title: Optimal image sample size for corneal nerve morphometry.
abstract: OBJECTIVE
  Arbitrary numbers of corneal confocal microscopy images have been used for analysis of corneal subbasal nerve parameters under the implicit assumption that these are a representative sample of the central corneal nerve plexus. The purpose of this study is to present a technique for quantifying the number of random central corneal images required to achieve an acceptable level of accuracy in the measurement of corneal nerve fiber length and branch density.
  
  
  METHODS
  Every possible combination of 2 to 16 images (where 16 was deemed the true mean) of the central corneal subbasal nerve plexus, not overlapping by more than 20%, were assessed for nerve fiber length and branch density in 20 subjects with type 2 diabetes and varying degrees of functional nerve deficit. Mean ratios were calculated to allow comparisons between and within subjects.
  
  
  RESULTS
  In assessing nerve branch density, eight randomly chosen images not overlapping by more than 20% produced an average that was within 30% of the true mean 95% of the time. A similar sampling strategy of five images was 13% within the true mean 80% of the time for corneal nerve fiber length.
  
  
  CONCLUSIONS
  The "sample combination analysis" presented here can be used to determine the sample size required for a desired level of accuracy of quantification of corneal subbasal nerve parameters. This technique may have applications in other biological sampling studies.
authors.affiliation: Institute of Health and Biomedical Innovation and School of Optometry, Queensland University of Technology, Kelvin Grove, Queensland, Australia. dimitrios.vagenas@qut.edu.au
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Vagenas
authors.forename: Nicola
authors.initials: N
authors.lastname: Pritchard
authors.forename: Katie
authors.initials: K
authors.lastname: Edwards
authors.forename: Ayda M
authors.initials: AM
authors.lastname: Shahidi
authors.forename: Geoff P
authors.initials: GP
authors.lastname: Sampson
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Russell
authors.forename: Rayaz A
authors.initials: RA
authors.lastname: Malik
authors.forename: Nathan
authors.initials: N
authors.lastname: Efron
mesh_terms: D003315:Cornea / Q000294:innervation*; D003924:Diabetes Mellitus, Type 2 / Q000150:complications / Q000175:diagnosis; D003930:Diabetic Retinopathy / Q000209:etiology / Q000473:pathology*; D006801:Humans; D007089:Image Enhancement / Q000592:standards*; D018613:Microscopy, Confocal / Q000379:methods / Q000592:standards*; D009411:Nerve Endings / Q000473:pathology; D009412:Nerve Fibers / Q000473:pathology*; D009882:Ophthalmic Nerve / Q000473:pathology*; D015203:Reproducibility of Results; D012720:Severity of Illness Index