title: Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.
abstract: BACKGROUND
  Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements.
  
  
  RESULTS
  We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided.
  
  
  CONCLUSIONS
  Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications.
authors.affiliation: Laboratory of Cancer Cell Biology and Genetics, Department of Human Oncology, Allegheny Singer Research Institute, Allegheny General Hospital, Pittsburgh, Pennsylvania 15212, USA. shackney@wpahs.org
authors.forename: Stanley
authors.initials: S
authors.lastname: Shackney
authors.forename: David R
authors.initials: DR
authors.lastname: Emlet
authors.forename: Agnese
authors.initials: A
authors.lastname: Pollice
authors.forename: Charles
authors.initials: C
authors.lastname: Smith
authors.forename: Kathryn
authors.initials: K
authors.lastname: Brown
authors.forename: Deborah
authors.initials: D
authors.lastname: Kociban
mesh_terms: D002449:Cell Aggregation; D002452:Cell Count; D002463:Cell Membrane Permeability; D048429:Cell Size; D004247:DNA / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods*; D017408:Guidelines as Topic; D006801:Humans; D047569:Laser Scanning Cytometry / Q000379:methods*; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology*; D012015:Reference Standards; D015203:Reproducibility of Results; D012107:Research Design; D016707:Tissue Fixation
chemical_list: D004247:DNA