1887

Chapter 19 : Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $30.00

Preview this chapter:
Zoom in
Zoomout

Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555818722/9781555818715_CH19-1.gif /docserver/preview/fulltext/10.1128/9781555818722/9781555818715_CH19-2.gif

Abstract:

The pioneering studies of Thomas et al. in the late 1950s first established that a cellular component of syngeneic bone marrow was capable of regenerating multilineage hematopoiesis in cancer patients receiving supralethal doses of radiation (1, 2). For the next 20 or more years, the majority of autologous and allogeneic hematopoietic stem/progenitor cell transplants were performed utilizing bone marrow as a source of stem cells (reviewed in reference 3). Circulating stem cells were also detectable in steady-state peripheral blood but were extremely rare, as evidenced by their low plating efficiency relative to marrow leukocytes in early colony-forming cell assays (4). Although blood stem/progenitor cells could be collected from steady-state peripheral blood by leukapheresis, the number of procedures required to obtain sufficient cells for transplant initially precluded their widespread use (5). With the development of more sophisticated colony-forming cell assays, a variety of reports in the mid-1980s clearly demonstrated the feasibility of obtaining clinically useful numbers of peripheral blood stem/progenitor cells from cancer patients recovering from chemotherapy (6–8). The availability of a number of hematopoietic cytokines used either singly or in combination with other cytokines and/or chemotherapy (9) has facilitated the harvesting of peripheral blood stem cells (PBSC) to the point where it is a preferred alternative to marrow for autologous and, increasingly, allogeneic transplantation (reviewed in reference 10).

Citation: Sutherland D, Keeney M. 2016. Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, p 182-198. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch19
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

Figures

Image of FIGURE 1
FIGURE 1

Enumeration of viable CD34 cells with the single-platform ISHAGE protocol (Stem-Kit) on a FACSCalibur cytometer equipped with CellQuest Pro, using 100 μl of a 24-hour-old PBSC sample diluted 1/10 with phosphate-buffered saline–1% bovine serum albumin and stained with CD34-FITC, CD45-PE, and 7-AAD (Stem-Kit) according to the manufacturer's instructions. After 25 min, the sample was lysed with 2 ml fresh NHCl. After 10 min at room temperature, 100 μl of Flow-count beads was added and the sample was immediately analyzed on the FACSCalibur cytometer running Cellquest Pro 5.2, as described previously ( ). Viable cells (7-AAD-) were gated in R8 of plots 8 and 9; only viable cells were analyzed by Boolean gating, as depicted in plots 2, 3, 4, and 6. The extra viability plot (plot 8) showing total CD34 cells (viable and nonviable) can be useful in samples containing large numbers of dead cells. This plot also confirms appropriate compensation between FL2 and FL3 PMTs, with the viable CD34 cells clearly visible and properly on-scale and dead cells excluded by R8. Viable lymphocytes (plot 6) are gated through R8 and R5 to set the lymph-blast region R4. As shown in the lower left quadrant of plot 5, the gate is set as a ‘live’ or ‘not’ gate prior to acquisition such that debris in this area resulting from the lyse–no-wash sample processing is excluded from the acquired list-mode file. Total beads are gated in R6 of plot 5 and singlet beads are identified in R7 of plot 7 (FSC versus time). A forward-scatter threshold or discriminator is set below the light scatter of the singlet beads so as to not exclude any beads from the data file (see plot 7). A total of 732 viable CD34 cells were counted in gate 4 (R8 and R1 through R4); 4,651 single beads were counted in gate 5 (R6 and R7), and the assayed bead concentration was 996/μl. Using expression editors, the sample contained 157 viable CD34 cells/μl and 14,212 viable CD45 cells/μl. CD34 cell viability was 54.46%.

Citation: Sutherland D, Keeney M. 2016. Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, p 182-198. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch19
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 2
FIGURE 2

Importance of viability dye (7-AAD) inclusion in the analysis of non-fresh samples. Analysis of the same sample as in Fig. 1 , except that viability discrimination with 7-AAD was NOT applied. When gating region R8 is expanded to include both viable and nonviable cells (plots 8 and 9) and region R4 is moved to include both live and dead lymphocytes (plot 6), both dead and live CD34 cells now cluster within the duplicate lymph-blast region R4 on plot 4. Both the absolute CD34 and CD45 counts are significantly increased versus the values obtained in Fig. 1 . Note the extra population of both CD34 cells (plot 4) and lymphocytes (plot 6) with reduced forward-angle light scatter; these are the dead CD34 and dead lymphocytes, respectively. Sample contains 286 total (viable + nonviable) CD34 cells/μl.

Citation: Sutherland D, Keeney M. 2016. Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, p 182-198. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch19
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 3
FIGURE 3

Enumeration of viable CD34 cells with Stem-Kit and Automated STEM-CXP software analysis on a Beckman-Coulter FC500. Fresh apheresis sample diluted 1/10 with phosphate-buffered saline–1% bovine serum albumin. Plot 1: all CD45 leukocytes, gated on 7-AAD negative (viable) events from histogram 8. Plots 2–4 are sequentially Boolean gated from plot 1. Plot 6 is gated on viable lymphocytes from plot 1 to allow the discriminator or threshold to be set on forward scatter. Total beads are gated as shown in plot 5 and Boolean gated on FL3 (stem count) versus time (plot 7) to enumerate singlets. A live gate in the bottom left corner of plot 5 is used to exclude debris from the list-mode file, as described for Fig. 1 . The leukocyte count (CD45, from plot 1) was 23.3 × 10/liter, and the absolute viable CD34 count was 23/μl (from plot 4). The overall CD45 viability was 95.1% (from plot 8).

Citation: Sutherland D, Keeney M. 2016. Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, p 182-198. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch19
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 4
FIGURE 4

Absolute viable CD34 cell counting with the ISHAGE single-platform protocol using TruCOUNT tubes and BD FACSCanto II. We used 100 μl of a fresh PB sample stained with CD45-FITC, CD34-PE, and 7-AAD in a TruCOUNT tube (SCE Kit). A threshold was established on FL-1 (CD45-FITC, plot 1) because the size of the TruCOUNT beads precludes the use of an FSC threshold. A debris-exclusion gate (R7) is also necessary to remove fluorescent debris from the list-mode file (debris visualized in lower left of R1). This allows an accurate determination of absolute CD45 cells to be made. The total number of beads in the list-mode file is obtained from R6 of plot 5 (gate G5 = R6). An alternative means of enumerating the beads is shown in plot 9. This plot can be useful if aggregates of CD34 cells are present or if other debris contaminates R6 such that the beads cannot be accurately delineated, as can be the case in some CD34-selected samples or in samples containing large amounts of platelet aggregates. Other aspects of the analysis were performed as for Fig. 1 . Canto II FCS3 data were exported as FCS2 files and analysis was performed using Cellquest Pro 6. Gate statistics were obtained from plot 1 (all events). The sample contained 113 viable CD34 cells/μl and 37,170 viable CD45 cells/μl.

Citation: Sutherland D, Keeney M. 2016. Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, p 182-198. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch19
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 5
FIGURE 5

Absolute CD34 cell counting in stabilized PB sample with the ISHAGE single-platform protocol using TruCOUNT tubes and BD FACSCanto II. We used 100 μl of a stabilized whole-PB sample stained with CD45-FITC, CD34-PE, and 7-AAD in a TruCOUNT tube (SCE Kit). A threshold was established on FL-1 (CD45-FITC, plot 1) as was a debris-exclusion gate (R7) as described in Fig. 4 . For analysis of stabilized (i.e., dead) samples, the viability gate P8 (plots 7 and 8) is fully opened so as to not exclude any cells (viable or nonviable) from analysis. The rest of the ISHAGE gating strategy is applied as shown in Fig. 4 .

Citation: Sutherland D, Keeney M. 2016. Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, p 182-198. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch19
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 6
FIGURE 6

Simultaneous detection of absolute viable CD34 and CD3 cells using single-platform ISHAGE protocol in the allotransplant setting. PBSC sample for allotransplant stained with CD45-FITC, CD34-PE, 7-AAD, and CD3-APC. CD34 cells enumerated through Boolean gates R8 and R1 through R4 as in Fig. 4 . CD3 cells enumerated through Boolean gates R10 (plot 9) and duplicate R4 (plot 6). TruCOUNT beads enumerated through gate R6 (plot 5) or alternatively with R9 (plot 9). Sample contained 39 viable CD34 cells/μl, 2,836 viable CD3 cells/μl, and 11,332 viable CD45 cells/μl.

Citation: Sutherland D, Keeney M. 2016. Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, p 182-198. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch19
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 7
FIGURE 7

Identifying CD34 cell subsets using the CPC Support Protocol ( ). PBSC sample stained with CD34-FITC and CD45–PE-Cy5. CD34 cells (1.01% of the gated CD45 events) were identified as described in Fig. 1 , plots 1–4, and displayed on CD34 versus FL2 to establish positive cell analysis region R5 (plot 1). Lymphocytes from the same list-mode file were also gated as described in Fig. 1 and displayed (plot 4) on a duplicate CD45 versus FL2 plot. Note the back-scattered lymphocytes (plot 4) have similar auto-fluorescence as gated CD34 cells (plot 1) and cluster parallel with horizontal axis, indicating optimized FL2/FL3 fluorescence compensation. Plots 2 and 5 show the staining of CD34 and lymphocytes, respectively, with CD90/Thy–1-PE. Plot 3 shows the light-scatter characteristics of the CD34 and CD90 cells. Plots 6 and 7 show the staining of the CD34 cells with CD133-PE and CD33-PE, respectively.

Citation: Sutherland D, Keeney M. 2016. Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, p 182-198. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch19
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 8
FIGURE 8

Identifying CD34 cell subsets in the marrow of a poor mobilizer. CD34 cells (2.00% of the total CD45 events) were identified as shown in plots 1–4 of Fig. 1 . Plots 1 and 2 show the equivalent of plots 3 and 4 from Fig. 1 . The majority of CD34 cells exhibit light-scatter characteristics of prelymphoid cells. An unstained control (no PE conjugate) of the gated CD34 cells from R4 (plot 2) was used to establish gating regions R5 and R7 (plot 3). Plots 4, 6, 7, and 8 show the staining of gated CD34 cells with CD90/Thy-1 (plot 4), CD133 (plot 6), CD38 (plot 7), and CD33 (plot 8). The light-scatter plot (plot 5) shows the light scatter of the CD34/CD90 cells. Most of the CD34 cells exhibit staining and light-scatter characteristics of primitive B cell progenitors or hematogones. Very few CD34 cells exhibit phenotype of primitive candidate engrafting cells (CD34, CD90, CD133, CD38).

Citation: Sutherland D, Keeney M. 2016. Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, p 182-198. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch19
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555818722.ch19
1. Thomas ED, Lochte Hl Jr, Lu JF, Ferrebee JW. 1957. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Eng J Med 257:491496.[CrossRef]
2. Thomas ED, Lochte Hl Jr, Cannon JH, Sahler OD, Ferrebee JW. 1959. Supralethal whole-body irradiation and isologous marrow transplantation in man. J Clin Invest 38:7091716.
3. Buckner CD. 1999. Autologous bone marrow transplants to hematopoietic stem cell support with peripheral blood stem cells: a historical perspective. J Hematother 8:233236.[CrossRef].[PubMed]
4. McCredie KB, Hersh EM, Freireich EJ. 1971. Cells capable of colony formation in the peripheral blood of man. Science 171:293294.[PubMed].[CrossRef]
5. Korbling M, Fleidner TM, Pflieger H. 1980. Collection of large quantities of granulocyte macrophage progenitor cells (CFUc) in man by means of continuous-flow leukapheresis. Scand J Haematol 24:2228.[PubMed].[CrossRef]
6. Juttner CA, To LB, Haylock DN, Branford A, Kimber RJ. 1985. Circulating autologous stem cells collected in very early remission from acute non-lymphoblastic leukemia produce prompt but incomplete hematopoietic reconstitution after high-dose melphalan or supralethal chemoradiotherapy. Br J Haematol 61:739745.[PubMed].[CrossRef]
7. Reiffers J, Bernard P, David B, Vezon H, Sarrat A, Marit G, Moulinier J, Broustet A. 1986. Successful autologous transplantation with peripheral blood hemopoietic cells in a patient with acute leukemia. Exp Hematol 14:312315.[PubMed]
8. Korbling M, Dorken B, Ho AD, Pezzuto A, Hunstein W, Fleidner TM. 1986. Autologous transplantation of blood derived hemopoietic stem cells after myeloablative therapy in a patient with Burketts lymphoma. Blood 67:529532.[PubMed]
9. Siena S, Bregni M, Brando B, Tarella C, Stern AC, Pieri A, Bonadonna G. 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 74:19051914.[PubMed]
10. To LB, Haylock DN, Simmons PJ, Juttner CA. 1997. The biology and clinical uses of blood stem cells. Blood 89:22332258.[PubMed]
11. Civin C, Strauss LC, Brovall C. Fackler MJ, Schwartz JF, Shaper JH. 1984. Antigenic analysis of hematopoiesis III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG1a cells. J Immunol 133:157165.[PubMed]
12. Tindle RW, Nichols RAB, Chan L, Campana D, Catovsky D, Birnie GD. 1985. A novel monoclonal antibody B1-3C5 recognizes myeloblasts and non-B non-T lymphoblasts in acute leukemias and CGL blast crises, and reacts with immature cells in normal bone marrow. Leuk Res 9:19.[PubMed].[CrossRef]
13. Andrews RG, Singer JW, Bernstein ID. 1986. Monoclonal antibody 12.8 recognizes a 115-Kd molecule present on both unipotent and multipotent colony-forming cells and their precursors. Blood 67:842845.[PubMed]
14. Berenson RJ, Andrews RG, Bensinger WI, Kalamasz D, Knitter G, Buckner CD, Bernstein ID. 1988. Antigen CD34-positive marrow cells engraft lethally irradiated baboons. J Clin Invest 81:951955.[CrossRef].[PubMed]
15. Berenson RJ, Bensinger WI, Hill RS, Andrews RG, Garcia-Lopez J, Kalamaz DF, Still BJ, Spitzer G, Buckner D, Bernstein ID, Thomas ED. 1991. Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. Blood 77:17171722.[PubMed]
16. Treleaven JG, Mehta J. 1992. Bone marrow and peripheral blood stem cell harvesting. J Hematother 1:215223.[CrossRef].[PubMed]
17. Smith A, Keating A. 1994. Peripheral blood progenitor cell transplantation: clinical, technical and economic considerations. J Hematother 3:331348.[CrossRef].[PubMed]
18. Wagner JE, Broxmeyer HE, Byrd RL, Zehnbauer B, Schmeckpeper B, Shah N, Griffin C, Emanuel PD, Zuckerman S, Cooper S, Carow C, Bias W, Santos GW. 1992. Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment. Blood 79:18741881.[PubMed]
19. Cairo MS, Wagner JE. 1997. Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation. Blood 90:46654678.[PubMed]
20. Vormoor J, Lapidot T, Pflumio F, Risdon G, Patterson B, Broxmeyer HE, Dick JE. 1994. Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 83:24892497.[PubMed]
21. Wang JC, Doedens M, Dick JE. 1997. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 89:39193924.[PubMed]
22. Civin CI, Trischman T, Fackler MJ, Bernstein I, Buhring H, Campos L, Greaves MF, Kamoun M, Katz D, Lansdorp P, Look T, Seed B, Sutherland DR, Tindle R, Uchanska-Zeigler B,. 1989. Summary of CD34 cluster workshop section, p 818825. In Knapp W, Dorken B, Gilks WR, Reiber EP, Schmidt RE, Stein H, von dem Borne AEGKr (ed), Leukocyte Typing IV. Oxford University Press, Oxford, United Kingdom.
23. Greaves MF, Titley I, Colman SM, Buhring H-J, Campos L, Castoldi GL, Garrido F, Gaudernack G, Girard J-P, Ingles-Esteve J, Invernizi R, Knapp W, Lansdorp PM, Lanza F, Merle-Beral H, Parravicini C, Razak K, Ruiz-Cabello F, Springer TA, van der Schoot CE, and Sutherland DR,. 1995. Report on the CD34 cluster workshop, p 840846. In Schlossman S, Boumsell L, Gilks W, Harlan JM, Kishimoto T, Morimoto C, Ritz J, Shaw S, Silverstein R, Springer T, Tedder TF, Todd RF (ed), Leukocyte Typing V. Oxford University Press, Oxford, United Kingdom.
24. Tricot G, Jagannath S, Vesole DH, Nelson J, Tindle S, Miller L, Cheson B, Crowley J, Barlogie B. 1995. Peripheral blood stem cell transplants for multiple myeloma: identification of favorable variables for rapid engraftment in 225 patients. Blood 85:588596.[PubMed]
25. Bender JG, To LB, Williams S, Schwartzberg LS. 1992. Defining a therapeutic dose of peripheral blood stem cells. J Hematother 1:329341.[CrossRef].[PubMed]
26. Chapple P, Prince HM, Quinn M, Bertoncello I, Juneja S, Wolf M, Januszewicz H, Brettell M, Gardyn J, Seymour C, Venter D. 1998. Peripheral blood CD34+ cell count reliably predicts autograft yield. Bone Marrow Transplant 22:125130.[CrossRef].[PubMed]
27. Luider J, Brown C, Selinger S, Quinlan D, Karlsson L, Ruether D, Stewart D, Klassen J, Russell JA. 1997. Factors influencing yields of progenitor cells for allogeneic transplantation: optimization of G-CSF dose, day of collection, and duration of leukapheresis. J Hematother 6:575580.[CrossRef].[PubMed]
28. Weaver CH, Hazelton B, Birch R, Palmer P, Allen C, Schwartzberg L, West W. 1995. An analysis of engraftment kinetics as a function of CD34 cell content of peripheral blood progenitor collections in 692 patients after the administration myeloablative chemotherapy. Blood 86:39613969.[PubMed]
29. Negrin RS, Atkinson K, Leemhuis T, Hanania E, Juttner C, Tierney D, Hu WW, Johnston LJ, Shizuru JA, Stockerl-Goldstein KE, Blume KG, Weissman IL, Bower S, Baynes R, Dansey R, Karanes C, Peters W, Klein J. 2000. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant 6:262271.[PubMed].[CrossRef]
30. Siena S, Bregni M, Brando B, Belli N, Ravagnani F, Gandola L, Stern AC, Lansdorp PM, Bonadonna G, Gianni AM. 1991. Flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients. Blood 77:400409.[PubMed]
31. Bender JG, Unverzagt K, Walker D,. 1994. Guidelines for determination of CD34 + cells by flow cytometry: application to the harvesting and transplantation of peripheral blood stem cells, p 3143. In Wunder E, Sovalat H, Henon PR, Serke S (ed), Hematopoietic Stem Cells: The Mulhouse Manual. AlphaMed Press, Dayton, OH.
32. Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. 1997. re: Towards a worldwide standard for CD34 + enumeration? Response to Letter to the Editor. J Hematother 6:8589.[CrossRef]
33. Marti GE, Johnsen HE, Sutherland DR, Serke S. 1998. A convergence of methods for a worldwide standard for CD34+ cell enumeration. Letter to the Editor. J Hematother 7:105109.[CrossRef].[PubMed]
34. Keeney M, Chin-Yee I, Gratama JW, Sutherland DR. 1998. Perspectives: Isotype controls in the analysis of lymphocytes and CD34+ stem/progenitor cells by flow cytometry—time to let go! Cytometry (Comm Clin Cytometry) 34:280283.[CrossRef]
35. Hulspas R, O'Gorman MRG, Wood BL, Gratama JW, Sutherland DR. 2009. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B 76B:355364.[CrossRef]
36. Roederer M. 2001. Spectral compensation for flow cytometry: visualization artifacts, limitations and caveats. Cytometry 45:194205.[CrossRef]
37. Sutherland DR, Keating A. 1992. The CD34 antigen: structure, biology and potential clinical applications. J Hematother 1:115129.[CrossRef].[PubMed]
38. Lanza R, Healy L, Sutherland DR. 2001. Structural and functional features of the CD34 antigen: an update. J Biol Reg Homeos Ag 15:113.
39. Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. 1996. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. J Hematother 5:213226.[CrossRef].[PubMed]
40. Gratama JW, Keeney M, Sutherland DR,. 1999. Enumeration of CD34+ hematopoietic stem and progenitor cells, p 6.4.16.4.22. In Robinson JP, Darzynkiewicz Z, Dean PN, Hibbs AR, Orfao A, Rabinovitch PS, Wheeless LL (ed), Current Protocols in Cytometry. John Wiley and Sons Inc, New York, NY.
41. Sutherland DR, Keeney M, Gratama JW,. 2003. Enumeration of CD34+ hematopoietic stem and progenitor cells, p 6.4.16.4.23. In Robinson JP, Darzynkiewicz Z, Dean PH, Dressler LG, Rabinovitch PS, Stewart CS, Tanke HJ, Wheeless LL (ed), Current Protocols in Cytometry. John Wiley and Sons Inc, New York, NY.
42. Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini F. 2001. Resting and activated endothelial cells are increased in the peripheral blood of cancer patients. Blood 97:36583661.[PubMed].[CrossRef]
43. Borowitz MJ, Guenther KL, Schultz KE, Stelzer GT. 1993. Immunophenotyping 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 100:534540.[PubMed].[CrossRef]
44. Sutherland DR, Keating A, Nayar R, Anania S, Stewart AK. 1994. Sensitive detection and enumeration of CD34+ cells in peripheral blood and cord blood by flow cytometry. Exp Hematol 22:10031010.[PubMed]
45. Sutherland DR, Yeo EL, Stewart AK, Nayar R, DiGiusto R, Hoffman R, Zanjani ED, Murray LJ. 1996. Identification of CD34+ subsets following glycoprotease selection: engraftment of CD34+/Thy-1+/Lin/stem cells in fetal sheep. Exp Hematol24:795806.[PubMed]
46. Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Sutherland DR. 1998. Single-platform flow cytometric absolute CD34+ cell counts based on the ISHAGE Guidelines. Cytometry (Comm Clin Cytom) 34:6167.[CrossRef]
47. Sutherland DR, Nayyar R, Acton E, Giftakis A, Dean S, Mosiman V. 2009. Comparison of two single-platform ISHAGE-based CD34 enumeration protocols on FACSCalibur and FACSCanto cytometers. Cytotherapy 11:595605.[CrossRef].[PubMed]
48. Gratama JW, Orfao A, Barnett D, Brando B, Huber A, Janossy G, Johnsen HE, Keeney M, Preijers F, Rothe G, Serke S, Sutherland DR, Van Der Schoot E, Schmitz G, Papa S. 1998. Flow cytometric enumeration of CD34+ hematopoietic progenitor cells. Cytometry (Comm Clin Cytom) 34:128142.[CrossRef]
49. Gratama JW, Kraan J, Keeney M, Mandy F, Sutherland DR, Wood BL. 2007. Enumeration of immunologically defined cell populations by flow cytometry; approved guideline, 2nd ed. Document H42-A2 27 No.16. Clinical and Laboratory Standards Institute, Wayne, PA.
50. Sutherland DR, Keeney M,. 2009. Enumeration of CD34+ cells by flow cytometry, p 538554. In Aremen EM, Loper K (ed), Cellular Therapy: Principles, Methods and Regulations. An American Association of Blood Bankers Cell Therapy Technical Manual. American Association of Blood Bankers, Bethesda, MD.
51. Brocklebank AM, Sparrow RL. 2001. Enumeration of CD34+ cells in cord blood: a variation on a single-platform flow cytometric method based on the ISHAGE gating strategy. Cytometry 46:254261.[PubMed].[CrossRef]
52. Keeney M, Chin-Yee I, Nayar R, Sutherland DR. 1999. Effect of fixatives on CD34+ cell enumeration. J Hematother Stem Cell Res 8:327329.[CrossRef].[PubMed]
53. Chang A, Ma DDF. 1996. The influence of flow cytometric gating strategy on the standardization of CD34+ cell quantitation: an Australian multicenter study. J Hematother 5:605616.[CrossRef].[PubMed]
54. Barnett D, Granger V, Kraan J, Whitby L, Reilly JT, Papa S, Gratama JW. 2000. Reduction of intra- and interlaboratory variation in CD34+ stem cell enumeration using stable test material, standard protocols and targeted training. CD34 Task Force of the European Working Group of Clinical Cell Analysis (EWGCCA). Br J Haematol 108:784792.[PubMed].[CrossRef]
55. Gratama JW, Kraan J, Keeney M, Sutherland DR, Granger V, Barnett D. 2003. Validation of the single-platform ISHAGE method for CD34+ hematopoietic stem and progenitor cell enumeration in an international multicenter study. Cytotherapy 5:5565.[CrossRef].[PubMed]
56. Whitby A, Whitby L, Fletcher M, Reilly JT, Sutherland DR, Keeney M, Barnett D. 2012. ISHAGE Protocol: are we doing it correctly? Cytometry B 82B:917.[CrossRef]
57. Walker I, Shehata N, Cantin C, Couture F, Dhedin N, Barty R, Foley R, Sutherland DR, Sigouin C, Schultz K, Mitchell D. 2004. Canadian multicenter pilot trial of haploidentical donor transplantation. Blood Cell Mol Dis 33:222236.[CrossRef]
58. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. 1992. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci USA 89:28042808.[PubMed].[CrossRef]
59. Craig W, Kay R, Cutler RL, Lansdorp PM. 1993. Expression of Thy-1 on human hematopoietic progenitor cells. J Exp Med 177:13311242.[PubMed].[CrossRef]
60. Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, Leary AG, Olweus J, Kearney J, Buck DW. 1997. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90:50025012.[PubMed]
61. Sutherland H, Eaves C, Eaves A, Dragowska W, Landsdorp PM. 1989. Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 74:15631570.[PubMed]
62. Terstappen LWMM, Huang S, Safford M, Lansdorp PM, Loken MR. 1991. Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+ CD38 progenitor cells. Blood 77:12181227.[PubMed]
63. Negrin RS, Atkinson K, Leemhuis T, Hanania E, Juttner C, Tierney D, Hu WW, Johnston LJ, Shizuru JA, Stockerl-Goldstein KE, Blume KG, Weissman IL, Bower S, Baynes R, Dansey R, Karanes C, Peters W, Klein J. 2000. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant 6:262271.[PubMed].[CrossRef]
64. Vose JM, Bierman PJ, Lynch JC, Atkinson K, Juttner C, Hanania CE, Bociek G, Armitage JO. 2001. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with recurrent indolent non-Hodgkin's lymphoma. Biol Blood Marrow Transplant 7:680687.[PubMed].[CrossRef]
65. Michallet M, Philip T, Philip I, Godinot H, Sebban C, Salles G, Thiebaut A, Biron P, Lopez F, Mazars P, Roubi N, Leemhuis T, Hanania E, Reading C, Fine G, Atkinson K, Juttner C, Coiffier B, Fiere D, Archimbaud E. 2000. Transplantation with selected autologous peripheral blood CD34+Thy1+ hematopoietic stem cells (HSCs) in multiple myeloma: impact of HSC dose on engraftment, safety, and immune reconstitution. Exp Hematol 28:858870.[PubMed].[CrossRef]
66. Chin-Yee IH, Keeney M, Stewart AK, Belch A, Bence-Buckler I, Couban S, Howson-Jan K, Rubinger M, Stewart D, Sutherland DR, Paragamian V, Bhatia M, Foley R. 2002. Optimizing parameters for peripheral blood leukapheresis after r-metHuG-CSF (filgrastim) and r-metHuSCF (ancestim) in patients with multiple myeloma: a temporal analysis of CD34+ absolute counts and subsets. Bone Marrow Transplant 30:851860.[CrossRef].[PubMed]
67. Watts MJ, Sullivan AM, Leverett D, Peniket AJ, Perry AR, Williams CD, Devereux S, Goldstone AH, Linch DC. 1998. Back-up bone marrow is frequently ineffective in patients with poor peripheral-blood stem-cell mobilization. J Clin Oncol 16:15541560.[PubMed]
68. Shaughnessy P, Chao N, Shapiro J, Walters K, McCarty J, Abhyankar S, Shayani S, Helmons P, Leather H, Pazzalia A, Pickard S. 2013. Pharmacoeconomics of hematopoietic stem cell mobilization: an overview of current evidence and gaps in the literature. Biol Blood Marrow Transplant 19:13011309.[CrossRef].[PubMed]
69. Basak GW, Mikala G, Koristek Z, Jaksic O, Basic-Kinda S, Cegledi A, Reti M, Masszi T, Mayer J, Giebel S, Hübel K, Labar B, Wiktor-Jedrzejczak W. 2011. Identification of prognostic factors for plerixafor-based hematopoietic stem cell mobilization. Am J Hematol 86:550553.[CrossRef].[PubMed]
70. Nademanee AP, DiPersio JF, Maziarz RT, Stadtmauer EA, Micallef IN, Stiff PJ, Hsu FJ, Bridger G, Bolwell BJ. 2012. Plerixafor plus granulocyte colony-stimulating factor versus placebo plus granulocyte colony-stimulating factor for mobilization of CD34+ hematopoietic stem cells in patients with multiple myeloma and low peripheral blood CD34+ cell count: results of a subset analysis of a randomized trial. Biol Blood Marrow Transplant 18:15641572.[CrossRef].[PubMed]

Tables

Generic image for table
TABLE 1

Logical gate setup for BD and Beckman instruments

Citation: Sutherland D, Keeney M. 2016. Standardized Flow Cytometry Assays for Enumerating CD34 Hematopoietic Stem Cells, p 182-198. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch19

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error