title: Optimization and limitations of use of cryopreserved peripheral blood mononuclear cells for functional and phenotypic T-cell characterization.
abstract: 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 degrees C for < or =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 degrees 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.
authors.affiliation: University of Colorado Denver School of Medicine, Denver, CO, USA. Adriana.Weinberg@uchsc.edu
authors.forename: Adriana
authors.initials: A
authors.lastname: Weinberg
authors.forename: Lin-Ye
authors.initials: LY
authors.lastname: Song
authors.forename: Cynthia
authors.initials: C
authors.lastname: Wilkening
authors.forename: Anne
authors.initials: A
authors.lastname: Sevin
authors.forename: Bruce
authors.initials: B
authors.lastname: Blais
authors.forename: Raul
authors.initials: R
authors.lastname: Louzao
authors.forename: Dana
authors.initials: D
authors.lastname: Stein
authors.forename: Patricia
authors.initials: P
authors.lastname: Defechereux
authors.forename: Deborah
authors.initials: D
authors.lastname: Durand
authors.forename: Eric
authors.initials: E
authors.lastname: Riedel
authors.forename: Nancy
authors.initials: N
authors.lastname: Raftery
authors.forename: Renee
authors.initials: R
authors.lastname: Jesser
authors.forename: Betty
authors.initials: B
authors.lastname: Brown
authors.forename: M Fran
authors.initials: MF
authors.lastname: Keller
authors.forename: Ruth
authors.initials: R
authors.lastname: Dickover
authors.forename: Elizabeth
authors.initials: E
authors.lastname: McFarland
authors.forename: Terence
authors.initials: T
authors.lastname: Fenton
mesh_terms: D001769:Blood / Q000276:immunology*; D002175:Candida / Q000276:immunology; D049109:Cell Proliferation; D002470:Cell Survival; D005434:Flow Cytometry / Q000379:methods; D005615:Freezing; D006805:Human Experimentation; D006801:Humans; D019041:L-Selectin / Q000032:analysis; D017493:Leukocyte Common Antigens / Q000032:analysis; D018655:Lymphocyte Count; D016131:Lymphocyte Subsets / Q000276:immunology; D011043:Pokeweed Mitogens / Q000276:immunology; D013048:Specimen Handling / Q000379:methods*; D013601:T-Lymphocytes / Q000276:immunology*; D013742:Tetanus / Q000276:immunology
chemical_list: D011043:Pokeweed Mitogens; D019041:L-Selectin; D017493:Leukocyte Common Antigens