title: Reproducibility of pixel-by-pixel analysis of Heidelberg retinal flowmetry images: the Thessaloniki Eye Study.
abstract: OBJECTIVE
  To identify the investigator effect in the analysis results of Heidelberg retinal flowmetry (HRF) images when pixel-by-pixel analysis is performed.
  
  
  METHODS
  Thirty-two of 732 HRF images were randomly selected from a population-based study. Pixel-by-pixel analysis was performed by two trained masked graders in the following way: a square window of 40 x 40 pixels or two windows of 30 x 30 pixels or four windows of 20 x 20 pixels free from blood vessels at the peripapillary retina were identified. Using a 1 x 1-pixel window, the grader performed pointwise analysis according to a specific protocol. The analysis process was performed by each observer three times (A, B, C) at 1-week intervals. The percentage of pixels with < 1 arbitrary unit of flow (zero flow), the 10th, 25th, 50th, 75th and 90th percentiles and mean flow values were calculated. The difference between the results of analyses B-A and C-A for all HRF parameters was estimated using the Wilcoxon signed rank test. Mixed-effect regression models were also used after controlling for grader effect and correlation within subjects.
  
  
  RESULTS
  There was no statistically significant difference between the results of analyses B-A and C-A or for any parameter in the mixed-effect regression models. Intraclass correlation was 0.9665 for the percentage of zero flow pixels.
  
  
  CONCLUSIONS
  Pixel-by-pixel analysis of HRF images by trained graders remains a highly reproducible method. No grader effect was found. If a precise protocol is followed, the results are independent of the exact placement of the analysis windows and the pointwise analysis of the identified and mapped retinal tissue.
authors.affiliation: II Department of Ophthalmology, Aristotle University of Thessaloniki, Thessaloniki, Greece.
authors.forename: Leonidas
authors.initials: L
authors.lastname: Mavroudis
authors.forename: Alon
authors.initials: A
authors.lastname: Harris
authors.forename: Fotis
authors.initials: F
authors.lastname: Topouzis
authors.forename: M Roy
authors.initials: MR
authors.lastname: Wilson
authors.forename: Fei
authors.initials: F
authors.lastname: Yu
authors.forename: Eleftherios
authors.initials: E
authors.lastname: Anastasopoulos
authors.forename: Archimidis
authors.initials: A
authors.lastname: Koskosas
authors.forename: Brent
authors.initials: B
authors.lastname: Siesky
authors.forename: Theofanis
authors.initials: T
authors.lastname: Pappas
authors.forename: Panayiota
authors.initials: P
authors.lastname: Founti
authors.forename: Anne L
authors.initials: AL
authors.lastname: Coleman
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D006801:Humans; D017078:Laser-Doppler Flowmetry; D016014:Linear Models; D008297:Male; D015588:Observer Variation; D012039:Regional Blood Flow; D015203:Reproducibility of Results; D012171:Retinal Vessels / Q000502:physiology*