Recombinant TechnologyQuantification of T-cell receptor excision circle DNA using fluorescence resonance energy transfer and the LightCycler system
Introduction
Myeloablative chemotherapy followed by allogeneic stem cell transplantation is associated with a prolonged and substantial, potentially detrimental period of T-cell immunodeficiency Keever et al., 1989, Bomberger et al., 1998. This depletion of T-cells contributes to an increased incidence of opportunistic infections, such as viral (e.g. cytomegalovirus) and fungal infections (e.g. invasive aspergillosis), (Socie et al., 1999). Reconstitution of T-cells depends on either the expansion of preexisting memory T-cells or the generation of new T-cells from haematopoietic stem cells (Bomberger et al., 1998). Complete reconstitution of immunity requires a thymus-dependent generation of de novo T-cells with a broad T-cell receptor repertoire (Heitger et al., 1997). Undergoing age-related involution, the thymus is gradually replaced by adipose tissue but still remains active in adults (Mackall and Gress, 1997). Recent studies have shown that the activity of the thymus is age-dependent and thymopoiesis is severely limited after childhood Douek et al., 1998, Small et al., 1999, Zhang et al., 1999. Factors that inhibit thymic function are graft-versus-host disease and direct damage from chemo- and or radiotherapy (Weinberg et al., 2001). Measuring thymopoietic capacity only on the basis of phenotyping of naı̈ve T-cells is of limited value as CD45RA+ T-cells may immediately convert into memory T-cells (Picker et al., 1993), may proliferate antigen independently (Soares et al., 1998) or may persist most of their life span (Tough and Sprent, 1994). An alternative approach to monitoring thymopoiesis is to measure the frequency of T-cell receptor excision circles (TRECs) among peripheral blood cells Douek et al., 1998, Douek et al., 2000.
T-cell receptor excision circles (TRECs) are generated during V(D)J gene recombination, a process responsible for diversity of the T-cell receptor repertoire (Al-Harthi et al., 2000). T-cell receptor excision circles are circular extrachromosomal DNA fragments which are stable, do not replicate with cellular proliferation and are thus diluted with every cell division. In consequence, the number of TRECs is a potential marker permitting estimates of thymic output (Douek et al., 1998).
In this study we developed and evaluated a sensitive, rapid and simple real-time PCR assay based on the LightCycler technique to quantify the amount of TRECs among peripheral blood cells. We compared the number of TRECs found in cord blood to the number of TRECs in blood specimens obtained from healthy individuals aged between 1 and 80 years (n=52) and patients (n=13) after allogeneic stem cell transplantation. Furthermore, we compared the results of the LightCycler assay to a conventional quantitative PCR based on the PCR-ELISA technique.
Section snippets
Patients
Patients were recruited from the University of Tuebingen, Medical Hospital (n=7) and Children's Hospital (n=6). The patients received bone marrow (n=8) or peripheral blood stem cells (n=5) from an allogeneic donor and were conditioned with either busulfan-cyclophosphamide (n=6), total body irradiation and cyclophosphamide (n=5) or total body irradiation, etoposide and cyclophosphamide (n=2). The transplantation was performed for the treatment of acute leukemia (n=8), chronic myelogenous
Results
For quantification of the δRec–ψJα Signal Joint TREC DNA, serially diluted plasmids ranging between 2×109 and 2×100 copies were amplified by LightCycler and PCR-ELISA assays. By LightCycler, a sensitivity of 2×101 copies was achieved (Fig. 1a), whereas by PCR-ELISA, a detection limit of 2×100 copies could be demonstrated. The LightCycler assay showed linearity between 2×109 and 2×101 copies of plasmid DNA; linear regression was obtained over the whole range (r=−1.0, error=0.218, slope=−3.484,
Discussion
We have developed a simple, rapid and very sensitive PCR protocol for the quantification of δRec–ψJα Signal Joint TREC DNA using the LightCycler technique. The assay was shown to have a sensitivity of 20 copies of plasmid δRec–ψJα Signal Joint TREC DNA. Previously described assays achieved a reproducible detection limit of 500 copies of target DNA (Al-Harthi et al., 2000).
Rapid amplification by alternating heated air and air of ambient temperature, and online quantification allowed the test to
Acknowledgements
We thank Dr. T. Orlikowski from the Department of Neonatology of the University of Tuebingen for the cord blood samples.
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