Precise CD34+quantification using a multi-parameter flow-cytometric methodwith fluorescent microparticles

Background: In recent years, peripheral blood stem cell transplantation has replaced autologous bone marrow transplantation to a large extent in patie nts with hematological malignancies. Optimal timing and adequate harvesting rely upon the precise quantification of mobilized CD34+ cells in blood and apheresis products. The quality assessment of collected stem cells include s viability tests, short-term culture assays and the determination of CD34 cells by flow cytometry. External quality controls have suggested the urge nt need for greater standardization of the enumeration of CD34+ cells. Mate rial and Methods: We evaluated a single-platform assay for CD34+ cell quant ification in peripheral blood and leukapheresis samples (n = 22) by using a four-parameter flow-cytometric method (forward and side angle light scatte r, CD34 PE, CD45 FITC) including Flow Count fluorospheres for direct quanti fication. Results: When processing times of 30 and 300 s were compared, the interassay coefficients of variation declined significantly from 11% and 1 2% in peripheral blood and leukapheresis to 4.7% and 5.2% (p = 0.003 and p = 0.016), respectively. The precision analysis of the flow-cytometric metho d (intra-assay variation) generated coefficients of variation, which droppe d from 8.1% and 8.5%, respectively, to 3.3% for both samples with regard to the processing time (p < 0.001). Conclusion: The data demonstrate the need of single-platform flow-cytometri c methods based on fluorescent microparticles. They showed a greater precis ion of CD34+ cell enumeration over the course of analysis time. Because of its clinical relevance in peripheral blood stem transplantation, these impr ovements should supplement the current guidelines.