keratin 8/18 RPE labeled, and unlabeled MCF-7 breast carcinoma cells. DNA was stained with PI (100 microM). Post-acquisition software compensation was applied to correct the spectral cross-talk of PI fluorescence. Secondly, the distribution of the Ki-67 (FITC) protein during the cell cycle (PI) of SiHa cervical carcinoma cells (no software compensation) was compared to the Ki-67 expression pattern of SiHa cells, simultaneously stained for keratin 8 (RPE), after applying software compensation. Finally, software compensation was used to compare the relative levels of PCNA and p53 expression in two clinical ovarian cancer ascites specimens, stained for PCNA or p53 (FITC), keratin 8/18 (RPE), and DNA (PI), with a known p53 status (positive and negative, respectively).
  
  
  RESULTS
  The Ki-67 cell cycle-dependent pattern of a triply stained sample (Ki-67 (FITC), keratin 8 (RPE), and DNA (PI)) is restored after software compensation and the results are comparable to the Ki-67 distribution of a sample stained solely for Ki-67 and DNA. P53 expression could only be resolved after using software compensation in the p53 positive ovarian ascites (OA) sample.
  
  
  CONCLUSIONS
  We conclude that software compensation is a robust and reliable post-acquisition method for the correction of RPE/PI spectral cross-talk, permitting better identification of weakly expressed proteins in heterogeneous clinical tumor samples stained for multiple cellular antigens and DNA using PI.
authors.affiliation: Department of Pathology, Leiden University Medical Center, P.O. Box 9600, Building 1, L1-Q, 2300 RC, Leiden, Netherlands. W.E.Corver@lumc.nl
authors.forename: Willem E
authors.initials: WE
authors.lastname: Corver
authors.forename: Gert Jan
authors.initials: GJ
authors.lastname: Fleuren
authors.forename: Cees J
authors.initials: CJ
authors.lastname: Cornelisse
mesh_terms: D004273:DNA, Neoplasm / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D010799:Phycoerythrin; D011419:Propidium; D012680:Sensitivity and Specificity; D012984:Software; D013194:Staining and Labeling / Q000379:methods; D014407:Tumor Cells, Cultured
chemical_list: D004273:DNA, Neoplasm; D010799:Phycoerythrin; D011419:Propidium; D016650:Fluorescein-5-isothiocyanate