Extended Data Fig. 5: Validation of IMC findings with IHC.

a, Relationship between fibrosis score and fibroblast metacluster abundance, visualized as a scatter plot. b, c, IHC for two markers across all disease groups. Haematoxylin–diaminobenzidine staining of CD163 (b) or MPO (c) in tissue from healthy and diseased lung, matching the patients in the IMC cohort. Scale bars, 200 μm. d, Analysis of IHC data. Example images demonstrating the process of colour decomposition that underlies the separation of the haematoxylin (nuclei) and diaminobenzidine (CD163 or MPO) (H-DAB) in lung tissues. Scale bars, 400 μm (long bars), 50 μm (short bars). e, Example images demonstrating the process of nuclei segmentation. Left column, original images in RGB space; middle column, resulting segmentation in which each nucleus has a random colour and the background is black; and right column, borders of segmented nuclei in red overlain on the original image. Scale bars, 200 μm. f, Example image section, demonstrating the process of quantification of the diaminobenzidine stain. Top, original image in RGB space; second panel, nuclei segmentation; third panel, numeric value of the DAB stain for each nucleus; and bottom, histogram of nuclei intensity in DAB stain, modelled as a Gaussian mixture with two components used to discretize nuclei into negative or positive for DAB on the basis of a threshold that best separates the two mixtures. Scale bars, 50 μm. g, Percentage of cells within an image that are positive for the respective DAB stain in IHC (left column) or positive for the respective marker in IMC data (right column). h, Comparison of the estimated effect sizes of change between disease groups estimated from IMC (x axis) or IHC (y axis) data for the two stains. The Pearson correlation coefficient and its significance are indicated. i, Analysis of lacunae with IHC data. Representative images of the amount of lacuna space in IHC data for healthy lungs and lungs of patients with late COVID-19. For each image, the original image and the segmented background space is shown along with a value denoting the amount of lacuna space for the image (fraction of the image without cells that represents the alveolar or capillary space). Scale bars, 200 μm. j, Quantification of lacuna space across MPO images in IHC. **P < 0.01; *P < 0.05, two-sided Mann–Whitney U-test, pairwise between groups, Benjamini–Hochberg FDR adjustment. k, Comparison of the estimated effect sizes of change in the amount of lacuna space between disease groups, estimated from IMC (x axis) or IHC (y-axis) data. For a, h, k, r, Pearson coefficient; P, its two-tailed P value.