Colony-Forming Unit Assay Adapted For Patient-Derived Bone Marrow and Peripheral Blood Mononuclear Cells

The colony-forming unit (CFU) assay is a foundational technique for studying hematologic diseases, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). This assay employs semisolid media supplemented with defined cytokine combinations to support three-dimensional growth and lineage-specific differentiation of erythroid and myeloid progenitor cells. However, patient-derived peripheral blood and bone marrow samples present technical challenges due to reduced viability, increased sensitivity to handling, and variability in colony growth. Furthermore, existing guidelines for identifying and counting colonies show substantial variability in defining colony size and lineage types, limiting reproducibility across studies. The protocol described herein is optimized for the growth and differentiation of hematopoietic stem and progenitor cells isolated from MDS and AML patient samples. The protocol emphasizes gentle cell handling, sterile processing of patient specimens, optimized culture conditions, and growth durations tailored to patient-derived cells. This protocol provides a practical framework for CFU assays using patient-derived samples and establishes a standard metric for consistent colony counting and analysis in hematologic malignancy research.