Method Description

This assay involves pre-analytical preparation of a pure cell population followed by analytical evaluation of the DNA. A modified peripheral blood mononuclear cells (PBMC) isolation is used to prepare a nearly pure population of CD3+ T cells (adults) or total lymphocytes (pediatrics) from whole blood. The resulting purity and cell counts are obtained from the TCD4 flow cytometric assay. The cells are then lysed with Proteinase K to a predetermined target concentration to release and expose the DNA for polymerase chain reaction (PCR). The genomic DNA and T-cell receptor excision circles (TREC) in the cell lysates are quantified in the real-time PCR assay, in triplicate, by using a fluorescent probe specific for the T-cell receptor delta-deletion TREC signal joint and a distinct fluorescent probe for the reference gene, albumin. There is one copy of TREC per CD3+ T cell, while there are 2 copies of albumin in every cell. A standard curve is used to determine the absolute quantity of TREC and albumin from the fluorescence intensities measured. The albumin counts are used to determine the cell counts in each reaction and to normalize the number of TREC copies to a standard reporting unit of copies per million CD3+ T cells. The pediatric TREC counts, though measured from total lymphocytes, can be adjusted to the same reporting units using the %CD3 purity from the flow cytometric assay.(Douek DC, Vescio RA, Betts MR, et al: Assessment of thymic output in adults after hematopoietic stem cell transplantation and prediction of T cell reconstitution. Lancet. 2000;355:1875-1881; Douek DC, Hill B: Personal Communication; 2005; Gaballa A, Clave E, Uhlin M, Toubert A, Arruda LCM: Evaluating thymic function after human hematopoietic stem cell transplantation in the personalized medicine era. Front Immunol. 2020 Jul 31;11:1341. doi: 10.3389/fimmu.2020.01341)