Fourier analysis of collagen bundle orientation in myocardial infarction scars

Abstract

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.

Keywords: Collagen; Fourier; Myocardial infarction; Orientation; Scar.

Fig. 1

Protocol for microphotograph of regions of interest in infarct scar samples. After sample visualization with light microscopy (H&E and Masson’s trichrome), regions of interest are defined, i.e., in the central area of the infarct scar. For each technique, 21 histological samples and 105 microphotographs (five per sample) were analyzed. Using histopathological references, the same regions are manually co-localized in each sample and microphotographs are taken using the described protocol. Finally, Masson’s trichrome microphotographs were optimized with digital removal of non-collagenous structures. Note that the Picrosirius red image (center left) depicts a macro photograph without polarized light microscopy. This macro caption as well as the Masson’s trichrome (upper) one have been edited for brightness and contrast to increase understandability. Bar (macro images) = 1 mm; bar (microphotographs) = 100 µm. H&E Hematoxylin–Eosin

Fig. 2

Protocol for CBO index measurement by Fourier analysis. a Microphotograph of a pig myocardial scar sample stained with H&E and visualized with confocal microscopy. A Best Fit filter has been applied. b Conversion to grey scale 16. c FFT has been applied to the image and the 2D power plot has been obtained. d The spectrum gain has been adjusted to obtain a more defined power plot. e Manual segmentation to select the central frequencies in the FFT power plot. f Automatic major and minor axis measurement after implementing an area filter range and Smoothing, Pre-Filter, and Convex Hull filters. bar = 100 µm. For each technique, 21 histological samples and 105 microphotographs (five per sample) were analyzed. CBO collagen bundle orientation, FFT fast Fourier transform. H&E Hematoxylin–Eosin

Fig. 3

Bland–Altman plots and limits of agreement for CBO index measurement by Fourier analysis. In each plot, the average collagen orientation index as measured by the two different methods being compared is presented in the x-axis, and differences in collagen orientation index measurements between these two methods are plotted in the y-axis. Limits of agreement are graphically represented (as dashed lines and shadowed area) and calculated as mean ± 2 SDs of the mean difference between the measurements. For each technique, 21 histological samples and 105 microphotographs (five per sample) were analyzed. a–d Plots for interrater reliability (reproducibility) of measurements in each technique. e–j Plots for consistency between staining and microscopy techniques. CBO  collagen bundle orientation, SDs standard deviations

Fig. 4

Examples of CBO index in infarct scarring with each staining technique. Samples were stained with Masson’s trichrome (visualized with light microscopy, a and e), optimized Masson’s trichrome with digital removal of non-collagenous structures (b and f), Picrosirius red (visualized with polarized light microscopy, c and g) and H&E (visualized with confocal microscopy, d and h). In the first row (a–d), a case of parallel-oriented collagen bundles is shown. Collagen orientation index for each image is 0.67, 0.52, 0.41, and 0.35. In the second row (e–h), a case of randomly oriented collagen bundles is depicted. Collagen orientation index for each image is 0.88, 0.8, 0.82, and 0.82. bar = 100 µm. For each technique, 21 histological samples and 105 microphotographs (five per sample) were analyzed. CBO collagen bundle orientation, H&E Hematoxylin–Eosin

Fig. 5

Histogram of CBO index measurements in the different techniques. a Histogram in Masson’s trichrome and examples of a parallel (a1) and random (a2) CBO index pattern in myocardial scar. Bin width: 0.05. Bin centers: 0.61, 0.67, 0.74, 0.79, 0.83, 0.88, 0.92 and 0.97. b Histogram in optimized Masson’s trichrome with digital removal of non-collagenous structures and examples of a parallel (b1) and random (b2) CBO index pattern in myocardial scar. Bin width: 0.075. Bin centers: 0.47, 0.49, 0.6, 0.66, 0.74, 0.82, 0.89 and 0.95. c Histogram in Picrosirius red and examples of a parallel (c1) and random (c2) CBO index pattern in myocardial scar. Bin width: 0.07. Bin centers: 0.4, 0.48, 0.55, 0.61, 0.69, 0.76, 0.82, 0.89 and 0.95. d Histogram in H&E + confocal and examples of a parallel (d1) and random (d2) CBO index pattern in myocardial scar. Bin width: 0.08. Bin centers: 0.28, 0.33, 0.4, 0.49, 0.57, 0.63, 0.73, 0.8, 0.87 and 0.93. For each technique, 21 histological samples and 105 microphotographs (five per sample) were analyzed. CBO collagen bundle orientation, H&E Hematoxylin and Eosin