Comparative measurement of collagen bundle orientation by Fourier analysis and semiquantitative evaluation: reliability and agreement in Masson's trichrome, Picrosirius red and confocal microscopy techniques

Abstract

Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin-Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image-Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E-confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland-Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong agreement with H&E-confocal (0.7 < ICC < 0.8). Comparison of measurements between the three techniques for the same observer showed an almost perfect agreement (ICC > 0.8), better with Fourier analysis than with semiquantitative evaluation (single and average). These results in nonpathological skin samples were also confirmed in a preliminary analysis in eight scleroderma skin samples. Our results show that Masson's trichrome and Picrosirius red are consistent with H&E-confocal for measuring collagen bundle orientation in histological samples and could thus be used indistinctly for this purpose. Fourier analysis is superior to average semiquantitative evaluation and should keep being used as the preferred method.

Effects of PARP-1 Deficiency and Histamine H4 Receptor Inhibition in an Inflammatory Model of Lung Fibrosis in Mice

“…A slight effect is reported in VUF treated mice. To confirm the assessment of lung collagen, the Picrosirius red was used as second staining method; this specific staining for collagen (Lattouf et al., 2014; Marcos-Garcés et al., 2017) confirms the results obtained with Azan staining (Figure 3B).…”

“…OD measurements of the aniline blue-stained collagen fibers were performed after selection of a correct threshold to eliminate aerial air spaces and bronchial/alveolar epithelium (Formigli et al., 2007). To confirm the assessment of lung collagen, the Picrosirius red staining was carried out; the sections were stained in 0.1% Direct Red 80/Sirius Red F3B (Sigma-Aldrich) in saturated picric acid at room temperature for 1 h, and then they were differentiated in 0.5% acetic acid, prior to dehydration, clearing, and mounting (Lattouf et al., 2014; Marcos-Garcés et al., 2017).…”

Effects of New NSAID-CAI Hybrid Compounds in Inflammation and Lung Fibrosis

“…The picrosirius red staining was carried out to confirm the assessment of lung collagen; the sections were stained in 0.1% Direct Red 80/Sirius Red F3B (Sigma-Aldrich) in saturated picric acid at room temperature for 1 h, and then they were differentiated in 0.5% acetic acid, prior to dehydration, clearing, and mounting [ 23 , 24 ].…”

Stereological and Morphometric Study of Type 3 Collagen Formation in the Cutaneous Wounds of Diabetic Mice Treated with Mesenchymal Stem Cells

“…The birefringent colour spectrum observed in Picrosirius Red staining, as red for type 1 collagen and green and yellow for type 3 collagen, as well as the identification of black elastic fibres in Weigert staining, are well documented in the literature [16,17]. According to these authors, type 1 collagen corresponds to the mature form of the fibre while type 3 collagen corresponds to the immature form; however, both types of fibre were observed to be intertwined in the histological sections evaluated, thus denoting the process of maturation of the fibres in progress (Figure 3).…”