Identification and characterisation of malignant cells using RT-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 immunoglobulin heavy chain (IgH) gene as clonal marker. All plasma cells with the phenotype CD38++/CD45RA- expressed the clonal marker, whereas it could not be detected in plasma cells with the phenotype CD38++/CD45RA+. A minor population of clonal 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 autograft from a patient with chronic myeloid leukemia (CML), was determined. The number of cells expressing BCR/ABL mRNA was nearly equal in the CD34+/CD38+ 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.
Rasmussen
T
T
Department of Haematology L, Herlev Hospital, University of Copenhagen, Denmark. thomas.r@inet.uni-c.dk
Honoré
L
L
Johnsen
H E
HE
D017931:DNA Primers; D005434:Flow Cytometry / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics*; D009101:Multiple Myeloma / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D017931:DNA Primers