Exploring a Flow Cytometry-Based CFU Assay for Functional Assessment of Human HSPCs: a Robust Alternative to Morphological Colony Evaluation

Assessment of hematopoietic stem and progenitor cells (HSPC) function is essential for both research and clinical applications. Traditional colony-forming unit (CFU) assays, based on morphological evaluation in semi-solid media, are labor-intensive and subject to operator bias, limiting reproducibility and standardization. We evaluated a commercially available, flow cytometry-based liquid CFU assay for quantifying HSPC proliferation and differentiation. Using a high-throughput platform, colonies were identified and classified by the expression of CD14, CD15, and CD235a (Glycophorin A). Results were compared with conventional semi-solid CFU assays. The flow-based assay demonstrated strong correlation with traditional methods, while reducing both hands-on time and inter-operator variability. The approach allowed objective, reproducible classification of BFU-E, CFU-G, CFU-GM, CFU-M, and CFU-GEMM colonies, and was compatible with clinically relevant sample types. The liquid CFU assay provides a standardized, efficient, and robust alternative for functional evaluation of human HSPCs, supporting its broad adoption in both research and clinical settings.