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Chapter 21 : Standardized Flow Cytometry Assays for Enumerating Hematopoietic Stem Cells
Recent studies have indicated that umbilical cord blood (CB) represents a rich source of CD34+ hematopoietic stem/progenitor cells, and consequently, there has been a proliferation of CB banks worldwide. Currently, the enumeration of CD34+ stem/progenitor cells by flow cytometry represents the most clinically useful surrogate marker of graft adequacy and provides crucial information to the transplant physician. While flow cytometric enumeration of CD34+ cells represents the most clinically useful assay of graft assessment, the assays that were initially developed were insufficiently robust, and interinstitutional variability in particular was problematic. An increasing array of ex vivo manipulations has been developed to engineer the graft to suit specific clinical requirements. Included in the latter are positive selection techniques to purify CD34+ cells and negative purging techniques to remove residual tumor cells in the autologous setting, or T lymphocytes in the allogeneic setting. Finally, there is widespread interest in the development of clinically useful ex vivo expansion methodologies and gene therapy protocols. At present, the authors feel that the methodologies based on the original International Society of Hematotherapy and Graft Engineering (ISHAGE) protocol and updated in Current Protocols in Cytometry (CPC) represent the most accurate and flexible protocols currently available to both clinical and research laboratories for the analysis of the increasingly wide variety of normal and abnormal hematopoietic samples.
Enumeration of CD34+ cells in a CB sample stained with CD34-PE and CD45-FITC (clones 581 and J33, respectively; Immunotech-Coulter) using the basic ISHAGE protocol ( 31 ). Plot A is gated on all events, plot B is gated on R1 events, plot C is gated on R1 and R2 events, and plot D is gated on R1, R2, and R3 events. Plot E is gated on all events, and plot F is gated lymphocytes back-scattered from R5 (plot A) to ensure optimal placement of lymphblast region R4 as described previously ( 31 ). Plots G and H show the same sample stained with isotype control IgG1-PE and CD45-FITC. Only two plots (equivalent to plots C and D) are shown for the control sample. No events satisfying the gating criteria of plots A to D are found in R4 of plot H. Gate statistics are obtained from plot B (CD45+ events).
Absolute viable CD34+-cell counting using the single-platform ISHAGE protocol ( 14 , 20 , 33 ) performed on 100 μl of a 24-h-old PBSC sample stained with CD34-FITC and CD45-PE. After 25 min, the sample was lysed with 2 ml of NH4Cl containing 1 μg of 7-AAD. After 10 min at room temperature, 100 μl of Flow-Count beads was added and the sample was analyzed immediately as described previously ( 14 , 20 ). Dead cells (7-AAD+) gated in R8 and were removed from analysis by logical gating as depicted in plots B, C, D, and F. A total of 456 viable CD34+ cells were counted in gate 4 (not R8 and R1 through R4), 4,082 beads were counted in gate 7 (R6 and R7), and the assayed bead concentration was 1,046/μl. The sample contains 117 viable CD34+ cells/μl.
Importance of removing dead cells in accurate enumeration of CD34+ cells by the single-platform ISHAGE protocol. The list mode file from the same 24-h-old sample as shown in Fig. 2 was analyzed without prior removal of nonviable (7-AAD+) cells. Note that most nonviable CD34+ cells and nonviable lymphocytes (plots D and F, respectively) form a second cluster characterized by lower FSC compared to their viable equivalents shown on the corresponding plots of Fig. 2 . The sample contains 162 total CD34+ cells/μl.
Absolute viable CD34+-cell counting with the ISHAGE single-platform protocol using TruCOUNT tubes. Fifty microliters of a fresh peripheral blood sample was stained with CD45-FITC and CD34-PE in a TruCOUNT tube. A threshold was established on FL1 (CD45-FITC) because the size of the TruCOUNT beads precludes the use of an FSC threshold. The total number of beads in the list mode file is obtained from gate 7 (R6 and R7). Gate statistics are obtained from plot A (all events). The sample contains 205 viable CD34+ cells/μl.
Identifying CD34+-cell subsets using the CPC support protocol ( 33 ) and PBSC sample stained with CD34-FITC and CD45-PE-Cy5. CD34+ cells (1.01% of the gated CD45+ events) were identified as described for Fig. 1 , plots A to D (plots A and B not shown), and displayed on CD34 versus FL2 to establish positive cell analysis R5 (plot E). Plot F displays gated lymphocytes (from plot B) on a CD45-versus-FL2 plot. Note that the back-scattered lymphocytes have autofluorescence similar to that of gated CD34+ cells (plot E) and cluster parallel with the horizontal axis, indicating optimized FL2/FL3 fluorescence compensation. Plots G and H show the staining of CD34+ cells and lymphocytes, respectively, with CD90/Thy-1-PE. Plots J and K show the staining of the CD34+ cells with CD133-PE and CD33-PE, respectively.
Identifying CD34+-cell subsets in the marrow of a poor mobilizer. CD34+ cells (2.00% of the total CD45+ events in G4) are identified in plots A to D (A and B not shown). 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 was used to establish gating R5 (plot E). Plots F, G, H, and J show the staining of gated CD34+ cells with CD90/Thy-1, AC133, CD38, and CD33, respectively. The lower right plot shows the light scatter of the CD34+ CD33- cells.