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

TL;DR

This paper introduces a flow cytometry-based CFU assay as a faster and more reliable alternative to traditional methods for evaluating human hematopoietic stem and progenitor cells.

Contribution

A novel flow cytometry-based liquid CFU assay is proposed as a standardized and efficient alternative to traditional morphological colony evaluation.

Findings

The flow-based assay strongly correlates with traditional CFU methods but reduces hands-on time and variability.

It enables objective classification of multiple colony types like BFU-E and CFU-GM.

The method is compatible with clinically relevant sample types and supports broader adoption.

Abstract

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.

The online version contains supplementary material available at 10.1007/s12015-025-11045-w.

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