title: Assessment of cardiac fibrosis: a morphometric method comparison for collagen quantification.
abstract: Fibrotic remodeling of the heart is a frequent condition linked to various diseases and cardiac dysfunction. Collagen quantification is an important objective in cardiac fibrosis research; however, a variety of different histological methods are currently used that may differ in accuracy. Here, frequently applied collagen quantification techniques were compared. A porcine model of early stage heart failure with preserved ejection fraction was used as an example. Semiautomated threshold analyses were imprecise, mainly due to inclusion of noncollagen structures or failure to detect certain collagen deposits. In contrast, collagen assessment by automated image analysis and light microscopy (LM)-stereology was more sensitive. Depending on the quantification method, the amount of estimated collagen varied and influenced intergroup comparisons. PicroSirius Red, Masson's trichrome, and Azan staining protocols yielded similar results, whereas the measured collagen area increased with increasing section thickness. Whereas none of the LM-based methods showed significant differences between the groups, electron microscopy (EM)-stereology revealed a significant collagen increase between cardiomyocytes in the experimental group, but not at other localizations. In conclusion, in contrast to the staining protocol, section thickness and the quantification method being used directly influence the estimated collagen content and thus, possibly, intergroup comparisons. EM in combination with stereology is a precise and sensitive method for collagen quantification if certain prerequisites are considered. For subtle fibrotic alterations, consideration of collagen localization may be necessary. Among LM methods, LM-stereology and automated image analysis are appropriate to quantify fibrotic changes, the latter depending on careful control of algorithm and comparable section staining.NEW & NOTEWORTHYDirect comparison of frequently applied histological fibrosis assessment techniques revealed a distinct relation of measured collagen and utilized quantification method as well as section thickness. Besides electron microscopy-stereology, which was precise and sensitive, light microscopy-stereology and automated image analysis proved to be appropriate for collagen quantification. Moreover, consideration of collagen localization might be important in revealing minor fibrotic changes.
authors.affiliation: Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany; schipke.julia@mh-hannover.de.|Cluster of Excellence REBIRTH (from Regenerative Biology to Reconstructive Therapy), Hannover, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Schipke
authors.affiliation: Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany.|Cluster of Excellence REBIRTH (from Regenerative Biology to Reconstructive Therapy), Hannover, Germany.|Biomedical Research in Endstage and Obstructive Lung Disease Hannover, German Center for Lung Research, Hannover, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Brandenberger
authors.affiliation: Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Rajces
authors.affiliation: Division of Cardiology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; and.
authors.forename: Martin
authors.initials: M
authors.lastname: Manninger
authors.affiliation: Department of Cardiology, Campus Virchow-Klinikum, Charite Universitätsmedizin, Berlin, Germany.
authors.forename: Alessio
authors.initials: A
authors.lastname: Alogna
authors.affiliation: Department of Cardiology, Campus Virchow-Klinikum, Charite Universitätsmedizin, Berlin, Germany.
authors.forename: Heiner
authors.initials: H
authors.lastname: Post
authors.affiliation: Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany.|Cluster of Excellence REBIRTH (from Regenerative Biology to Reconstructive Therapy), Hannover, Germany.|Biomedical Research in Endstage and Obstructive Lung Disease Hannover, German Center for Lung Research, Hannover, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Mühlfeld
mesh_terms: D000818:Animals; D001391:Azo Compounds / Q000032:analysis; D003094:Collagen / Q000032:analysis*; D005260:Female; D005355:Fibrosis / Q000473:pathology; D007090:Image Interpretation, Computer-Assisted / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D009206:Myocardium / Q000737:chemistry* / Q000473:pathology*; D013552:Swine
chemical_list: D001391:Azo Compounds; C009798:C.I. direct red 80; D003094:Collagen
keywords: automated image analysis; cardiac fibrosis; collagen quantification; stereology