Thank you everyone for pointing out my error. Our lab does use 8G12 (HPCA-2) to check the purity of our Miltenyi human 34 selections. -- Paul Weiss Flow Cytometry Specialist University of Illinois at Chicago 900 S Ashland Ave Chicago, IL 60607 Phone: 312-355-0123 Fax: 312-413-7963 pweiss1@uic.edu > From: "D. Robert Sutherland" <rob.sutherland@utoronto.ca> > Date: Tue, 23 Oct 2001 00:46:52 +0100 > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > Subject: Re: Miltenyi magnetic beads and CD34+ > > > 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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