Stability of T cell phenotype and functional assays following heparinized umbilical cord blood collection

Abstract

Umbilical cord blood has been used for a wide variety of immunologic investigations including assessments of developmental perturbations by antenatal exposures. Recent advances in multiparameter flow cytometry have allowed finer characterization of lymphocyte phenotype and function, revealing important differences between the fetal and adult immune systems. The degree of variability between human subjects confounds the ability to draw firm conclusions. Artifacts resulting from processing techniques exacerbate this variability. The unpredictable nature of deliveries, especially of premature infants, makes it difficult to control variables such as timing of umbilical cord mononuclear cell (UCMC) isolation and method of collection. Additionally, in multicenter studies dependent on central processing, delays are inevitable. However, little available literature describes systematic testing of the degree to which processing variations affect UCMC phenotype and function. Using multiparameter flow cytometry, we tested the effect of collection technique and length of time prior to UCMC isolation on T cell phenotype and function, with the goal of creating a standardized operating procedure for a multicenter investigation. The study also provides a benchmark data set including extensive surface and functional phenotyping of umbilical cord T cells. UCMC isolation delay of up to 24 h produced similar T cell phenotype and function as tested by in vitro SEB stimulation. There were few statistically significant differences between time points based on data medians. We conclude that, for the purpose of immunologic investigations, a 24-h time delay from sample collection to mononuclear cell isolation does not introduce a significant degree of variation in T cell phenotype and function when adhering to strict standard operating procedures.

Figure 1

Mononuclear cell count and viability measured prior to cryopreservation (a) and after thawing (b). Cell numbers and percents are shown for processing time points of 0, 24, and 48 h. Each symbol represents a single subject. Comparisons were made between processing delay of 0 h (T0), 24 h (T24), and 48 h (T48) using two-tailed paired t tests. Differences between time points were not significant.

Figure 2

Mononuclear cell population frequencies measured by flow cytometry. Cord blood mononuclear cells were subtyped and expressed as a percentage of total events using live/dead, anti-CD235 (RBC marker), anti-CD14 (monocytes marker), anti-CD56 (NK marker), and anti-CD3 (T cell marker). Each symbol represents a single subject. Lines show medians within boxed interquartile range. Statistical comparisons were performed using Wilcoxon signed-rank test between samples isolated after delay of 0 h (T0), 24 h (T24), and 48 h (T48). There were no significant differences after a 24-h delay, but a significant decrease in CD56hi and CD3+ frequencies was observed after 48-h delay (P < 0.05).

Figure 3

T cell subset frequencies measured by flow cytometry. Cord blood T cells were identified using anti-CD3+ antibody. Frequencies of CD3+ T cell subsets were further identified using anti-CD4, anti-CD8, and anti-TCRγδ. Frequencies are expressed as percentages of CD3+ events. Each symbol represents an individual subject. Lines show medians within boxed interquartile range. Statistical comparisons were performed using Wilcoxon signed-rank test between samples isolated after delay of 0 h (T0), 24 h (T24), and 48 h (T48). No significant differences were detected after delay to processing of 24 or 48 h.

Figure 4

Cord blood CD41 T cell cytokine production following in vitro stimulation. Cord blood CD4+ T cells were stimulated in vitro with SEB, permeabilized and stained for intracellular cytokines IFNγ, TNFα, IL2, IL4/5/13, MIP1β, and IL17. Only events within the CD69+ gate were considered cytokine-positive. Frequencies are expressed as percentages of total CD4+ events. Each symbol represents a single subject. Statistical comparisons were performed using Wilcoxon signed-rank test between samples isolated after delay of 0 h (T0), 24 h (T24), and 48 h (T48). Significant differences were found at T24 and T48 for IL2 and MIP1β (P < 0.05) only.

Figure 5

Cord blood CD8+ T cell cytokine production following in vitro stimulation. Cord blood CD8+ T cells were stimulated in vitro with SEB, permeabilized and stained for intracellular cytokines IFNγ, TNFα, IL2, MIP1β. Only events within the CD69+ gate were considered cytokine-positive. Frequencies are expressed as percentages of total CD8+ events. Each symbol represents a single subject. Statistical comparisons were performed using Wilcoxon signed-rank test between samples isolated after delay of 0 h (T0), 24 h (T24), and 48 h (T48). Significant differences were found at T24 and T48 for IL2 (P < 0.05) only.