INTRODUCTION: Studying pediatric patients includes challenges such as difficulty obtaining samples, limited normal controls and small volumes. Standardized flow cytometry (FC) lymphocyte immunophenotyping panels are commercially available and normal adult ranges are published by the ONE Study group; no comparable pediatric control data exist. Our objective is to establish a reference dataset for pediatric studies using standardized, comprehensive FC panels. Our group collaborated with the local clinical laboratory to obtain normal pediatric samples with the goal of establishing comprehensive pediatric reference data. This study also provides a pilot study for clinical implementation.

METHODS: Using 700uL of EDTA blood, DuraClone FC phenotyping was performed on healthy pediatric controls aged from 66 days to 16 years (n=10). Samples were tested in five, 10-colour FC T and B cell panels. Acquisition was performed on a Navios cytometer. The markers programmed cell death protein 1 (PD-1), a measure of T cell exhaustion, and gamma/delta (γδ) T cells and associated markers Vd1 and Vd2, were analysed.

RESULTS: Preliminary analyses show age related trends in PD-1+ T cells with increasing PD-1+ CD4 T cells with age (R²=0.701); this association is much weaker for CD8 T cells. The number of CD3+ T cells, including γδ T cells, decreases with age, while the % of each population of cells remains constant. There is a trend indicating decreasing ratio of V delta 1 to V delta 2 γδ T cells with age.

CONCLUSION: Duraclone is a rapid, standardized immunophenotyping method using only small blood volumes. These results begin to establish useful reference data for pediatric patients and suggest these FC panels show promise for application in the clinical laboratory. Our use of fresh whole blood differs from published pediatric reference range studies utilizing frozen cells and ensures populations are not lost/altered during mononuclear cell isolation or thaw. We continue to explore age-related trends and test additional samples. This study highlights the value of partnership between research and clinical laboratories.

CITATION INFORMATION: Sosniuk M., Halpin A., Wizniak J., Szkotak A., Urschel S., Ionescu L., West L. What's Normal? An Evaluation of Pediatric T Cell Markers in Healthy Controls Am J Transplant. 2017;17 (suppl 3).

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