Outline of sample processing and analysis pipeline for spatial transcriptomics. (A) Workflow of preparing human BM trephines for Xenium in situ analysis. (B) DV200 measurements for BM trephines obtained from diagnostic laboratory (n = 5) and formalin-fixed, paraffin-embedded in-house with (n = 8) and without EDTA-based decalcification (n = 16). Error bars represent mean ± standard error of the mean. ∗∗∗P = .0007; ∗∗∗∗P < .0001 (by 1-way analysis of variance [ANOVA] and Dunnett multiple comparisons test). (C-E) Xenium data of a BM trephine collected from a patient with relapsed myeloma (RM-1) using a human multitissue and cancer panel; uniform manifold approximation and projection (UMAP) of Xenium cells (C); spatial plot (scale bar, 1 mm) of panel C with cell type labels derived from published scRNA-seq data (D)¹⁹; and H&E images (E) of PCs (i), pericytes and endothelial cells (ii), megakaryocytes (iii), and erythrocytes (iv), overlaid with transcript expression for listed genes. Each dot represents a single transcript (scale bar, 10 μm). (F) Pixel-level analysis of a disease-free BM trephine (Ctrl-1) with FICTURE using a reference single-cell gene expression profile to define cell types.²¹ Magnification of the white-boxed areas (P1 and P2) focusing on endosteal, vascular, and hematopoietic cell–rich microenvironments. Factors are colored based on cell identity inferred from the reference gene expression profile¹⁹ (scale bars, 1 mm [overview] and 100 μm [magnified areas]). Ba, basophil; Eo, eosinophil; H&E, hematoxylin and eosin; Ma, mast cell; RCA, rolling circle amplification; VSMC, vascular smooth muscle cell.