Flow Cytometric Detection of Intracellular Th1/Th2 Cytokines Using Whole Blood: Validation of Immunologic Biomarker for Use in Epidemiologic Studies

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Abstract
Few biological markers of immune function have been thoroughly validated for use in epidemiologic studies that involve delayed sample processing and analysis. Here, we report our validation results for flow cytometric detection of intracellular T-helper 1/T-helper 2 (Th1/Th2) cytokines using 500 μL of whole blood obtained from children and adults. The detection of Th1/Th2 cytokine profiles by flow cytometry is a practical and mechanistically relevant assay because dysregulated cytokine production has been observed in many immune-mediated disorders, including cancer. We evaluated the intraassay and intraindividual and interindividual variability and the effects of a 24- to 72-hour delayed analysis on Th1 and Th2 end points. We compared the distributions of %CD4 lymphocytes, %Th1, and %Th2 in young children (age 1 year, n = 50) and adults (age 25–52 years, n = 16). Subjects sampled monthly for up to 1 year showed minimal variation in CD4, Th1, and Th2 end points. Delayed analysis of samples (up to 24 hours) resulted in no significant differences in the expression of CD4, Th1, and Th2; however, at 48 and 72 hours, all end points differed significantly from baseline (P < 0.01). A random effects model confirmed that interindividual variability was much greater than intraindividual variability for CD4 and Th1. Compared with adults, children had marginally higher %CD4, similar %Th2, but significantly lower %Th1 (P < 0.01). These results show that flow cytometric detection of CD4, Th1, and Th2 markers using whole blood is reproducible and that these biomarkers can be effectively used in human population studies that involve transported samples, delayed processing and analysis, and limited blood volumes.