title: Estimation of immune cell densities in immune cell conglomerates: an approach for high-throughput quantification.
abstract: BACKGROUND
  Determining the correct number of positive immune cells in immunohistological sections of colorectal cancer and other tumor entities is emerging as an important clinical predictor and therapy selector for an individual patient. This task is usually obstructed by cell conglomerates of various sizes. We here show that at least in colorectal cancer the inclusion of immune cell conglomerates is indispensable for estimating reliable patient cell counts. Integrating virtual microscopy and image processing principally allows the high-throughput evaluation of complete tissue slides.
  
  
  RESULTS
  For such large-scale systems we demonstrate a robust quantitative image processing algorithm for the reproducible quantification of cell conglomerates on CD3 positive T cells in colorectal cancer. While isolated cells (28 to 80 microm(2)) are counted directly, the number of cells contained in a conglomerate is estimated by dividing the area of the conglomerate in thin tissues sections (< or =6 microm) by the median area covered by an isolated T cell which we determined as 58 microm(2). We applied our algorithm to large numbers of CD3 positive T cell conglomerates and compared the results to cell counts obtained manually by two independent observers. While especially for high cell counts, the manual counting showed a deviation of up to 400 cells/mm(2) (41% variation), algorithm-determined T cell numbers generally lay in between the manually observed cell numbers but with perfect reproducibility.
  
  
  CONCLUSIONS
  In summary, we recommend our approach as an objective and robust strategy for quantifying immune cell densities in immunohistological sections which can be directly implemented into automated full slide image processing systems.
authors.affiliation: Medical Oncology, National Center for Tumor Diseases, University of Heidelberg, Heidelberg, Germany.
authors.forename: Niels
authors.initials: N
authors.lastname: Halama
authors.forename: Inka
authors.initials: I
authors.lastname: Zoernig
authors.forename: Anna
authors.initials: A
authors.lastname: Spille
authors.forename: Kathi
authors.initials: K
authors.lastname: Westphal
authors.forename: Peter
authors.initials: P
authors.lastname: Schirmacher
authors.forename: Dirk
authors.initials: D
authors.lastname: Jaeger
authors.forename: Niels
authors.initials: N
authors.lastname: Grabe
mesh_terms: D000465:Algorithms; D017252:CD3 Complex / Q000096:biosynthesis; D002452:Cell Count; D003106:Colon / Q000378:metabolism / Q000473:pathology; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000473:pathology*; D001330:Electronic Data Processing; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods*; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008853:Microscopy / Q000379:methods; D015233:Models, Statistical; D011379:Prognosis; D015203:Reproducibility of Results; D012984:Software; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D017252:CD3 Complex