Detection Limit of Monoclonal B-Cells Using Multiplex PCR and Laser-Induced Fluorescence Capillary Electrophoresis
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Background: The identification of monoclonality has been widely used for making diagnoses of lymphoproliferative lesions. Awareness of the sensitivity and detection limit of the technique used would be important for the data to be convincing. Methods: We investigated the minimum require¬ment of cells and sensitivity of gel electrophoresis (GE) and laser-induced fluorescence capillary electrophoresis (LFCE) for identifying IgH gene rearrangement using BIOMED-2 protocols. DNA extracted from Raji cells were diluted serially with peripheral blood mononuclear cells (PBMNCs) DNA. DNA from mixtures of diffuse large B-cell lymphoma (DLBCL) and reactive lymph nodes were also serially diluted. Results: For Raji cells, the detection limit was 62 and 16 cell-equiva¬lents for GE and LFCE, respectively. In the condition with PBMNCs mixture, 2.5% and 1.25% of clonal cells was the minimum requirement for GE and LFCE, respectively. In 23% of DLBCL cells in tissue section, the detection limit was 120 and 12 cell-equivalents for GE and LFCE, respec-tively. In 3.2% of DLBCL cells, that was 1,200 and 120 cell-equivalents for GE and LFCE, respec-tively. Conclusions: These results show that LFCE method is more sensitive than GE and the sensitivity of clonality detection can be influenced by the amount of admixed normal lymphoid cells.
Abstract
Background: The identification of monoclonality has been widely used for making diagnoses of lymphoproliferative lesions. Awareness of the sensitivity and detection limit of the technique used would be important for the data to be convincing. Methods: We investigated the minimum require¬ment of cells and sensitivity of gel electrophoresis (GE) and laser-induced fluorescence capillary electrophoresis (LFCE) for identifying IgH gene rearrangement using BIOMED-2 protocols. DNA extracted from Raji cells were diluted serially with peripheral blood mononuclear cells (PBMNCs) DNA. DNA from mixtures of diffuse large B-cell lymphoma (DLBCL) and reactive lymph nodes were also serially diluted. Results: For Raji cells, the detection limit was 62 and 16 cell-equiva¬lents for GE and LFCE, respectively. In the condition with PBMNCs mixture, 2.5% and 1.25% of clonal cells was the minimum requirement for GE and LFCE, respectively. In 23% of DLBCL cells in tissue section, the detection limit was 120 and 12 cell-equivalents for GE and LFCE, respec-tively. In 3.2% of DLBCL cells, that was 1,200 and 120 cell-equivalents for GE and LFCE, respec-tively. Conclusions: These results show that LFCE method is more sensitive than GE and the sensitivity of clonality detection can be influenced by the amount of admixed normal lymphoid cells.
참고문헌 (16)
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