A rapid restriction fragment length polymorphism polymerase chain reaction-based diagnostic method for identification...

Identification of a clonal proliferation of lymphocytes is central to the diagnosis of lymphoma compared with a reactive lymphoproliferation. We propose a novel diagnostic technique based on restriction fragment length polymorphism (RFLP) of amplified polymerase chain reaction (PCR) products of the T-cell receptor -gamma (TCR-gamma) gene rearrangement to rapidly identify monoclonality in T-cell lymphomas and improve diagnosis of malignancy.

DNA from peripheral blood mononuclear cells (PBMCs) of 10 healthy volunteers and 7 T-cell lymphoma patients were isolated and the TCR-gamma was amplified with consensus primers for the different variable (V) and joining (J) segments. Restriction digests were done using BstN1 and the fragments separated via gel electrophoresis. Verification was by Southern analysis.

Restriction digests of the 10 healthy controls show a characteristic nine-band digest pattern whereas the restriction digests of the 7 T-cell lymphomas each show altered banding patterns completely distinct from the normal nine-band pattern (Fisher exact test = 0.00005). Sensitivity assays demonstrate the test can detect clonal populations representing 2% of total. This method also enables identification of particular clonal populations. The entire procedure can be performed in one day, does not require radioactivity, and requires only small quantities of specimens.

This RFLP-PCR-based diagnostic method for T-cell lymphomas is specific, sensitive, efficient, and reproducible, and enables the identification of clonally expanded populations of T lymphocytes. It offers the ability to identify particular clonal populations, as with Southern analysis, combined with the benefits of a PCR method.