Identification and characterisation of clonal incomplete T-cell-receptor Vdelta2-Ddelta3/Ddelta2-Ddelta3 rearrangements by denaturing high-performance liquid chromatography and subsequent fragment collection: implications for minimal residual disease monitoring in childhood acute lymphoblastic leukemia

Incomplete T-cell-receptor delta (TCR-delta) 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-delta targets. Clonality of PCR amplified TCR-delta 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. Vdelta2-Ddelta3 and Ddelta2-Ddelta3 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 degrees 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.