Quantification of T-cell receptor excision circle DNA using fluorescence resonance energy transfer and the LightCycler system

T-cell receptor excision circles (TRECs) are circular, stable extrachromosomal DNA fragments and are generated during V(D)J gene recombination, a process responsible for diversity of the T-cell receptor repertoire. Here we describe a sensitive, rapid and easy to perform real-time PCR assay based on the LightCycler technique for the quantification of TRECs among peripheral blood cells and a comparison of this assay with conventional PCR-ELISA. By LightCycler, a sensitivity of 20 copies of plasmid deltaRec-psiJa Signal Joint TREC DNA was achieved whereas by PCR-ELISA, a detection limit of 2 copies was demonstrated. In blood samples from healthy individuals (n = 52) a median TREC count of 1.6 x 10(4) copies [range 2 x 10(1) to 2 x 10(5)]/2 x 10(5) peripheral blood mononuclear cells (PBMNCs) and in cord blood, a median TREC count of 1.45 x 10(5) copies [range 1.2 x 10(5) to 1.6 x 10(5)]/2 x 10(5) PBMNCs) could be detected. No significant difference was found in 15 individuals when unfractionated PBMNCs (median count of 1.1 x 10(5) copies/2 x 10(5) PBMNCs) or magnetic associated cell sorter (MACS)-sorted CD45RA+ T-cells (median count of 2.5 x 10(5) copies/2 x 10(5) cells) were analyzed for TREC counts. In addition, we examined the number of deltaRec-psiJa Signal Joint TREC in paediatric (n = 6) and adult patients (n = 7) after allogeneic stem cell transplantation. In children, we observed a median TREC count of 5.7 x 10(4) copies/2 x 10(5) PBMNCs after 1 x 10(4) and 5.6 x 10(4) copies/2 x 10(5) PBMNCs after 2 years, and in adults, a median count of 3.6 x 10(4) copies/2 x 10(5) PBMNCs after 1 x 10(4) and 1.1 x 10(4) copies/2 x 10(5) PBMNCs after 2 years. In conclusion, the LightCycler-based real-time PCR assay described offers a very sensitive and rapid tool for the quantification of TREC DNA. (C) 2002 Elsevier Science B.V. All rights reserved.