Correspondence: Farid Azmoudeh-Ardalan, Pathology Department, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Dr Gharib St, Tehran 14197–33141, Iran. E-mail: azmoudeh@sina.tums.ac.ir (Received 23 October 2011; accepted 15 February 2012) True volumetric method for flow cytometric enumeration of CD34 + stem cells and its agreement with a standard bead-based single-platform protocol SAMIRA MORTAZAVI 1 , FARID AZMOUDEH ARDALAN 1 , SEYYED REZA SAFAEE NODEHI 2 , FAHIMEH FIROUZJAIE KARDER 3 & NAFISEHSADAT MIRALIAKBARI 3 1 Pathology Department, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran, 2 Oncology and Hematology Department,Vali-e-Asr Hospital, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran, and 3 Flow Cytometry Division,Vali-e-Asr Clinical Laboratory, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran Abstract Background aims. Stem cells are commonly enumerated with bead-based methods in blood and marrow progenitor cell transplantation centers. We compared the International Society of Hematotherapy and Graft Engineering (ISHAGE) bead-based method with a true volumetric one that obviates the use of fluorescent beads for enumeration. Methods. From 31 samples, including 15 peripheral blood samples and 16 leukapheresis products, CD34 + cells were enumerated with the single-platform bead-based ISHAGE method and a true volumetric method. After exclusion of two outliers, one from the peripheral blood group and the other from the leukapheresis group, the results were compared. Results. In the periph- eral blood category, no significant difference was observed. However, a proportional systematic error was seen in the leukapheresis group. The systematic error was corrected in the leukapheresis group using a regression line equation. The 95% confidence interval of differences was [–5.83, 2.18] for the peripheral blood and [–38.40, 38.77] for the leukapheresis group after correction of the systematic error. Conclusions. The true volumetric method is a simple and reliable approach that can be used instead of the more popular bead-based procedures. Key Words: CD34 antigen, enumeration, flow cytometry, stem cell, transplantation Introduction CD34 + stem cell enumeration is of great impor- tance in blood and marrow transplantation from both prognostic and therapeutic points of view (1–3). The exact time of leukapheresis after granulocyte– macrophage colony-stimulating factor (GM-CSF) administration is also determined by CD34 + cell enumeration (4,5). There are several methods for CD34 counting (6,7). The determination of colony-forming units by cell culture is the historical method (2,8,9); however, because it is a very labor-intensive and lengthy pro- cess, it has been almost completely abandoned in routine practice (2,10). Currently, flow cytometric procedures, standardized by International Society of Hematotherapy and Graft Engineering (ISHAGE), are more popular for CD34 enumeration (2,10–13). Both single- and dual-platform methodologies have been suggested for CD34 enumeration (5,14). In dual-platform procedures, the total cell count is performed by a hematologic cell counter, and the CD34 percentage is determined flow cytometrically. The major disadvantage of these methods is reduced reproducibility resulting from utilization of two instruments with additive imprecisions (3,15,16). Therefore, single-platform procedures are the meth- ods of choice for CD34 enumeration (5). Most sin- gle-platform procedures are based on adding a known concentration of fluorescent beads and simultaneous enumeration of both beads and CD34 + cells in one sample (1,15,16). The beads are added to all tubes in an additional step. Although the use of commer- cial tubes containing a known concentration of pre- dispensed fluorescent beads (e.g. Trucount tubes, Becton-Dickinson, San Jose, CA) can reduce impre- cision substantially, this variation has not disappeared completely (1,11,15,17,18). One of the limitations of bead-based methods is the summative imprecision of Cytotherapy, 2012; 14: 621–629 ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2012.667875