title: A simple correction for cell autofluorescence for multiparameter cell-based analysis of human solid tumors.
abstract: BACKGROUND
  Corrections that have been proposed to minimize the unwanted contribution of cell autofluorescence to the total fluorescence signal often require either specialized instrumentation or the sacrifice of a data channel so as to perform a measurement that can be used to correct for autofluorescence in individual cells. Here we propose a simple cell by cell correction for autofluorescence that is suitable for multiparameter laser scanning cytometry (LSC) studies in human solid tumors that relies on the ratio of mean autofluorescence to mean total cell fluorescence (mean Flauto/mean Fltotal). This approach assumes a correlation between the autofluorescence component and the total signal in individual cells. This correction does not require specialized instrumentation, and does not sacrifice a data channel in multiparameter studies. A potential disadvantage is that errors may be introduced by the assumption of a correlation between the two components of the total fluorescence signal in individual cells in samples in which no such correlation exists.
  
  
  METHODS
  Distributions of cell autofluorescence and total Her-2/neu cell fluorescence were obtained separately by LSC in three human breast cancer cell lines and in three samples of primary human lung cancer. In the breast cancer cell lines, autofluorescence measurements and Her-2/neu measurements were also obtained on the same cells.
  
  
  RESULTS
  We show that there is a partial correlation between autofluorescence and total Her-2/neu/FITC fluorescence in individual cells in the three breast cancer cell lines. We also show that the results of a ratio-based autofluorescence correction agree with those based on a true cell by cell correction. Computer simulation studies suggest that in samples with no correlation between the autofluorescence component and the true probe/dye fluorescence component, the ratio correction produces robust estimates of the mean true fluorescence signal, with relatively small but systematic underestimates of the coefficient of variation of such measurements under conditions commonly encountered in the measurement of human solid tumors.
  
  
  CONCLUSIONS
  A simple cell by cell correction for autofluorescence based on the ratio of mean Flauto to mean Fltotal can be applied in cell samples in which there is a correlation between cell autofluorescence and true probe/dye fluorescence in individual cells. In cell samples that lack this correlation, or in which it is not known whether such a correlation exists, this correction can be used with the reservation that there is a systematic but relatively small underestimation of the degree of variability of the measurements.
authors.affiliation: Laboratory of Cancer Cell Biology and Genetics, Department of Human Oncology, Allegheny Singer Research Institute, Allegheny General Hospital, Pittsburgh, Pennsylvania 15212, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Smith
authors.forename: Agnese
authors.initials: A
authors.lastname: Pollice
authors.forename: David
authors.initials: D
authors.lastname: Emlet
authors.forename: Stanley E
authors.initials: SE
authors.lastname: Shackney
mesh_terms: D001943:Breast Neoplasms / Q000737:chemistry / Q000473:pathology*; D045744:Cell Line, Tumor; D003198:Computer Simulation; D005434:Flow Cytometry / Q000379:methods* / Q000706:statistics & numerical data; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis; D005455:Fluorescent Antibody Technique / Q000379:methods / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D006801:Humans; D047569:Laser Scanning Cytometry / Q000379:methods* / Q000706:statistics & numerical data; D008175:Lung Neoplasms / Q000737:chemistry / Q000473:pathology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis
chemical_list: D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D018719:Erb-b2 Receptor Tyrosine Kinases