Fourier analysis of collagen bundle orientation in myocardial infarction scars.

Collagen bundle orientation (CBO) in myocardial infarct scars plays a major role in scar mechanics and complications after infarction. We aim to compare four histopathological methods for CBO measurement in myocardial scarring. Myocardial infarction was induced in 21 pigs by balloon coronary occlusion. Scar samples were obtained at 4 weeks, stained with Masson's trichrome, Picrosirius red, and Hematoxylin-Eosin (H&E), and photographed using light, polarized light microscopy, and confocal microscopy, respectively. Masson's trichrome images were also optimized to remove non-collagenous structures. Two observers measured CBO by means of a semi-automated, Fourier analysis protocol. Interrater reliability and comparability between techniques were studied by the intraclass correlation coefficient (ICC) and Bland-Altman (B&A) plots and limits of agreement. Fourier analysis showed an almost perfect interrater reliability for each technique (ICC ≥ 0.95, p < 0.001 in all cases). CBO showed more randomly oriented values in Masson's trichrome and worse comparability with other techniques (ICC vs. Picrosirius red: 0.79 [0.47-0.91], p = 0.001; vs. H&E-confocal: 0.70 [0.26-0.88], p = 0.005). However, optimized Masson's trichrome showed almost perfect agreement with Picrosirius red (ICC 0.84 [0.6-0.94], p < 0.001) and H&E-confocal (ICC 0.81 [0.54-0.92], p < 0.001), as well as these latter techniques between each other (ICC 0.84 [0.60-0.93], p < 0.001). In summary, a semi-automated, Fourier-based method can provide highly reproducible CBO measurements in four different histopathological techniques. Masson's trichrome tends to provide more randomly oriented CBO index values, probably due to non-specific visualization of non-collagenous structures. However, optimization of Masson's trichrome microphotographs to remove non-collagenous components provides an almost perfect comparability between this technique, Picrosirius red and H&E-confocal.