Identification and characterisation of malignant cells using PT-PCR on single flow-sorted cells

Abstract

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.