Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study

Abstract

Background: Cryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and peripheral blood mononuclear cells.

Results: Paired samples of PBMC were processed freshly, or after cryopreservation in a Nalgene container (NC) or a controlled-rate freezer (CRF). Although there were some differences between the methods, cryopreservation inhibited PHA-induced IL-10 secretion and Der f 1-induced IL-2 secretion, and augmented PHA-induced IL-2 secretion and spontaneous secretion of TNF-alpha. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-gamma, TNF-alpha) by PHA-stimulated cord blood mononuclear cells. With the exception of PHA-induced IL-13, results from fresh and cryopreserved cord blood samples were not significantly correlated. Finally, in reproducibility studies involving processing of identical cell samples in up to 4 separate laboratories, variances in cytokine responses of fresh cells stimulated at separate sites did not exceed those in cryopreserved cells stimulated at a central site.

Conclusion: Collectively, these studies indicate that cryopreservation can affect mononuclear cell cytokine response profiles, and that IL-10 secretion and antigen-induced responses may be especially vulnerable. These studies also demonstrate that mononuclear cell responses can be standardized for performance in a small number of laboratories for multicenter studies, and underscore the importance of measuring reproducibility and of testing whether cryopreservation techniques alter specific immunologic outcomes.

Figures

Figure 1

Effect of cryopreservation on PBMC secretion of IL-10 and IL-2. Mean response ratios were calculated for IL-10 (A) and IL-2 (B) for cells cryopreserved with either the Nalgene or constant rate freezer methods. Whiskers represent the 95% confidence intervals for difference. See methods for details of statistical analysis.

Figure 2

Effect of cryopreservation on PBMC cytokine responses. Data points represent cytokine responses by the same subjects' PBMC either cultured fresh or after cryopreservation. A, C. cells were stimulated for Each data point represents the comparison of the same subject at the same time point. Data shown include up to 3 time points per subject. The dotted line represents perfect agreement or concordance, and the solid line is best fit to data. Rho = Pearson correlation; CCC = Concordance Correlation Coefficient. See methods for details of statistical analysis. * p < 0.05; ** p < 0.01; ***p < 0.001.

Figure 3

Effect of cryopreservation on CBMC cytokine responses. Data points represent cytokine responses by the same subjects' CBMC either cultured fresh or after cryopreservation. The dotted line represents perfect agreement or concordance, and the solid line is best fit to data. Rho = Pearson correlation; CCC = Concordance Correlation Coefficient. See methods for details of statistical analysis. * p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

Reproducibility of cytokine responses of fresh vs. cryopreserved cells. Data points represent cytokine responses from replicate PBMC cultures. The dotted line represents perfect agreement or concordance, and the solid line is best fit to data. Rho = Pearson correlation; CCC = Concordance Correlation Coefficient. See methods for details of statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Four-site reproducibility study design.

Figure 6

Reproducibility of cytokine responses in central vs. peripheral processing protocols. Cytokine responses from PHA, TT and LPS stimulated cells cultured at each local site as fresh cells or cultured centrally following cryopreservation and shipment from local sites. Units are pg/ml. Three separate experiments are shown as runs 1–3.