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(American Journal of Pathology. 2001;159:2031-2043.)
© 2001 American Society for Investigative Pathology

Technical Advance

Simultaneous Evaluation of T- and B-Cell Clonality, t(11;14) and t(14;18), in a Single Reaction by a Four-Color Multiplex Polymerase Chain Reaction Assay and Automated High-Resolution Fragment Analysis

A Method for the Rapid Molecular Diagnosis of Lymphoproliferative Disorders Applicable to Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissues, Blood, and Bone Marrow Aspirates

Verena S. Meier, Alex Rufle and Fred Gudat

From the Institute for Pathology, University of Basel, Basel, Switzerland

Current polymerase chain reaction (PCR) methods for the molecular diagnosis of B- and T-cell lymphomas by determination of clonality of immunoglobulin heavy chain (IgH) and T-cell receptor-{gamma} rearrangements and by detection of the chromosomal translocations t(14;18) and t(11;14), require several laborious and costly PCR assays for each of these diagnostic tests. We have developed a multiplex PCR assay for the simultaneous determination of B- and T-cell clonality and the detection of the chromosomal translocations t(14;18) and t(11;14) in a single reaction, using four-color fluorescence and automated high-resolution fragment analysis. The 26 primers combined in the multiplex PCR correspond to the sequences of >90% of the 69 variables and 6 join IgH genes and 100% of the T-cell receptor-{gamma} variables and join genes that could participate in the respective rearrangements. In addition, they detect the major and the minor breakpoint regions of the t(14;18) and the major breakpoint region of the t(11;14), and amplify the ß-globin gene as an internal control. The specificity of the multiplex PCR was confirmedby analysis of 39 T-cell lymphomas and 58 B-cell lymphomas, including 11 mantle cell lymphomas bearing the t(11;14) and 25 follicular lymphomas bearing the t(14;18), with known rearrangements and/or translocations. Fifteen samples of reactive lymphadenitis remained negative.




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