Multi-color immune-phenotyping of CD34 subsets reveals unexpected differences between various stem cell sources. 
Flow cytometric routine CD34 analysis enumerates hematopoietic stem and progenitor cells irrespective of their subpopulations although this might predict engraftment dynamics and immune reconstitution.  We established a multi-color CD34 assay containing CD133, CD45RA, CD10, CD38 and CD33.  We examined PBSC, donor bone marrow (BMd) and BM of patients 1 year after allografting (BM1y) regarding their CD34 subset composition, which differed significantly amongst those materials: the early CD45RA(-)CD133(+)CD38(low) subpopulations were significantly more frequent in PBSC than in BMd, and very low in BM1y.  Vice versa, clearly more committed CD34 stages prevailed in BM, particularly in BM1y where the proportion of multi-lymphoid and CD38(++) B-lymphoid precursors was highest (mean 59%).  CD33 was expressed at different intensity on CD45RA(±)CD133(±) subsets allowing discrimination of earlier from more committed myeloid precursors.  Compared with conventional CD34(+) cell enumeration, the presented multi-color phenotyping is a qualitative approach defining different CD34 subtypes in any CD34 source.  Its potential impact to predict engraftment kinetics and immune reconstitution has to be evaluated in future studies.