Clin. Lab. Haem. 1999, 21, 301–308 Guideline for the flow cytometric enumeration of CD34 + haematopoietic stem cells PREPARED BY THE CD34 + HAEMATOPOIETIC STEM CELL WORKING PARTY* *D. Barnett, G. Janossy, A. Lubenko, E. Matutes, A. Newland & J.T. Reilly Members of the General Haematology Task Force of the British Committee for Standards in Haematology: J.T. Reilly (Chairman), B.J. Bain (Secretary), R. Amos, I. Cavill, C. Chapman, K. Hyde, E. Matutes, J. Parker-Williams, I.D. Walker Introduction It is well established that the 3% of cells in the bone marrow which express the CD34 antigen, a heavily glycosylated mucin-like structure, are capable of reconstituting long- term, multilineage haematopoiesis after myeloablative therapy (Berenson et al. 1988; Andrews et al. 1992). CD34 + cells are also found in the peripheral blood of normal indi- viduals, but are extremely rare (approximately 0.01–0.05% of total nucleated cells). However, current treatment and mobilization regimes, including chemotherapy and/or haematopoietic growth factors, can significantly increase circulating CD34 + stem cell numbers in patients and heal- thy donors. Peripheral blood stem cells (PBSC) have now virtually replaced bone marrow as the primary source of stem cells for autologous transplantation (Gratwohl, Her- mans & Baldomero 1996) and the procedure is being increasingly used for allogeneic transplantation between HLA-identical siblings (Russell, Gratwohl & Schmitz 1996). Potential advantages of PBSC transplantation include a more rapid haematopoietic reconstitution, lower risk of contamination by tumour cells, reduced hospitalization costs, increased numbers of T-lymphocytes and NK cells that may reduce post-transplant relapse, and the elim- ination of a need for general anaesthesia (Dreger et al. 1994; Ager et al. 1995). In addition, the PBSC product is also more suitable for ex vivo manipulation, such as CD34 + cell selection (Brugger, Henschler & Heimfeld 1994), tumour purging (Ross et al. 1995) and gene transfer (Bregni et al. 1992). Transplant centres routinely rely on CD34 + cell quanti- fication by flow cytometry to determine the optimal timing and to confirm the adequacy of PBSC harvests (Haas et al. 1994). In contrast, assessment of haematopoietic pro- Correspondence: BCSH Secretary, British Society for Haematology, 2 Carleton House Terrace, London SW1Y 5AF, UK. Whilst the advice and information in these guidelines is believed to be true and accurate at the time of going to press, neither the authors nor the publishers can accept any legal responsibility or liability for any errors or omissions that may be made. © 1999 Blackwell Science Limited genitor cells (HPC) in colony-forming assays, although cor- relating with CD34 levels (Siena et al. 1991), is handicapped by a lack of reproducibility and the prolonged assay time. A minimum threshold dose, in adults, of between 2 and 5 × 10 6 CD34 + cells/kg body weight has been observed in multiple clinical settings to result in adequate engraftment (Krause et al. 1996), although the lack of standardization of the assay prevents a more exact definition of threshold level (Bender et al. 1992). This lack of standardization with respect to reagents and gating stra- tegies, as well as to the use of different haematology instru- ments to derive absolute total nucleated cell counts has contributed to the widespread inter-laboratory variation (Barnett et al. 1998). As a result, there is an urgent need for a nationally agreed protocol to enable standardization of the assay and to enable comparison of clinical and lab- oratory data. This guideline provides recommendations for: (1) specimen collection and frequency of CD34 testing; (2) monoclonal antibody (mAb) selection; (3) cell separation, lysing and counting techniques; (4) gating strategies; (5) isotype controls; (6) determination of absolute counts; (7) instrument quality control; (8) data analysis; (9) data reporting; (10) data storage; and (11) quality assurance. Finally, this guideline will also give a brief overview of several new technologies that still require evaluation. Specimen collection and frequency of CD34 testing All peripheral blood specimens should be collected by vene- puncture into either 0.34 M di- or tri-potassium ethylene- diaminetetraacetic acid (K 2 EDTA or K 3 EDTA) anticoagulant. The total WBC must be obtained within 6 h of vene- puncture. If a specimen is referred to a central laboratory for analysis resulting in a delay of over 6 h, then the result of a total WBC must accompany the sample. All samples must be labelled with the patient’s surname and fore- name(s), a unique patient identifier such as the hos- pital reference number and date of birth as well as 301