title: Cytometric detection of DNA amplified with fluorescent primers: applications to analysis of clonal bcl-2 and IgH gene rearrangements in malignant lymphomas.
abstract: Follicular lymphomas comprise almost two thirds of the US adult non-Hodgkin's lymphomas (NHL) and are the most common malignancy of B-lineage lymphocytes. Polymerase chain reaction (PCR) protocols have been developed to detect the t(14;18) translocation, which juxtaposes the bcl-2 proto-oncogene to the Ig heavy-chain (IgH) gene in 85% of follicular lymphomas and monoclonal rearrangements of the IgH gene in B-cell NHL that lack bcl-2 rearrangements. We used PCR to amplify bcl-2 and IgH rearrangements in DNA from patients with lymphoproliferative disorders and analyzed the products in parallel by gel electrophoresis and flow cytometry, which detected PCR products incorporating fluoresceinated oligonucleotide primers by sequence-specific capture to oligonucleotide-coated magnetic beads. Overall, flow cytometry was superior to electrophoresis of ethidium-bromide-stained agarose gels for detection of products of nested PCR to detect intergenic rearrangements involving bcl-2 and single primer-pair amplification of clonal rearrangement of IgH. Flow cytometric analysis detected bcl-2 translocations in 12 of 13 CD10+ B-cell lymphomas and clonal IgH rearrangements in 14 of 17 monoclonal B-cell populations. In contrast, analysis by gel electrophoresis detected bcl-2 translocations in only 10 of 13 CD10+ and clonal IgH gene rearrangements in only 9 of 17 monoclonal B-cell populations. Flow cytometric analysis was more sensitive than gel electrophoresis and could detect a 16-fold greater dilution of a bcl-2-amplified product than gel electrophoresis. Similarly, flow cytometry could detect an amplification product when template DNA was diluted 10,000-fold, whereas gel electrophoresis only detected amplification products when template was subjected to dilution between 100- and 1,000-fold. This shows the utility of flow cytometry for the analysis of DNA amplification products incorporating fluorochrome-labeled primers as a rapid, objective alternative to conventional strategies. Because current-generation clinical laboratories emphasize automation, flow cytometric analysis of PCR-amplified products shows increased analytic sensitivity and offers a vehicle for automation of DNA amplification tests.
authors.affiliation: Department of Pathology, University of Louisville, KY 40202.
authors.forename: R L
authors.initials: RL
authors.lastname: Barker
authors.forename: C A
authors.initials: CA
authors.lastname: Worth
authors.forename: S C
authors.initials: SC
authors.lastname: Peiper
mesh_terms: D001483:Base Sequence; D001853:Bone Marrow / Q000276:immunology; D002460:Cell Line; D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D016130:Immunophenotyping; D016393:Lymphoma, B-Cell / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D010920:Placenta / Q000276:immunology; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2; D011519:Proto-Oncogenes; D014178:Translocation, Genetic
chemical_list: D017931:DNA Primers; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D007143:Immunoglobulin Heavy Chains; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011505:Protein-Tyrosine Kinases