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Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): "Instituto de Salud Carlos III" and "Fondos Europeos de Desarrollo Regional FEDER" and Conselleria de Educación – Generalitat Valenciana. Introduction After acute myocardial infarction (AMI), the lack of oxygen and nutrients leads to cardiomyocyte necrosis and eventually to the formation of a collagen-based scar. Infarct scar characteristics, such as collagen bundle orientation, have a relevant influence on scar mechanics, the occurrence of cardiac arrhythmias, left ventricular dilation or aneurysm formation, wall stiffness, and the development of wall rupture or heart failure. However, the most adequate method for collagen bundle orientation (CBO) measurement in myocardial scar is not established. Purpose We aim to compare the measurement of collagen bundle orientation in infarct scar by Fourier analysis in three different histopathological techniques. Methods Juvenile swine (n=21) were subjected, by means of percutaneous balloon inflation, to a transient 90-min occlusion of mid left anterior descending artery followed by one month of reperfusion (chronic AMI group). Samples were obtained from the infarcted zone and stained with Masson's trichrome, Picrosirius red and Haematoxylin-Eosin (H-E) standard protocols. Five microphotographs of the myocardial scar were taken at 200x magnification with light, polarised and confocal microscopy, respectively. A single observer measured CBO by means of Fast Fourier Transform analysis using a semi-automated protocol. Comparability between techniques was studied by the Intraclass Correlation Coefficient (ICC), the coefficient of variation (CV) and the Bland-Altman (B&A) plots and limits of agreement. Results Measurement of CBO in Masson's trichrome tended to show higher (more "random-oriented") values than in Picrosirius and H-E+confocal techniques (ICC 0.79 and 0.7, p=0.001 and 0.005; B&A 0.29 to -0.02 and 0.43 to 0.01; CV 6.97% and 12.98%, respectively). However, measurement of CBO in Picrosirius and H-E+confocal techniques showed an "almost perfect" agreement (ICC 0.84, p<0.001; B&A 0.28 to -0.09; CV 17.33%). Selective staining and/or visualization of collagen in these latter techniques may underlie our findings, contrary to non-selective Masson's trichrome. Conclusion Picrosirius red staining (visualized with polarised microscopy) and Haematoxylin-Eosin (visualized with confocal microscopy) are comparable in terms of collagen bundle orientation measurement by Fourier analysis in an animal model of chronic infarct scar. Masson's trichrome (visualized with light microscopy) tends to show more "random-oriented" values, potentially due to non-specific staining and visualization of non-collagenous structures such as cells, and should not be recommended for this specific purpose. [ABSTRACT FROM AUTHOR]