AML infiltration induces proliferation of bone marrow MSPCs. (A) Exemplary procedure for quantification of CD271. First, tile images were fused into larger images (1). Second, a color deconvolution was performed (ImageJ, IHC Profiler plugin) to obtain separate hematoxylin and DAB images. The hematoxylin image was thresholded to obtain a mask of the total bone region (2). The DAB image (3) was inverted and thresholded to obtain a mask of the positive DAB regions (4). Scale bars, 1 mm. (B) Representative images of bone marrow biopsy samples of non-leukemic donors (controls) and AML patients stained for CD271⁺ MSPCs (brown) and counterstained with hematoxylin (blue). Scale bars, 400 µm. (C) Quantification of CD271⁺ bone marrow MSPCs in controls (n = 58) and AML patients (n = 36); CD271⁺ area is calculated by CD271⁺ stained area divided by total tissue surface. (D) Left: representative image from bone marrow after silver staining representing reticular fibers. Right: image converted by trainable Weka segmentation showing 3 different classes: red, reticular fiber; green, tissue; purple, background. Scale bars, 50 µm. (E) Representative images of bone marrow biopsy samples from controls and AML patients stained for reticular fibers with silver staining (black fiber) and counterstained with the fast-red nuclear solution. Scale bars, 100 µm. (F) Quantification of reticular fibers in control (n = 19) and AML (n = 37) bone marrow by calculating positive silver stained area divided by total tissue surface (n = 56). Data are shown as mean ± standard error of the mean (SEM). *P < .05; **P < .01 (determined by Mann-Whitney U test).