BACKGROUND:Accuracy of bone marrow (BM) blast count in low-risk myelodysplastic syndromes (MDS) still remains a challenge though it is essential for prognosis. We investigated whether the enumeration of CD34+ myeloid cells by flow cytometry immunophenotyping (FCI) could be used as a consistent parameter for clinical MDS studies. METHODS:Six clinical centers entered the study and information on their FCI protocols was recorded. Sixty-seven flow cytometry listmodes from BM samples of patients with low-risk MDS with <5% BM blasts were exchanged among participants in two different rounds. Interlaboratory variations on the quantification of CD34+ myeloid cells were calculated and strategies to solve differences were evaluated. RESULTS:An overall "very good" agreement on CD34+ cell count among participants (intraclass correlation coefficient = 0.720) was observed, but agreement was "low" in 22 files. No single parameter could fully explain all discrepancies, but 3 technical issues were identified as relevant: the use of the CD34/CD45/CD117/HLA-DR mAb combination, acquisition of ≥50,000 events and a low percentage of debris/aggregates. The frequency of discordant results increased with the accumulation of pitfalls (none, 16%; 1 pitfall, 40%; 2 pitfalls, 83%; P = 0.006). Finally, the use of a common gating strategy for analysis increased the percentage of files with "very good" agreement to 100%. CONCLUSIONS:Prevention of specific technical pitfalls is mandatory to reach a good reproducibility of CD34+ cell count among centers. These recommendations set the basis for laboratory standardization and enable the use of CD34+ cell enumeration as additional information in low-risk MDS patients. © 2017 International Clinical Cytometry Society.