Large-scale prospective T cell function assays in shipped, unfrozen blood samples: experiences from the multicenter TRIGR trial

Abstract

Broad consensus assigns T lymphocytes fundamental roles in inflammatory, infectious, and autoimmune diseases. However, clinical investigations have lacked fully characterized and validated procedures, equivalent to those of widely practiced biochemical tests with established clinical roles, for measuring core T cell functions. The Trial to Reduce Insulin-dependent diabetes mellitus in the Genetically at Risk (TRIGR) type 1 diabetes prevention trial used consecutive measurements of T cell proliferative responses in prospectively collected fresh heparinized blood samples shipped by courier within North America. In this article, we report on the quality control implications of this simple and pragmatic shipping practice and the interpretation of positive- and negative-control analytes in our assay. We used polyclonal and postvaccination responses in 4,919 samples to analyze the development of T cell immunocompetence. We have found that the vast majority of the samples were viable up to 3 days from the blood draw, yet meaningful responses were found in a proportion of those with longer travel times. Furthermore, the shipping time of uncooled samples significantly decreased both the viabilities of the samples and the unstimulated cell counts in the viable samples. Also, subject age was significantly associated with the number of unstimulated cells and T cell proliferation to positive activators. Finally, we observed a pattern of statistically significant increases in T cell responses to tetanus toxin around the timing of infant vaccinations. This assay platform and shipping protocol satisfy the criteria for robust and reproducible long-term measurements of human T cell function, comparable to those of established blood biochemical tests. We present a stable technology for prospective disease-relevant T cell analysis in immunological diseases, vaccination medicine, and measurement of herd immunity.

FIG 1

Effect of transit time on the percentages of samples passing lab QC. A generalized estimating equation (GEE) accounting for repeated measures of the same subject showed that a trend effect of transit time on the proportion of samples passing QC was highly significant (P < 0.0001).

FIG 2

Decreasing median counts of unstimulated cells as shipping time increases, changes which were statistically significant using the Cuzick nonparametric test for trend (P = 0.005).

FIG 3

Replicate values for [³H]thymidine incorporation in unstimulated and stimulated microcultures, presented on the log₁₀ scale (cpm), show high concordance between wells. The Pearson correlation coefficients were 0.95 for cells alone, 0.94 for PHA, 0.91 for tetanus toxin, and 0.89 for ovalbumin (OVA). Each of the four correlations are significantly different from a rho of 0 (P < 0.0001).

FIG 4

Counts of cells alone (cpm), split by the year of the sample collection and age of subject (follow-up visit), with interquartile ranges, means, and medians. The graph excludes outliers and the relatively small number of results from 2002 to 2003 and 2008 to improve resolution of the other years. Counts of unstimulated PBMCs increase with subject age, and variability is reduced over the course of the study.

FIG 5

Proportion of samples which did not proliferate to the expected level for all three positive-control analytes falls during infancy, suggesting that an appropriate T cell response develops rapidly between 3 and 6 months of age.

FIG 6

Percentage of samples which proliferated to at least a stimulation index of 10 in response to PHA, by shipping time. The mean percentages were >90% up to and including 4 days in shipping. The trend over shipping time was statistically significant (Cuzick nonparametric test for trend, P = 0.0015).

FIG 7

Minimal progression of polyclonal proliferative purely T cell (anti-CD3) and broad lymphocyte lectin (PHA) responses from infancy to childhood. Horizontal lines mark different reference thresholds for positive reactions: SIs of 1.5, 4 (expected threshold for anti-CD3), and 10 (expected threshold for PHA).

FIG 8

The development of tetanus toxoid responses corresponds with the recommended vaccination schedules in the United States and Canada at 2, 4, 6, and 18 months. Means and medians are presented by diamonds and lines, respectively, while stimulation indices of 1.5 and 4 (expected threshold for tetanus toxoid) are shown by horizontal lines.