Thymic output: Assessment of CD4⁺ recent thymic emigrants and T-Cell receptor excision circles in infants

CD4⁺ recent thymic emigrants (CD4⁺ RTEs) constitute a subset of T cells recently generated in the thymus and exported into peripheral blood. CD4⁺ RTEs have increased copy numbers of T-cell receptor excision circles (TREC). They are characterized by the expression of CD31 on naïve CD4 T-cells. We aimed to validate a flow-cytometry assay to enumerate CD4⁺ RTEs and assess its performance in relation to TREC measurement.

CD4⁺ RTEs cell count in peripheral blood was measured to determine sample stability, precision, linearity, and to establish reference ranges. TRECs were measured using qPCR assay performed with DNA isolated from peripheral blood. CD4⁺ RTEs, TRECs, and flow cytometry results for major T-cell markers were assessed in 50 infants less than 2 years of age.

Inter-and intra-assay precisions (% CV) were 1.5-12.2 and 1.5-7.0, respectively. Linearity studies showed that the results are linear over a range of 0.7 to 403.0 CD4⁺ RTEs/μL of blood. There was 84% agreement (42 of 50) between CD4⁺ RTEs and TRECs qualitative results for the infant samples. CD4⁺ RTEs reference ranges in 17 healthy children was in agreement with published data, while that of the healthy adults were 51-609 cells/μL of blood.

The validation results provide acceptable measures of the CD4⁺ RTEs test performance within CAP/CLIA frameworks. CD4⁺ RTEs and TRECs assays show high agreement in the infant population. The CD4⁺ RTEs test can be used as a confirmation for the TREC results along with or as an alternative to T-cell phenotyping in infants with repeatedly low TRECs concentrations. © 2015 International Clinical Cytometry Society.