title: "Virtual flow cytometry" of immunostained lymphocytes on microscopic tissue slides: iHCFlow tissue cytometry.
abstract: BACKGROUND
  A method and approach is developed for fully automated measurements of immunostained lymphocytes in tissue sections by means of digital color microscopy and patent pending advanced cell analysis. The validation data for population statistic measurements of immunostained lymphocytes in tissue sections using tissue cytometry (TC) is presented. The report is the first to describe the conversion of immunohistochemistry (IHC) data to a flow cytometry-like two parameter dot-plot display, hence the technique is also a virtual flow cytometry. We believe this approach is a paradigm shift, as well as novel, and called the system iHCFlow TC. Seven issues related to technical obstacles to virtual flow cytometry (FC) are identified.
  
  
  METHODS
  Segmentation of a 512 x 474 RGB image and tabular display of statistical results table took 12-15 s using proprietary developed algorithms. We used a panel of seven antibodies for validation on 14 cases of mantle cell lymphoma giving percentage positive, total lymphocytes, and staining density. A total of 2,027 image frames with 810,800 cell objects (COBs) were evaluated. Antibodies to CD3, CD4, CD8, Bcl-1, Ki-67, CD20, CD5 were subjected to virtual FC on tissue. The results of TC were compared with manual counts of expert observers and with the results of flow cytometric immunophenotyping of the same specimen.
  
  
  RESULTS
  The correlation coefficient and 95% confidence interval by linear regression analysis yielded a high concordance between manual human results (M), FC results, and TC results per antibody, (r = 0.9365 M vs. TC, r= 0.9537 FC vs. TC). The technical issues were resolved and the solutions and results were evaluated and presented.
  
  
  CONCLUSIONS
  These results suggest the new technology of TC by iHCFlow could be a clinically valid surrogate for both M and FC analysis when only tissue IHC is available for diagnosis and prognosis. The application for cancer diagnosis, monitoring, and prognosis is for objective, rapid, automated counting of immunostained cells in tissues with percentage results. We report a new paradigm in TC that converts IHC staining of lymphocytes to automated results and a flow cytometry-like report. The dot plot histogram display is familiar, intuitive, informative, and provides the pathologists with an automated tool to rapidly characterize the staining and size distribution of the immunoreactive as well as the negative cell population in the tissue. This systems tool is a major improvement over existing ones and satisfies fully the criteria to perform Cytomics (Ecker and Tarnok, Cytometry A 2005;65:1; Ecker and Steiner, Cytometry A 2004;59:182-190; Ecker et al., Cytometry A 2004;59:172-181).
authors.affiliation: H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida, USA. cualinhd@moffitt.usf.edu
authors.forename: Hernani D
authors.initials: HD
authors.lastname: Cualing
authors.forename: Eric
authors.initials: E
authors.lastname: Zhong
authors.forename: Lynn
authors.initials: L
authors.lastname: Moscinski
mesh_terms: D001402:B-Lymphocytes / Q000166:cytology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology; D016001:Confidence Intervals; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D008214:Lymphocytes / Q000166:cytology*; D020522:Lymphoma, Mantle-Cell / Q000473:pathology; D015203:Reproducibility of Results; D012984:Software; D013194:Staining and Labeling; D018709:Statistics, Nonparametric