Hi Denice Hong and Paul Weiss, Before specifically addressing the original questions, it is important to note that the CD34 antibody used in the Miltenyi device is the class II epitope-specific monoclonal antibody QBEnd10 and NOT (as Paul suggested) the class III epitope-specific antibody 8G12 (commercially known as 'HPCA2' from BD Biosystems). The QBEnd10 epitope is located at the far N-terminus of the 'flag-pole-like' CD34 molecule. This epitope is composed of a linear stretch of 6 to 8 amino acid residues and as such is expressed on all glycoforms of the mucin-like CD34 glycoprotein. Again, in contrast to Paul's suggestion, it is NOT possible to assess the purity of the selected CD34+ cells with 'HPCA-1'. There are several reasons for this: First, HPCA-1 (aka clone My10), detects a class I (sialic acid-dependent) epitope (also in the far N-terminus) on CD34. As such, it probably cannot detect all glycoforms of the mucin-like CD34 molecule on all cells that express them. Furthermore, since My10's binding to CD34 depends at least in part on the presence of specific negatively charged sialic acid residues, its conjugation with similarly negatively charged fluorochromes such as FITC efffectively destroys its already weak binding affinity for its epitope. Additionally, the class II epitope is 'nested' within the class 1 sialic acid dependent epitopes at the far N-terminus of the CD34 molecule and even if it were possible to conjugate the class 1 antibodies such as My10 with large (non-charged) fluorochromes such as phycoerythrin, the presence of QBEnd10 (and paramagnetic beads) sterically hinder the binding of large macromolecular complexes such as antibody:PE conjugates. So, what can be used? Class III epitope-specific antibodies are used since they detect a 'common' non-glycosylation-dependent epitope of CD34. The class III epitope(s) are sufficiently spatially separated from the class I and class II epitopes that 'orbital steering' (the Lunar equivalent of steric hinderance) is not a problem. Furthermore, they can be conjugated with a variety of fluorochromes without loss of high avidity binding. Recommended reagents include clones 581 (from Beckman-Coulter) and HPCA-2. These issues have been detailed is several publications (The CD34 antigen: Structure, biology and potential clinical applications. J. Hematotherapy, 1: 115-129, 1992, The ISHAGE Guidelines For CD34+ Cell Determination By Flow Cytometry. J. Hematotherapy 3: 213-226, 1996, Structural and functional features of the CD34 antigen: an update. Journal of Biological Regulators and Homeostatic Agents 15: 1-13, 2001). We have performed several flow analyses on CD34+ cells purified by the Miltenyi device and as Paul indicated, the presence of the paramagnetic microbeads on the selected cells does not affect the light scatter properties (at least) of the cells. Indeed, the Miltenyi beads are so small that they can only be visualised (I am told) by electron microscopy. In our experience, we regularly see in excess of 97% purity of CD34+ cells with this device. Since we are using this device in haplo-identical allo-transplant studies, we also interested in enumerating the residual CD3+ T cells in the selected samples. To do this we perform simultaneous absolute CD34+ cell and CD3+ cell enumeration in a single tube using a variation of the single platform ISHAGE/CPC protocol (Enumeration of CD34+ Hematopoietic Stem and Progenitor Cells. Current Protocols in Cytometry Unit 6.4.1-6.4.22, 199). Our experience indicates that very few CD3+ cells are found in the CD34+ fraction; indeed data thus far shows that considerably less than 10e5 CD3+ cells/Kg patient weight end up in the graft! Examples (using either Beckman-Coulter or BD Biosystems instruments) of how these analyses are performed using Beckman-Coulter's Stem-Kit (CD45FITC, CD34PE and Flow Count beads) supplemented with CD3PE:Cy5 can be found at the ISHAGE web-site (www.ishage.org/committees/Committees/Graft_Evaluation/graft.htm). A few final words of caution; if you hope to obtain any reliable flow data whatsoever on CD34+ cells selected with the Miltenyi device, it is very important to keep the selected CD34+ cell fraction cold (on ice) from the moment it is eluted from the column, through the staining proceedure and until the sample is anlaysed by flow. Failure to follow these simple guidelines will almost certainly result in the generation of large aggregates of CD34+ cells, consequent to the upregulation of adhesion molecules on the CD34+ cells. The latter in turn is due to the fact that class II (and class I) CD34 antibodies stimulate a metabolism-dependent upregulation of adhesion molecule expression. The presence of aggregates destroys the ability of any flow method, whether based upon 'two platform' (%CD34+ from cytometry multiplied by the absolute leukocyte count from a hematology analyser) or 'single platform' (bead-based absolute CD34+ cell counting) to accurately enumerate the selected cells. Because of aggregates, viability assessments are also futile since for example, a single dead cell within an aggregate of cells will result in all cells in the aggregate being excluded from analysis. However, in the allotransplant setting, since the residual CD3+ cells do not get trapped in the CD34+ cell aggregates, it is possible, using single platform methods, to accurately assess the absolute CD3+ cell content in the graft. I hope this helps. D. Robert Sutherland, Department of Medical Oncology and Hematology Princess Margaret Hospital, The University Health Network, Toronto.
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