Single spectral flow cytometry panel for simultaneous detection of hematopoietic stem, progenitor, and mature lineages in mouse bone marrow

Hematopoiesis occurs in the bone marrow of adult mammals and is supported by hematopoietic stem cells that sustain lifelong blood cell production. Pathological conditions can disrupt HSC differentiation, causing anemia, immunodeficiency, and other cytopenias. Therefore, precise and simultaneous identification of hematopoietic populations from stem to mature cells is essential for understanding disease mechanisms and developing targeted therapies. In order to study these alterations, flow cytometry is generally the reference technique. However, most cytometry panels are designed to study a specific population, leaving out potential discoveries on other populations from the same microenvironment. Here we present the design of a 19-marker spectral flow cytometry panel capable of simultaneously identifying HSPCs, erythroid, myeloid and lymphoid cells within a single murine bone marrow sample. This integrated approach replaces multiple conventional panels and enables comprehensive mapping of hematopoietic differentiation from a single assay. Validation confirmed accurate detection of long-term and short-term HSCs, multipotent progenitors, common myeloid and lymphoid progenitors, and erythroid populations from proerythroblasts to reticulocytes. UMAP visualization captured the continuous trajectory of differentiation. We validated our method on aged and β-thalassemic mouse models showing a clear detection of hematopoiesis and erythropoiesis alterations respectively. This panel provides a robust and flexible tool suitable for both basic research and translational studies.