BACKGROUND:Quantitation of peripheral blood (PB) CD34(+) cells is now an established method for timing PBPC harvesting. Recent refinements to the dual-platform ISHAGE gating strategy for CD34(+) cells has seen the introduction of microbeads to enable absolute counting of cells on a single instrument platform. This eliminates the need for total WBCC performed on an automated hematology analyzer and potentially increases the analytical precision of the methodology. At the same time, alternative methods for CD34(+) cell enumeration have started to emerge, notably microvolume fluorimetry, which forms the basis of the fully-automated STELLer CD34 method using the Imagn 2000. METHODS:We performed a three-way evaluation of these methods. Sixty-eight samples of PB from 42 patients undergoing PBPC mobilization were analyzed by all three methods and correlations between all three calculated. The two-platform ISHAGE method was used as the reference method. RESULTS:Precision and linearity of the single-platform and STELLer CD34 assays were excellent. Correlation with the dual-platform reference method was also excellent (single-platform method slope = 1.03, intercept = -0.03 and R(2) = 0.9325, STELLer CD34 assay slope = 0.827, intercept = 4.27, R(2) =0.8215). Bias, determined by Bland-Altman analysis, was 1.16 and -1.62 for single platform and STELLer CD34 assay respectively. CONCLUSION:The three methods of CD34(+) cell enumeration gave equivalent results. The single-platform methodology negated the need for a separate white cell analyzer, while the STELLer CD34 methodology was technically the simplest.