Methodology article

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

Wayne G Shreffler¹ , Cynthia M Visness² , Melissa Burger³ , William W Cruikshank⁴ , Howard M Lederman⁵ , Maite de la Morena⁶ , Kristine Grindle³ , Agustin Calatroni² , Hugh A Sampson¹ and James E Gern³

¹  Mount Sinai School of Medicine, Division of Pediatric Allergy & Immunology, New York, NY, USA

²  Rho Inc., Chapel Hill, NC, USA

³  Department of Pediatrics, University of Wisconsin Hospital, Madison, WI, USA

⁴  Pulmonary Center, Boston University School of Medicine, Boston, MA, USA

⁵  Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

⁶  Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA

author email corresponding author email

BMC Immunology 2006, 7:29doi:10.1186/1471-2172-7-29

Published:	12 December 2006

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-α. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-γ, TNF-α) 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.