title: A modification of the staining technique of reticular fibres for image analysis of the cardiac collagen network.
abstract: BACKGROUND
  Silver stain of reticular fibres demonstrates the fine structure of the cardiac collagen network. However, nuclei are also stained with current techniques, a drawback that makes computer image analysis difficult. To solve this problem our study was designed to modify Gomori's method. Reactive concentrations and action times represent the core of that modification. Only stromal tissue is stained. The technique was tested for repeatability and reproducibility to assess the precision of the measurement procedure in the assessment of myocardial collagen in a consecutive series of myocardial samples from patients with and without heart muscle disease. In addition, we checked the reliability of the method by comparing our results with the point-counting method (PCM).
  
  
  METHODS
  The right ventricular myocardium was fixed in 10% buffered formaldehyde and routinely processed. Paraffin sections (4 microm) were stained with several modifications of Gomori's method. Variable concentrations of potassium permanganate, silver solutions and different oxidation times were tried. A field from each sample was digitized. Additionally, the technique was tested for repeatability and reproducibility.
  
  
  RESULTS
  We obtained absence of background and nuclear staining together with a highly contrasted image of the collagen network with 3 min oxidation with 2% potassium permanganate and a silver concentration of 1% during 5 min. In this way, it was very easy to perform acquisition, thresholding and area measurement without any further manual processing of the image.
  
  
  CONCLUSIONS
  This technique appears very helpful for the quantitative study of the cardiac collagen network by means of computerized image analysis systems.
authors.affiliation: Pathology Department, Hospital de Pediatría JP Garrahan, Buenos Aires, Argentina.
authors.forename: Oscar Antonio
authors.initials: OA
authors.lastname: Schwint
authors.forename: María
authors.initials: M
authors.lastname: Labraga
authors.forename: Claudio Osvaldo
authors.initials: CO
authors.lastname: Cervino
authors.forename: Matías
authors.initials: M
authors.lastname: Haffar
authors.forename: Pablo Hernán
authors.initials: PH
authors.lastname: Sequeiros
authors.forename: Hernán Javier Aldana
authors.initials: HJ
authors.lastname: Marcos
mesh_terms: D000293:Adolescent; D000328:Adult; D003094:Collagen / Q000648:ultrastructure*; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007223:Infant; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000648:ultrastructure*; D015203:Reproducibility of Results; D012155:Reticulin / Q000648:ultrastructure*; D012680:Sensitivity and Specificity; D016622:Silver Staining / Q000379:methods*
chemical_list: D012155:Reticulin; D003094:Collagen