title: Rapid quantification of myocardial fibrosis: a new macro-based automated analysis.
abstract: BACKGROUND
  Fibrosis is associated with various cardiac pathologies and dysfunction. Current quantification methods are time-consuming and laborious. We describe a semi-automated quantification technique for myocardial fibrosis and validated this using traditional methods.
  
  
  METHODS
  Pulmonary Hypertension (PH) was induced in adult Wistar rats by subcutaneous monocrotaline (MCT) injection(40 mg/kg). Cryosections of myocardial tissue (5 μm) of PH rats (n = 9) and controls (n = 9) were stained using Picrosirius red and scanned with a digital microscopic MIRAX slide scanner. From these sections 21 images were taken randomly of each heart. Using ImageJ software a macro for automated image analysis of the amount of fibrosis was developed. For comparison, fibrosis was quantified using traditional polarisation microscopy. Both methods were correlated and validated against stereology as the gold standard. Furthermore, the method was tested in paraffin-embedded human tissues.
  
  
  RESULTS
  Automated analysis showed a significant increase of fibrosis in PH hearts vs. control. Automated analysis correlated with traditional polarisation and stereology analysis (r(2) = 0.92 and r(2) = 0.95 respectively). In human heart, lungs, kidney, and liver, a similar correlation with stereology (r(2) = 0.91) was observed. Time required for automated analysis was 22% and 33% of the time needed for stereology and polarisation analysis respectively.
  
  
  CONCLUSIONS
  Automated quantification of fibrosis is feasible, objective, and time-efficient.
authors.affiliation: Department of Pulmonary Diseases, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Awal M
authors.initials: AM
authors.lastname: Hadi
authors.forename: Koen T B
authors.initials: KT
authors.lastname: Mouchaers
authors.forename: Ingrid
authors.initials: I
authors.lastname: Schalij
authors.forename: Katrien
authors.initials: K
authors.lastname: Grunberg
authors.forename: Gerrit A
authors.initials: GA
authors.lastname: Meijer
authors.forename: Anton
authors.initials: A
authors.lastname: Vonk-Noordegraaf
authors.forename: Willem J
authors.initials: WJ
authors.lastname: van der Laarse
authors.forename: Jeroen A M
authors.initials: JA
authors.lastname: Beliën
mesh_terms: D000818:Animals; D001331:Automation; D001391:Azo Compounds / Q000378:metabolism; D001835:Body Weight; D004562:Electrocardiography; D005355:Fibrosis; D006439:Hemodynamics; D006801:Humans; D006976:Hypertension, Pulmonary / Q000000981:diagnostic imaging / Q000473:pathology / Q000503:physiopathology; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008859:Microscopy, Polarization; D016686:Monocrotaline; D009206:Myocardium / Q000473:pathology*; D009929:Organ Size; D016612:Paraffin Embedding; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014463:Ultrasonography
chemical_list: D001391:Azo Compounds; C009798:C.I. direct red 80; D016686:Monocrotaline