Quantitative Real-time PCR Method for Detection of B-lymphocyte Monoclonality by Comparison of Kappa and Lambda Immunoglobulin Light Chain Expression

Background: An abnormal IgLkappa:IgLlambda ratio has long been used as a clinical criterion for non-Hodgkin B-cell lymphomas. As a first step toward a quantitative real-time PCR-based multimarker diagnostic analysis of lymphomas, we have developed a method for determination of IgLkappa:IgLlambda ratio in clinical samples.

Methods: Light-up probe-based real-time PCR was used to quantify IgLkappa and IgLlambda 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.