title: Quantitative analysis of disseminated tumor cells in the bone marrow by automated fluorescence image analysis.
abstract: Accurate quantification of disseminated tumor cells in hematological samples is of fundamental importance in clinical oncology. However, even highly standardized protocols allow only a rough estimation of the total analyzed cell number, as sample processing may have adverse effects on the number of cells available for analysis. The fluorescence-based microscopic scanning system (MetaCyte) detects, counts, captures, and relocates immunolabeled tumor cells in hematopoietic samples. We report on a cell-counting approach that has been implemented into the scanning system to precisely quantify the number of cells per slide. The cell-counting function, which was designed to determine the number of all nucleated (DAPI-stained) cells on the slide, allows an accurate counting of the tumor cells and the total number of cells analyzed in the given microscopic sample. The reliability of the cell-counting approach was demonstrated by the analysis of DAPI-stained images with 18-1,363 nucleated cells. A good correlation (r(2) = 0.965) between the manually and automatically gained results was observed. The counting accuracy could even be optimized after implementing a correction factor. To prove or disprove an interslide variation, routine bone marrow cytospin preparations from neuroblastoma patients were immunostained for GD2/FITC and counterstained with DAPI. Automatic cell counting of cytospin preparations from the same patients showed significant differences in the total cell number (up to 67% cell loss during preparation, with a maximum interslide difference of 4.7 x 10(5) mononuclear cells). We conclude that determination of the tumor cell content in hematopoietic samples is only reliable when it is performed together with accurate cell counting.
authors.affiliation: CCRI, St. Anna Kinderspital, Vienna, Austria.
authors.forename: G
authors.initials: G
authors.lastname: Méhes
authors.forename: T
authors.initials: T
authors.lastname: Lörch
authors.forename: P F
authors.initials: PF
authors.lastname: Ambros
mesh_terms: D001331:Automation; D001854:Bone Marrow Cells / Q000737:chemistry; D019046:Bone Marrow Neoplasms / Q000473:pathology* / Q000556:secondary*; D002452:Cell Count; D005455:Fluorescent Antibody Technique; D005732:Gangliosides / Q000032:analysis / Q000276:immunology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence / Q000379:methods*; D009447:Neuroblastoma / Q000473:pathology; D015203:Reproducibility of Results
chemical_list: D005732:Gangliosides; C019403:ganglioside, GD2