Enumeration of CD34+ Hematopoietic Stem and Progenitor Cells

D. Robert Sutherland1, Michael Keeney2, Jan W. Gratama3

1 The University Health Network, Toronto, Ontario, 2 London Health Sciences Center, London, Ontario, 3 Daniel den Hoed Cancer Center, Rotterdam
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.4
DOI:  10.1002/0471142956.cy0604s25
Online Posting Date:  August, 2003
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Abstract

CD34 is the first documented cell surface antigen whose expression within the hematopoetic system is restricted to stem and progenitor cells of all lineages. Transplantation of CD34+ cells has created a need for accurate and rapid assessment of CD34+ cell concentrations in peripheral blood as well as for the optimal timing of apheresis sessions to insure the harvest of sufficient CD34+ cells. However, accurate enumeration of these cells represents a rareā€event analysis, a demanding and somewhat complicated procedure. This comprehensive unit presents the clearest and most definitive methods yet available for CD34+ enumeration by flow cytometry, evaluates a number of commercially available procedures, and provides invaluable supporting information and detailed analysis which we believe is found in no other publication. All the possible problems are discussed and examples provided, allowing the clinical hematology lab to be certain that analysis of CD34+ cells is performed in an appropriate manner.

Keywords: CD34; hematopoietic stem cells; enumeration; multicolor analysis; absolute counts; viability staining

     
 
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Table of Contents

  • Basic Protocol 1: Enumeration of Absolute Numbers of CD34+ Cells
  • Support Protocol 1: Quantitation of Residual CD3+ T Lymphocytes in T Cell–Depleted Allogeneic Stem/Progenitor Cell Grafts
  • Support Protocol 2: Immunological Characterization of CD34+ Cells
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enumeration of Absolute Numbers of CD34+ Cells

  Materials
  • Sample of interest: peripheral blood, apheresis, cord blood, bone marrow, selected CD34+ cells, or cryopreserved and thawed samples
  • Phycoerythrin‐conjugated CD34 monoclonal antibody (CD34‐PE MAb), appropriately titered (unit 4.1)
  • Fluorescein isothiocyanate–conjugated CD45 monoclonal antibody (CD45‐FITC MAb), appropriately titered (unit 4.1)
  • 1 × ammonium chloride lysing solution ( appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A) supplemented with 1% human serum albumen (or similar; PBS/HSA)
  • Flow‐Count counting beads (Beckman Coulter)
  • 12 × 75–mm polypropylene tubes
  • Flow cytometer with at least three fluorescence detectors and appropriate filter sets for detection of FITC, PE, and if required, 7‐AAD
  • Additional reagents and equipment for assessing leukocyte count ( appendix 3A)

Support Protocol 1: Quantitation of Residual CD3+ T Lymphocytes in T Cell–Depleted Allogeneic Stem/Progenitor Cell Grafts

  • PE‐Cy5‐conjugated CD3 monoclonal antibody, appropriately titered (unit 4.1)
NOTE: Several fluorochromes other than PE‐Cy5 may be used as conjugate for the CD3 MAb. Here, PE‐Cy5 is selected because of its high signal‐to‐noise ratio and minimal spectral overlap with PE. For data acquisition with a Beckman Coulter XL instrument with three fluorescence detectors, the use of CD3‐PE‐Cy5 requires replacement of the 620‐nm red fluorescence band‐pass filter by a 675‐nm band‐pass filter. On an XL with four fluorescence detectors, CD45‐PE‐Cy5 is detected in the far‐red fluorescence channel.

Support Protocol 2: Immunological Characterization of CD34+ Cells

  • Phycoerythrin‐conjugated monoclonal antibody identifying marker of interest (CDx‐PE MAb), appropriately titered (unit 4.1)
  • Fluorescein isothiocyanate–conjugated anti‐CD34 monoclonal antibody (CD34‐FITC MAb), appropriately titered (unit 4.1)
  • PE‐Cy5‐conjugated anti‐CD45 monoclonal antibody (CD45‐PE‐Cy5 MAb), appropriately titered (unit 4.1)
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Figures

Videos

Literature Cited

Literature Cited
   Allen, D.S., Keeney, M., Howson‐Jan, K., Popma, J., Weir, K., Bhatia, M., Sutherland, D.R., and Chin‐Yee, I.H. 2002. Number of viable CD34+ cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation. Bone Marrow Transplant. 29:967‐972.
   Bender, J.G., Unverzagt, K., and Walker, D. 1994. Guidelines for determination of CD34+ cells by flow cytometry: Application to the harvesting and transplantation of peripheral blood stem cells. In Hematopoietic Stem Cells: The Mulhouse Manual (E. Wunder, H. Sovalat, P.R. Henon, and S. Serke, eds.) pp. 31‐43. AlphaMed Press, Dayton, Ohio.
   Borowitz, M.J., Guenther, K.L., Schultz, K.E., and Stelzer, G.T. 1993. Immuno‐phenotyping of acute leukemia by flow cytometry: Use of CD45 and right angle light scatter to gate on leukemic blasts in three color analysis. Am. J. Clin. Pathol. 100:534‐540.
   Brando, B., Göhde Jr., W., Scarpati, B., and D'Avanzo G. 2001. The ‘vanishing counting bead’ phenomenon: Effect on absolute CD34+ cell counting in phosphate‐buffered saline‐diluted leukapheresis samples. Cytometry 43:154‐160.
   Brocklebank, A.M. and Sparrow, R.L. 2001. Enumeration of CD34+ cells in cord blood: A variation on a single‐platform flow cytometric method based on the ISHAGE gating strategy. Cytometry 46:254‐261.
   Chapple, P., Prince, H.M., Quinn, M., Bertoncello, I., Juneja, S., Wolf, M., Januszewicz, H., Brettell, M., Gardyn, J., Seymour, C., and Venter, D. 1998. Peripheral blood CD34+ cell count reliably predicts autograft yield. Bone Marrow Transplant. 22:125‐130.
   Gratama, J.W., Kraan, J., Levering, W., Van Bockstaele, D.R., Rijkers, G.T., and Van der Schoot, C.E. 1997. Analysis of variation in results of CD34+ hematopoietic progenitor cell enumeration in a multicenter study. Cytometry 30:109‐117.
   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. (for the European Working Group on Clinical Cell Analysis). 1998. Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. Cytometry 34:128‐142.
   Gratama, J.W., Menéndez, P., Kraan, J., and Orfao, A. 2000. Loss of CD34+ hematopoietic progenitor cells due to washing can be reduced by the use of fixative‐free erythrocyte lysing reagents. J. Immunol. Methods 239:13‐23.
   Gutensohn, K., Carrero, I., Krueger, W., Kroeger, N., Schafer, P, and Luedemann, K. 1999. Semi‐automated flow cytometric analysis of CD34‐expressing hematopoietic cells in peripheral blood progenitor cell apheresis products. Transfusion 39:1220.
   Johnsen, H.E. and Knudsen, L.M. (for the Nordic Stem Cell Laboratory Group) 1996. Nordic flow cytometry standards for CD34+ cell enumeration in blood and leukapheresis products: Report from the second Nordic workshop. J. Hematother. 5:237‐245.
   Keeney, M., Chin‐Yee, I., Weir, K., Popma, J., Nayar, R., and Sutherland, D.R. 1998a. Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. Cytometry 34:61‐70.
   Keeney, M., Gratama, J.W., Chin‐Yee, I.H., and Sutherland, D.R. 1998b. Isotype controls in the analysis of lymphocytes and CD34+ stem/progenitor cells by flow cytometry—time to let go! Cytometry 34:280‐283.
   Owens, M.A. and Loken, M.R. 1995. Peripheral blood stem cell quantitation. In Flow Cytometric Principles for Clinical Laboratory Practice, pp. 111‐128. Wiley‐Liss, New York.
   Siena, S., Bregni, M., Brando, B., Ravagnani, F., Bonadonna, G., and Gianni, A.M. 1989. Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high‐dose cyclophosphamide‐treated patients: Enhancement by intravenous recombinant human granulocyte‐macrophage colony‐stimulating factor. Blood 74:1905‐1914.
   Siena, S., Bregni, M., Brando, B., Belli, N., Ravagnani, F., Gandola, L., Stern, A.C., Lansdorp, P.M., Bonadonna, G., and Gianni, A.M. 1991. Flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients. Blood 77:400‐409.
   Stewart, A.K., Imrie, K., Keating, A., Anania, S., Nayar, R., and Sutherland, D.R. 1995. Optimizing the CD34+,Thy‐1+ stem cell content of peripheral blood collections. Exp. Hematol. 23:1619‐1627.
   Sutherland, D.R. and Keating, A. 1992. The CD34 antigen: Structure, biology and potential clinical applications. J. Hematother. 1:115‐129.
   Sutherland, D.R., Keating, A., Nayar, R., Anania, S., and Stewart, A.K. 1994. Sensitive detection and enumeration of CD34+ cells in peripheral and cord blood by flow cytometry. Exp. Hematol. 22:1003‐1010.
   Sutherland, D.R., Anderson, L., Keeney, M., Nayar, R., and Chin‐Yee, I. 1996a. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. J. Hematother. 5:213‐226.
   Sutherland, D.R., Anderson, L., Keeney, M., Nayar, R., and Chin‐Yee, I. 1996b. QBEnd10 (CD34) antibody is unsuitable for routine use in the ISHAGE CD34+ cell determination assay. J. Hematother. 5:601‐603.
   Sutherland, D.R., Anderson, L., Keeney, M., Nayar, R., and Chin‐Yee, I. 1997. Re: Toward a worldwide standard for CD34+ enumeration (letter). J. Hematother. 6:85‐89.
   Terstappen, L.W.M.M., Huang, S., Safford, M., Lansdorp, P.M., and Loken, M.R. 1991. Sequential generations of hematopoietic colonies derived from single nonlineage‐committed CD34+CD38− progenitor cells. Blood 77:1218‐1227.
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