title: Surface antigen expression on CD34+ cord blood cells: comparative analysis by flow cytometry and limiting dilution (LD) RT-PCR of chymopapain-treated or untreated cells.
abstract: Expression of antigens coexpressed on cord blood (CB) CD34+ cells was evaluated by flow cytometry analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Antigen expression was also comparatively analyzed by flow cytometry and limiting dilution (LD) RT-PCR to investigate effects of chymopapain on epitopes of several cell surface markers: LD RT-PCR allows detection of the expression of antigens degraded by chymopapain which are not identified by flow cytometry. Monoclonal antibodies (MoAbs) that recognize chymopapain resistant epitopes on several coexpressed cell surface markers were identified: these included MoAbs directed against CD11a, CD13, CD18, CD38, CD45RO, CD51, HLA-DR, Thy-1, c-kit, flt-3 (STK-1), and mdr-1. Interestingly, chymopapain treatment caused enhanced staining with MoAbs against HLA-DR, Thy-1, flt-3, mdr-1, and CD51. The frequency (LD RT-PCR) of CD18, CD38, Thy-1, and c-kit RT-PCR signals on pure sorted CD34+ CD18-, CD34+ CD38-, CD34+ Thy-1-, and CD34+ c-kit- cells, respectively, was similar in corresponding subsets treated or not with chymopapain. In contrast, the frequency of CD33 RT-PCR signals on sorted CD34+ CD33- cells was higher in chymopapain-treated samples than in untreated samples and thus confirmed at the transcriptional level that the epitope recognized by anti-CD33 is chymopapain sensitive. Our findings extend data on the phenotypic profile of CB CD34+ cells and show that several key cell surface markers of hematopoietic progenitor cells are chymopapain resistant. In addition, the results of the present study demonstrate that the RT-PCR can be applied to the analysis of multiple RNA species in small numbers of hematopoietic progenitor cells and show that LD RT-PCR allows the identification and frequency determination of rare cells which are undetectable by flow cytometry.
authors.affiliation: Department of Medicine, Eberhard-Karls-University Tübingen, Germany.
authors.forename: B L
authors.initials: BL
authors.lastname: Ziegler
authors.forename: S J
authors.initials: SJ
authors.lastname: Thoma
authors.forename: C P
authors.initials: CP
authors.lastname: Lamping
authors.forename: M
authors.initials: M
authors.lastname: Valtieri
authors.forename: R
authors.initials: R
authors.lastname: Müller
authors.forename: P
authors.initials: P
authors.lastname: Samoggia
authors.forename: H J
authors.initials: HJ
authors.lastname: Bühring
authors.forename: C
authors.initials: C
authors.lastname: Peschle
authors.forename: T M
authors.initials: TM
authors.lastname: Fliedner
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D018952:Antigens, CD34 / Q000032:analysis* / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects*; D002469:Cell Separation / Q000379:methods*; D002917:Chymopapain / Q000494:pharmacology*; D003114:Colony-Forming Units Assay; D000939:Epitopes / Q000187:drug effects / Q000276:immunology; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007231:Infant, Newborn; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D000939:Epitopes; D002917:Chymopapain