Immunocharacterization of the AML, B-ALL, CML, and control BM. (A) Visualization of the quantitative BM immunocharacterization pipeline. FFPE BM tissue blocks of AML patients (n = 69) and age- and sex-matched controls (n = 12) were retrieved from the Helsinki Biobank. TMAs were constructed from duplicate punches (1 mm in diameter) from each subject. TMAs were cut onto tissue slides and stained with mIHC consisting of ≤4 primary antibodies detected with fluorescence dyes and 4′,6-diamidino-2-phenylindole (DAPI) counterstain as well as 2 primary antibodies detected with chromogenic probes and hematoxylin counterstain. Tissue slides were scanned after both staining procedure and corresponding images registered to ensure that the location of individual cells is matched in parallel stainings. Following cell segmentation, marker colocalization and intensity were quantified in identified cells. (B) Immune cells (as a proportion of all cells in a TMA spot) and their immunophenotypes (as a proportion of their parent immune cell) derived from mIHC and computerized image analysis are plotted on a heatmap and organized by hierarchical clustering using Spearman correlation distance and the Ward linkage (ward.D2) method. Immunologic parameters are arranged in rows and patients in columns. Red denotes higher and blue lower proportions. (C) Using 10-fold crossvalidated elastic net–regularized logistic regression analysis, 4 subtype-specific classifiers were developed to identify AML, B-ALL, and CML patients and controls. Classifiers were developed with a training group (n = 94) and assessed with a test group (n = 95). The accuracy of the classifiers was evaluated on the test group with receiver-operator curves (AUC).