Identification and characterisation of clonal incomplete T-cell-receptor Vδ2–Dδ3/Dδ2-Dδ3 rearrangements by denaturing high-performance liquid chromatography and subsequent fragment collection: implications for minimal residual disease monitoring in childh

Incomplete T-cell-receptor δ (TCR-δ) rearrangements are widely used for detection of minimal residual disease in childhood acute lymphoblastic leukemia. In a substantial number of cases both alleles are rearranged and polymerase chain reaction (PCR) products have either to be cloned or excised and reamplified from acrylamide gels. Here we describe a novel HPLC-based method for identification and characterization of clonal TCR-δ targets. Clonality of PCR amplified TCR-δ products was examined on a high-resolution micropellicular DNASep matrix (WAVE Nucleic Acid Fragment Analysis System, Transgenomic) and subsequently classified into clonal, biclonal or negative samples. Vδ2-Dδ3 and Dδ2-Dδ3 rearrangements were analyzed by denaturing high-performance liquid chromatography (DHPLC), using triethylammonium acetate as an ion-pairing reagent, with a linear acetonitrile gradient at 50 °C. Biclonal elution profiles consisted of two individual homoduplex peaks and one heteroduplex peak unique for each patient sample. For characterization of biclonal rearrangements DHPLC separated samples were subjected to a second run and individual clonal peaks were collected. A software-controlled fragment collector was arranged in tandem with the HPLC system for this purpose and purified PCR products were collected in a time-dependent manner. Fractions were air dried and subsequently sequenced directly. The specificity of the observed patient specific sequences was tested via real time quantitative PCR on a LightCycler system.