BACKGROUND: Quantification of CD34+ stem and progenitor cells is predominantly performed by flow cytometric analysis of cells prepared by whole blood staining and red cell lysis. This method also includes cell washing, which is thought to cause the destruction and loss of some of the nucleated cells (NCs). To address this cell loss and its influence on the outcome of enumeration, three techniques for preparing cells for quantification of CD34+ cells were compared. STUDY DESIGN AND METHODS: Blood (n=179), bone marrow (n=60), and leukapheresis components (n=64) were examined by the use of density separation of mononuclear cells (MNCs) and two red cell-lysis procedures (wash and no- wash). Cell counts were determined in the original materials and after cell preparation. Absolute CD34+ cell counts were calculated using the flow cytometry-analyzed proportions of CD34+ cells and the various white cell counts. RESULTS: Depending on the cell source and the cell preparation chosen, the loss of NCs ranged between 12 percent and 89 percent of the original white cell number. This loss of NCs was exclusively due to cell washing and predominantly affected granulocytic cells. Analysis of the flow cytometry data revealed that the relative CD34+ values in blood and bone marrow were roughly threefold higher in density separated MNCs than in those that underwent the lyse-and-wash procedure. Calculation of absolute CD34+ cell counts confirmed that the MNC procedure underestimated the CD34+ cell content by a median of 26 percent (blood), 21 percent (bone marrow), and 5 percent (leukapheresis component) when compared with the median yield from analysis and cell counting performed after the lyse-and-wash procedure. On the other hand, the conventional lysis procedure, which applies the original white cell counts for CD34+ quantification, was shown to overestimate the CD34+ cell content by a median of 1.2-fold, 1.33-fold, and 1.13- fold, respectively. CONCLUSION: Neither density separation nor the whole-blood lysis procedure seems appropriate for optimal CD34+ quantification.