Introduction: Accurate enumeration of CD34+ stem cells is vital for clinical decision-making, especially in transplantation settings. External Quality Assessment (EQA) is crucial for ensuring methodological consistency across laboratories. Flow cytometry protocols, particularly single vs. dual-platform setups, may impact standardization.

Aim: To compare single and dual-platform assays for CD34+ stem cell enumeration over four consecutive EQA campaigns (2022–2025).

Methods: A total of 346 EQA samples were analysed by 22 Belgian clinical laboratories across four Sciensano EQA campaigns (2022–2025), using 17 distinct specimens. The samples were provided by Bio-Techne® (Status Flow Pro) and processed according to standard CD34+ enumeration protocols. Two analytical parameters were evaluated: absolute CD34+ cell count (cells/μL) and percentage of CD34+ cells within white blood cells. Linear mixed-effects models were applied using RStudio version 2025.05.1 to assess the influence of platform type (single vs. dual), while controlling for variability attributable to laboratory and sample-level effects.

Results:

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• For absolute CD34+ count, the single-platform approach showed an effect estimate of 0.046 cells/μL (SE = 0.39; t = 0.12). Sample-level variability was pronounced (SD = 12.03), with moderate inter-laboratory variability (SD = 0.68).
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• For CD34+ percentage: the platform effect was negligible (estimate = 0.0032%; SE = 0.0063; t = 0.52), with minimal laboratory variability (SD = 0.011) and expected sample-level dispersion (SD = 0.20). 
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• For both parameters, the platform did not significantly impact CD34+ stem cell enumeration results (p > 0.05).
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Conclusions: The choice between single and dual-platform methods does not significantly affect CD34+ cell enumeration results in an EQA setting. Both approaches yield comparable results across laboratories, supporting methodological flexibility and confirming consistency in inter-laboratory CD34+ reporting.