CD19 column preselection increases sensitivity of lymphoma cell detection by pcr for the t(14;18) breakpoint

The t(14;18), which disregulates bcl-2, is found in 85% of patients with follic ular lymphoma and 35% of patients with diffuse large cell lymphoma. Nested PCR can reliably detect cells harboring the t(14;18) at a concentration of one tumor cell in 104 normal cells. The sensitivity of PCR-based detection methods is limited by the decreased efficiency of PCR as the amount of genomic DNA in the reaction increases above 1.5μg. 1.5μg of DNA represents aproximately 2.5 X 105 cells; therefore, if the ratio of tumor cells to normal cells is 1: 106, the probability of representing one or more tumor cells in the 1.5/ig of DNA is 22%. We have found that the sensitivity of nested PCR for reliable detection of B-cell lymphomas that contain the t(14;18) can be increased through an initial column preselection to concentrate CD 19+ cells. To mimic tumor cell contamination in peripheral blood, we mixed H₂ cells [a non-Hodgkin's lymphoma cell line with the t(14;18) major break point cluster region] with normal white blood cells obtained through leukapheresis. The ratios of H₂ cells to normal cells ranged from 103 to 10s. From half of each cell mixture, CD19+ cells were selected using a column containing CD19 monoclonal antibody conjugated to magnetic beads. H₂ cells, like other Bcell lymphomas, are CD19+, but only 1.6% of the normal white blood cells were CD19+. The t(14;18) breakpoint was detected unambiguously by nested PCR after CD19 selection for all H₂:normal cell ratios to a concentration of 1: 106. For non-selected cell mixtures, the t(14;18) breakpoint was not detected reliably by nested PCR for H₂:normal cell ratios lower than 104.