# 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.

## Key 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.