Precise CD34+ Quantification Using a Multi-Parameter Flow-Cytometric Method with Fluorescent Microparticles

Abstract

Background: In recent years, peripheral blood stem cell transplantation has replaced autologous bone marrow transplantation to a large extent in patients 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 includes viability tests, short-term culture assays and the determination of CD34+ cells by flow cytometry. External quality controls have suggested the urgent need for greater standardization of the enumeration of CD34+ cells. Material and Methods: We evaluated a single-platform assay for CD34+ cell quantification in peripheral blood and leukapheresis samples (n = 22) by using a four-parameter flow-cytometric method (forward and side angle light scatter, CD34 PE, CD45 FITC) including Flow Count fluorospheres for direct quantification. Results: When processing times of 30 and 300 s were compared, the inter-assay coefficients of variation declined significantly from 11% and 12% 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 method (intra-assay variation) generated coefficients of variation, which dropped 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-cytometric methods based on fluorescent microparticles. They showed a greater precision of CD34+ cell enumeration over the course of analysis time. Because of its clinical relevance in peripheral blood stem transplantation, these improvements should supplement the current guidelines.