Identification and characterisation of malignant cells using PT-PCR on single flow-sorted cells
In an attempt to optimise stem cell graft evaluation we have developed a method of quantifying the number of cells in a phenotypically defined population of cells, expressing a gene of interest by combining an RT-PCR method working on whole single cells with flow cytometry. The clinical potential is illustrated by two examples. First, the phenotypes of clonal cells in the bone marrow (BM) of a patient with multiple myeloma (MM), were determined by sorting cells phenotypically defined by their expression of surface antigens and then performing RT-PCR on the individual sorted cells using the rearranged immunogiobulin heavy chain (IgH) gene as clonai marker. All plasma cells with the phenotype CD38++/CD45RA-expressed the clonai marker, whereas it could not be detected in plasma cells with the phenotype CD38++/CD45RA+ A minor population of clonai cells with the CD38+CD45RA- phenotype was found. Second, the number of committed (CD34+/CD38+) and non-committed (CD34+/CD38-) stem cells, expressing the chimeric fusion gene p210 BCR/ABL in the autografi from a patient with chronic myeloid leukemia (CML), was determined. The number of cells expressing BCR/ABL mRNA was nearly equal in the CD34VCD38+ and CD34+CD38- compartment (8.1 and 8.5%). The method presented can easily be applied to determine the phenotype of malignant cells, where a unique mRNA species exist. Furthermore, the method allows one to predict the outcome of antibody mediated purging experiment.
Keywordssingle cells RT-PCR IgH gene rearrangements BCR/ABL multiple myeloma chronic myeloid leukaemia
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- 4.Rasmussen T, Johnsen HE. A quantitative analysis for CD34 mRNA+ haematopoietic stem cells.Int J Hematol 1996;64: Supplement 1, 28 (Abstract).Google Scholar
- 7.Sambrook J, Fritch EF, Maniatis T.Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York, 1989.Google Scholar
- 10.Sanz I. Multiple mechanisms participate in the generation of diversity of human H chain CDR3 regions. Jtmmunol 1991;147:17200.Google Scholar
- 11.Hoppe BL, Conti-Tronconi BM, Horton RM. Gel-loading dyes compatible with PCR.Biotechniques 1996;12:679–6800.Google Scholar