The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4 ؉ and CD8 ؉ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at ؊70°C for <3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at ؊70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.

The undecapeptide substance P (SP) is a member of the tachykinin family of neurotransmitters, which has a pivotal role in the regulation of inflammatory and immune responses. One of the major barriers to the study of the in vivo role of SP in a number of immune disorders is the accurate measurement of SP in fluids. This is reflected in the variability of reported SP levels in serum and plasma of humans in both healthy and diseased states. This study was initiated in order to identify sources of variability by the comparative evaluation of the influences of sample preparation and analytical detection methods on the measurement of SP in plasma. The results indicate that sample preparation (peptide extraction versus no extraction) and the choice of analytical method for SP quantitation may yield significantly different values and may contribute to the variability in SP values reported in the literature. These results further emphasize the need for careful consideration in the selection of methods for SP quantitation, as well as caution in the interpretation and comparison of data reported in the literature.