Research paper Measuring the TREC ratio in dried blood spot samples: Intra- and inter-filter paper cards reproducibility ☆ P.O. Lang a, b, ⁎, S. Govind a , M. Dramé c , R. Aspinall a a Translational Medicine Research group, Cranfield Health, Cranfield University, Cranfield, UK b Nescens Centre of preventive medicine, Clinic of Genolier, Genolier, Switzerland c Methodological assistance unit, Department of Research and Innovation, Robert Debré Hospital, Reims teaching Hospitals, Reims, France article info abstract Article history: Received 19 November 2012 Received in revised form 6 December 2012 Accepted 10 December 2012 Available online 9 January 2013 The level of T-cell receptor excision circles (TREC), which decline with advancing age in normal individuals, has recently gained interest as a reference marker for studies on premature or early immunosenescence under particular health conditions. In order to facilitate translational studies at population and clinical levels, essential for the understanding of how changes in TREC levels are associated with responsiveness of the immune system, we have developed and optimized a real-time polymerase chain reaction (qPCR) assay which quantifies the TREC ratio from dried blood spot (DBS) samples. The present study considers a fully automated procedure to purify DNA and amplify sequences of interests by means of qPCR from DBS samples collected in healthy adults. Both TREC:PBMC (peripheral blood mononuclear cell) and TREC:T-cell ratios were compared for intra- and inter individual reproducibility. Furthermore, the impact of the length of storage on the quality of the DNA generated was also analyzed. In conclusion we describe a fully automated procedure for extracting DNA and qPCR set up, which offers a high-precision, robust qPCR assay for the quantification of both TREC:T-cell ratio and TREC:PBMC from DBS samples. © 2012 Elsevier B.V. All rights reserved. Keywords: TREC ratio Thymic TREC Dried blood spots Reproducibility Real time PCR Quantitative PCR 1. Introduction To ensure longevity and healthy life the maintenance of appropriate immunity is fundamental. Age- or disease- related changes of the immune system are of particular im- portance, contributing to the higher incidence and severity of infectious diseases, reduced efficacy of vaccination and possibly autoimmunity and cancer (Schneider, 2010). While these changes can affect many components of both the innate as well as adaptive immunity (Arnold et al., 2011) one of the most prominent features of the immune senescence process is changes in the composition of the T-cell compartment and particularly the decrease in antigen-inexperienced naïve T-cells (Lang et al., 2012a). This is also obvious in individuals suffering from combined immunodeficiency (Arnold et al., 2011; van Zelm et al., 2011; Lang et al., 2012a), in HIV-infected and lymphopenic cancer patients and at the end-stage of chronic renal diseases (Douek et al., 2000; Hazenberg et al., 2000, 2002; Betjes et al., 2011). Predicting individual immune responsiveness to any antigen using biological markers that distinguish between healthy and “immunologically vulnerable” states is desirable. To establish early identification of such individuals including an estimation of future complication risk, standardized and reliable assays are required. One of the newly explored methods involves quantification of circular DNA products that are generated during T-lymphocyte development with the creation of T-cell receptors (TCR) (van Zelm et al., 2011). This receptor is employed by naïve T-lymphocytes to recognize foreign anti- gens. In order to create a boarder repertoire of TCR molecules, each immature T-lymphocyte during intra-thymic develop- ment undergoes unique somatic rearrangements in its TCR loci (Lang et al., 2012a). During this rearrangement process, the intervening DNA sequences are deleted and circularized into Journal of Immunological Methods 389 (2013) 1–8 ☆ Conflict of interest: None of the authors declare conflict of interests for this manuscript. ⁎ Corresponding author at: Nescens, Centre of preventive medicine, c/o Clinique of Genolier, Route du Muids 3, CH-1272 Genolier, Switzerland. Tel.: + 41 22 366 93 09; fax: + 41 22 366 93 49. E-mail address: polang@nescens.com (P.O. Lang). 0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jim.2012.12.003 Contents lists available at SciVerse ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim