Quantitative analysis of disseminated tumor cells in the bone marrow by automated fluorescence image analysis

Accurate quantification of disseminated tumor cells in hematological sample s is of fundamental importance in clinical oncology. However, even highly s tandardized protocols allow only a rough estimation of the total analyzed c ell number, as sample processing may have adverse effects on the number of cells available for analysis. The fluorescence-based microscopic scanning s ystem (MetaCyte) detects, counts, captures, and relocates immunolabeled tum or cells in hematopoietic samples. We report on a cell-counting approach th at has been implemented into the scanning system to precisely quantify the number of cells per slide. The cell-counting function, which was designed t o 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 cell s analyzed in the given microscopic sample. The reliability of the cell-cou nting 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 ma nually and automatically gained results was observed. The counting accuracy could even be optimized after implementing a correction factor. To prove o r disprove an interslide variation, routine bone marrow cytospin preparatio ns from neuroblastoma patients were immunostained for GD2/FITC and counters tained 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 o f 4.7 x 10(5) mononuclear cells), We conclude that determination of the tum or cell content in hematopoietic samples is only reliable when it is perfor med together with accurate cell counting. Cytometry (Comm. Clin. Cytometry) 42:357-362, 2000. (C) 2000 Wiley-Liss, Inc.