Editing TREC ASSAY (section)

== Introduction ==
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T-cell receptor excision circles (TRECs) and K-deleting&nbsp;recombination excision circles (KRECs) are small circularized DNA elements that are excised in a proportion of T- and B-cells, respectively, during a genomic DNA recombination process, leading to the formation of a highly diversified repertoire of T- and B-cell receptors. They have no function, but because they are stable and cannot be replicated, they are diluted after each cell division, thus persisting only in one of the two daughter cells. Therefore, their levels in the peripheral blood can be assumed as an estimate of the thymic and bone marrow output.

While the TREC assay has been largely used over the last 15 years to&nbsp;evaluate the extent of thymic output,1 the KREC assay, which was initially developed to measure B-cell proliferation and its contribution to B-cell homeostasis in health and disease,2 has been only recently proposed as a marker of bone marrow output.3,4 Here, we describe the method we developed for the simultaneous quantification of both TRECs and KRECs.4 With this combined method, the variability associated to DNA quantification by real-time PCR is eliminated by the use of a unique standard curve obtained by diluting a triple-insert plasmid containing fragments of TRECs, KRECs and T-cell receptor alpha constant (TCRAC) gene in a 1:1:1 ratio. This allows a more accurate evaluation of the TREC and KREC copy number. Furthermore, the simultaneous quantification of the two targets in the same reaction allows reagent cost reduction.

The proposed TREC/KREC assay can be useful to measure the extent of&nbsp;T- and B-cell neo-production in children or adults with Severe Combined Immunodeficiency (SCID),4 Common Variable Immunodeficiency,5 autoimmune diseases,6-8 and HIV infection.9 Furthermore, it can be used to monitor the immune recovery after hematopoietic stem cell transplantation,10 enzyme replacement,11 and antiviral9 or immunomodulating therapies.6-8 Finally, because SCID patients are recognized using TREC assay despite the underlying genetic defects, and agammaglobulinemia patients can be identified using KREC quantification, the TREC/KREC assay can be also used to detect immunodeficiencies in newborn screening programs.12 In this case, the test must be performed on DNA extracted from small spots of blood blotted and dried on filter paper, must be highly sensitive and specific for the target diseases, as well as highly reproducible and cost-effective. The introduction of KREC quantification in the test should improve&nbsp;performances of newborn screening for immunodeficiencies, which has routinely been performed in the some parts of the USA (WI, MA, CA) since 2008 when Wisconsin became the first to introduce the analysis of
TRECs in its postnatal screening program.13</div>