Methodological contributions for the morphometric study of the lung: Approximation to the ideal sample size and quantification of collagen fiber

Background A morphometric study of the rat lung was done to determine the importance, within the precision of a morphometric study, of the sample size in relation to the quality of the image and to propose a method for the quantification of lung collagen fiber. Methods Sixty Wistar rats, divided into two age groups consisting of adult and old rats, were used. The left lungs were studied and processed for light microscopy. Methylene blue, resorcin-fuchsin, and Sirius red stainings were performed. The variables were quantified automatically. In the sections stained with methylene blue, the variables alveolar chord, wall thickness, mean linear intercept index, tissue density, and internal alveolar perimeter were quantified, in two series, one with ×40 magnification (panoramic image) and the other with ×100 magnification. In the sections stained with resorcin-fuchsin, elastic fiber was quantified and the result related with that obtained for the variable tissue density. The results were compared statistically, and those obtained with different magnifications for single variables were related by using the correlation test; the misclassification indices were also calculated. In the sections stained with Sirius red, the surface that was birefringent to polarized light was quantified and related to the collagen fiber; the results were compared statistically. Results Concerning results obtained for single variables with different magnifications good, correlation indices were obtained (r ≥ 0.62) for all but the wall thickness (r = 0.3). The results obtained for the panoramic images were the highest. The misclassification index was lower for the panoramic images. Significant differences (P < 0.5) were not found when comparing the mean birefringent surface in the two groups of animals. Conclusion The results obtained lead us to consider that, despite the fact that the quality of the panoramic images is poorer, the results for these images are more accurate, possibly because a greater number of structures was analyzed. The measurement of the birefringent surfaces of the sections stained with Sirius red may be used in the study of lung collagen fiber. Anat. Rec. 247:501–511, 1997. © 1997 Wiley-Liss, Inc.