Quantitative Real-Time PCR Method for Detection of B-Lymphocyte Monoclonality by Comparison of κ and λ Immunoglobulin Light Chain Expression

Background: An abnormal IgL:IgL ratio has long been used as a clinical criterion for non-Hodgkin B-cell lymphomas. As a first step toward a quantitative realtime PCR-based multimarker diagnostic analysis of lymphomas, we have developed a method for determination of IgL:IgL ratio in clinical samples. Methods: Light-up probe-based real-time PCR was used to quantify IgL and IgL cDNA from 32 clinical samples. The samples were also investigated by routine immunohistochemical analysis and flow cytometry. Results: Of 32 suspected non-Hodgkin lymphoma samples analyzed, 28 were correctly assigned from real-time PCR measurements assuming invariant PCR efficiencies in the biological samples. Four samples were false negatives. One was a T-cell lymphoma, one was a diffuse large B-cell lymphoma, and one was reanalyzed and found lymphoma-positive by in situ calibration, which takes into account sample-specific PCR inhibition. Twelve of the samples were fine-needle aspirates, and these were all correctly assigned. Conclusions: This work is a first step toward analyzing clinical samples by quantitative light-up probe-based real-time PCR. Quantitative real-time PCR appears suitable for high-throughput testing of cancers by measuring expression of tumor markers in fine-needle aspirates. The rapid expansion in knowledge of the human genome and the development of techniques for analysis of nucleic acids have opened new possibilities for diagnostics. In our first attempt to use quantitative real-time PCR (Q-PCR) 5 for detection of malignant tumors, we have focused on the analysis of non-Hodgkin lymphomas (NHLs).