title: Variable-Size Bead Layer as Standard Reference for Endothelial Microscopes.
abstract: OBJECTIVE
  For morphometric analysis of the cell mosaic of corneal endothelium, checking accuracy and precision of instrumentation is a key step. In this study, a standard reference sample is proposed, developed to reproduce the cornea with its shape and the endothelium with its intrinsic variability in the cell size.
  
  
  METHODS
  A polystyrene bead layer (representing the endothelium) was deposited on a lens (representing the cornea). Bead diameters were 20, 25, and 30 μm (fractions in number 55%, 30%, and 15%, respectively). Bead density and hexagonality were simulated to obtain the expected true values and measured using a slit-lamp endothelial microscope applied to 1) a Takagi 700GL slit lamp at 40× magnification (recommended standard setup) and 2) a Takagi 2ZL slit lamp at 25× magnification.
  
  
  RESULTS
  The simulation provided the expected bead density 2001 mm and hexagonality 47%. At 40×, density and hexagonality were measured to be 2009 mm (SD 93 mm) and 45% (SD 3%). At 25× on a different slit lamp, the comparison between measured and expected densities provided the factor 1.526 to resize the image and to use the current algorithms of the slit-lamp endothelial microscope for cell recognition.
  
  
  CONCLUSIONS
  A variable-size polystyrene bead layer on a lens is proposed as a standard sample mimicking the real shape of the cornea and the variability of cell size and cell arrangement of corneal endothelium. The sample is suggested to evaluate accuracy and precision of cell density and hexagonality obtained by different endothelial microscopes, including a slit-lamp endothelial microscope applied to different slit lamps, also at different magnifications.
authors.affiliation: *Materials Science Department, University of Milano-Bicocca, Milan, Italy; †COMiB, University of Milano-Bicocca, Milan, Italy; and ‡National Interuniversity Consortium of Materials Science and Technology INSTM, Via Giusti 9, Florence, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Tufo
authors.forename: Erica
authors.initials: E
authors.lastname: Prazzoli
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Ferraro
authors.forename: Federica
authors.initials: F
authors.lastname: Cozza
authors.forename: Alessandro
authors.initials: A
authors.lastname: Borghesi
authors.forename: Silvia
authors.initials: S
authors.lastname: Tavazzi
mesh_terms: D000328:Adult; D002452:Cell Count; D004728:Endothelium, Corneal / Q000166:cytology*; D005260:Female; D006801:Humans; D008297:Male; D008863:Microspheres; D011137:Polystyrenes; D012015:Reference Standards; D015203:Reproducibility of Results; D000072776:Slit Lamp Microscopy / Q000592:standards*; D055815:Young Adult
chemical_list: D011137:Polystyrenes