for the European Working Group on Clinical Cell Analysis
Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells†
Article first published online: 6 DEC 1998
Copyright © 1998 Wiley-Liss, Inc.
Volume 34, Issue 3, pages 128–142, 15 June 1998
How to Cite
Gratama, J. W., Orfao, A., Barnett, D., Brando, B., Huber, A., Janossy, G., Johnsen, H. E., Keeney, M., Marti, G. E., Preijers, F., Rothe, G., Serke, S., Sutherland, D. R., Van der Schoot, C. E., Schmitz, G. and Papa, S. (1998), Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. Cytometry, 34: 128–142. doi: 10.1002/(SICI)1097-0320(19980615)34:3<128::AID-CYTO3>3.0.CO;2-D
The European Working Group on Clinical Cell Analysis (EWGCCA) is a collaborative initiative of scientists active in the field of clinical cell analysis from 13 European countries. The goal of this working group, which is open to scientists active in this field, is the evaluation, refinement and standardization of new analytical techniques for clinical cell analysis. The group is divided into subsections depending on the different tasks and types of analytical techniques, i.e., flow and image cytometry or molecular biology. The core members of the EWGCCA are: G. Schmitz (Regensburg, Germany), coordinator; B. Autran (Paris, France); B. Brando (Milan, Italy); J.L. D'hautcourt (Boussu, Belgium); J.W. Gratama (Rotterdam, the Netherlands); A. Huber (Aarau, Switzerland); G. Janossy (London, UK); H.E. Johnsen (Copenhagen, Denmark); J. Kappelmayer (Debrecen, Hungary); R. Lenkei (Stockholm, Sweden); A. Orfao (Salamanca, Spain); S. Papa (Urbino, Italy); M. Papamichail (Athens, Greece); T. Töttermann (Uppsala, Sweden); G. Rothe (Regensburg, Germany); B. Zupanska (Warsaw, Poland).
The comments of Gerald E. Marti are his personal scientific opinions and do not reflect the policy of the Food and Drug Administration.
- Issue published online: 26 DEC 2002
- Article first published online: 6 DEC 1998
- Manuscript Accepted: 26 MAR 1998
- Manuscript Received: 29 DEC 1997
- Concerted Action. Grant Number: BMH4-97-2611
- hematopoietic stem cells;
- flow cytometry;
The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/μl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in the CD34+ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34+ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34+ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies. Cytometry (Comm. Clin. Cytometry) 34: 128–142, 1998. © 1998 Wiley-Liss, Inc.