SURFACE-ANTIGEN EXPRESSION ON CD34(-BLOOD CELLS - COMPARATIVE-ANALYSIS BY FLOW-CYTOMETRY AND LIMITING DILUTION (LD) RT-PCR OF CHYMOPAPAIN-TREATED OR UNTREATED CELLS() CORD)

Expression of antigens coexpressed on cord blood (CB) CD34(+) cells wa s evaluated by flow cytometry analysis and reverse transcriptase polym erase chain reaction (RT-PCR). Antigen expression was also comparative ly analyzed by flow cytometry and limiting dilution (LD) RT-PCR to inv estigate effects of chymopapain on epitopes of several cell surface ma rkers: LD RT-PCR allows detection of the expression of antigens degrad ed by chymopapain which are not identified by flow cytometry. Monoclon al antibodies (MoAbs) that recognize chymopapain resistant epitopes on several coexpressed cell surface markers were identified: these inclu ded MoAbs directed against CD11a, CD13, CD18, CD38, CD45R0, CD51, HLA- DR, Thy-1, c-kit, flt-3 (STK-1), and mdr-l. Interestingly, chymopapain treatment caused enhanced staining with MoAbs against HLA-DR, Thy-1, flt-3, mdr-l, and CD51. The frequency (LD RT-PCR) of CD18, CD38, Thy-1 , and c-kit RT-PCR signals on pure sorted CD34(+) CD18(-), CD34(+) CD3 8(-), CD34(+) Thy-1(-), and CD34(+) c-kit(-) cells, respectively, was similar in correspending subsets treated or not with chymopapain. In c ontrast, the frequency of CD33 RT-PCR signals on sorted CD34(+)CD33(-) cells was higher in chymopapain-treated samples than in untreated sam ples 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 sever al key cell surface markers of hematopoietic progenitor cells are chym opapain resistant. In addition, the results of the present study demon strate that the RT-PCR can be applied to the analysis of multiple RNA species in small numbers of hematopoietic progenitor cells and show th at LD RT-PCR allows the identification and frequency determination of rare cells which are undetectable by flow cytometry. (C) 1996 Wiley-Li ss, Inc.